Solène Irion
email: solene.irion@univ-littoral.fr

Urania Christaki

Hugo Berthelot

Stéphane L'helguen

Ludwig Jardillier

Small phytoplankton contribute greatly to CO2-fixation after the diatom bloom in the Southern Ocean

Keywords: CO2-fixation, NanoSIMS, Southern Ocean, small phytoplankton, diatoms

Côte d'Opale, la Région Hauts-de-France et le projet MOBYDICK (LEFE-CYBER 2017-2018, ANR 2018-2021).

Mes premiers remerciements s'adressent à mes deux directeurs de thèse. Merci pour la qualité scientifique de votre encadrement, vous avez écrit un projet de thèse novateur et ambitieux qui m'a immédiatement séduite. Merci pour la confiance que vous m'avez accordée, vous avez su m'accompagner avec bienveillance.

Merci à Hugo pour tes précieux conseils et ta bonne humeur lors des sessions NanoSIMS ; sans tes compétences, la partie NanoSIMS aurait été bien plus laborieuse.

Merci à l'équipe du LOG, en particulier à Ingrid et Ioli avec lesquelles j'ai toujours pris plaisir à travailler et discuter.

Merci à tous les participants à la campagne MOBYDICK, qui ont été d'une grande aide en mer et ont permis de partager un jeu de données de qualité. Je tiens à remercier en particulier Céline Dimer, qui a réalisé tous les prélèvements et traitements en laboratoire pour les analyses HPLC. Merci également à Bernard Quéguiner et Ingrid Obernosterer pour avoir porté le projet MOBYDICK.

Merci à toute l'équipe de l'ESE, qui m'a toujours accueillie chaleureusement, tout particulièrement à Purificación López-García et David Moreira pour avoir mis l'équipement de la plateforme UNICELL à ma disposition, ainsi qu'à Paola Bertolino, Hélène Timpano et Maria Ciobanu dont l'aide m'a été précieuse pour me former à la préparation des librairies de séquençage ou dans l'utilisation du cytomètre en flux.

Merci à Peter Magee pour les relectures et corrections des manuscrits en anglais.

Merci à mes stagiaires, Océane et Sébastien pour m'avoir aidée dans les comptages FISH et dans l'analyse de certaines images NanoSIMS.

Enfin, merci à tous mes proches sans qui cette thèse n'aurait pas été possible. Merci notamment à toutes les personnes qui ont pris soin de mon fils, Léandre, quand je n'étais pas disponible. Vous m'avez permis de profiter pleinement de cette expérience enrichissante, je vous en suis infiniment reconnaissante.

Résumé

Cette thèse s'intéresse à la diversité du petit phytoplancton (<20 µm) et à sa contribution à la fixation du carbone dans des écosystèmes marins contrastés : la région productive du plateau de Kerguelen d'une part et les zones à faible productivité en dehors du plateau, d'autre part. Le petit phytoplancton domine les communautés phytoplanctoniques tout au long de l'année en dehors du plateau, où les faibles concentrations en fer limitent la production primaire. Le plateau de Kerguelen, naturellement fertilisé en fer, est quant à lui caractérisé par le développement au printemps-été de blooms massifs de diatomées en chaîne et de grande taille. Depuis la découverte du mécanisme de fertilisation naturelle en fer sur le plateau, l'attention de la communauté scientifique s'est focalisée sur les diatomées de grande taille, qui favoriseraient la séquestration du carbone sur la zone. Toutefois, les données satellitaires suggéraient que le petit phytoplancton dominait les communautés phytoplanctoniques en dehors du bloom sur l'ensemble de la zone. Le premier objectif de ce travail visait à obtenir une image fine de la diversité du petit et grand phytoplancton après le bloom de diatomées (Mars 2018). Un fragment de l'ADNr 18S des communautés planctoniques de petite (0.2-20 µm) et grande taille (20-100 µm), collectées à plusieurs profondeurs, a été séquencé par la méthode Illumina MiSeq. Les séquences ainsi obtenues ont permis de déterminer la diversité taxonomique moléculaire du petit et grand phytoplancton. En surface, les diatomées étaient majoritaires dans la grande fraction de taille tandis que Phaeocystis antarctica était particulièrement important dans la petite fraction sur l'ensemble de la zone d'étude. Dans leur ensemble, les communautés de petit phytoplancton différaient sur et en dehors du plateau. Des concentrations élevées en acide silicique en dehors du plateau favorisaient la présence d'un assemblage varié de diatomées de petite taille, tandis que de fortes concentrations en ammonium sur le plateau pourraient favoriser le développement de picophytoplancton du genre Micromonas. L'utilisation de marqueurs pigmentaires chémotaxonomiques a permis de décrire la succession temporelle des communautés phytoplanctoniques sur le plateau, dominées par les diatomées du début au déclin du bloom, tandis que la contribution du petit phytoplancton augmentait fortement après le bloom (moins de 10% à 53% de la chlorophylle). Le deuxième objectif de cette thèse était d'établir la contribution relative de différents groupes phytoplanctoniques à la fixation globale de carbone, en prenant en compte les différences inter-et intra-groupe dans l'activité métabolique de cellules individuelles. Pour ce faire, des communautés planctoniques naturelles ont été incubées en présence d'un traceur isotopique (NaH 13 CO3) en reproduisant les conditions in situ. La fixation du carbone au niveau cellulaire a été mesurée par imagerie NanoSIMS et SIMS. Les grandes diatomées (> 20 µm) montraient des taux de croissance faibles et variables d'une cellule à l'autre, avec 19±13% de diatomées inactives. Inversement, les petites cellules, appartenant à des taxons phylogénétiques éloignés (prymnesiophytes, prasinophytes et petites diatomées) étaient majoritairement en croissance active (>98%). Par conséquent, le petit phytoplancton contribuait de 41 à 70% à la fixation du carbone sur l'ensemble de la zone après le bloom. Tandis que le petit phytoplancton contribuait de façon importante à la fixation de carbone et à la biomasse chlorophyllienne en surface, les diatomées dominaient dans les données pigmentaires et de séquençage en dessous de 200 m, indiquant leur export préférentiel par sédimentation directe. Cependant, un faisceau d'indices suggère que le phytoplancton de petite taille, en particulier Phaeocystis, pourrait participer à l'export de carbone par agrégation, ainsi que via les réseaux trophiques et la production de pelotes fécales des brouteurs.

Résumé détaillé en français Introduction

La diversité du phytoplancton

Les organismes phytoplanctoniques sont des microbes photoautotrophes qui vivent dans la couche euphotique des eaux douces ou des milieux marins. Au cours de la photosynthèse oxygénique, ils utilisent l'énergie lumineuse pour convertir le carbone inorganique en molécules de carbone organique riches en énergie. Ainsi, ils modulent activement les concentrations atmosphériques de dioxyde de carbone et d'oxygène. Le phytoplancton est composé d'une large gamme de taxons, des cyanobactéries aux eucaryotes, dont la taille varie considérablement (<1 à >100 µm), avec des représentants dans 4 des 6 super-groupes de l'arbre de vie eucaryote décrit par [START_REF] Adl | The new higher level classification of eukaryotes with emphasis on the taxonomy of protists[END_REF] (Fig. A2). Les diversités phylogénétique, de taille et de métabolisme des communautés de phytoplancton affectent la structure du réseau trophique et le cycle biogéochimique du carbone dans les océans.

Historiquement, 4 000 à 25 000 espèces de phytoplancton ont été décrites par observation morphologique au microscope [START_REF] Sournia | Marine phytoplankton: how many species in the world ocean?[END_REF][START_REF] Falkowski | The Evolution of Modern Eukaryotic Phytoplankton[END_REF]. Ce nombre sous-estime probablement largement l'étendue réelle de la diversité du phytoplancton et les approches moléculaires basées sur le séquençage du gène ARNr 18S ont révélé que le nombre réel de taxons de phytoplancton est systématiquement environ 10 fois plus élevé que les estimations basées sur la morphologie [START_REF] Pierella Karlusich | Phytoplankton in the Tara Ocean[END_REF]. Compte tenu de l'étendue de la complexité liée à l'évaluation de la diversité taxonomique du phytoplancton, le phytoplancton est souvent classé en fonction de la taille des cellules. La taille des cellules du phytoplancton s'étend sur au moins 9 ordres de grandeur, d'un volume cellulaire d'environ 0,1 µm 3 pour les plus petites cyanobactéries à plus de 10 8 µm 3 pour les plus grandes diatomées (Fig. A3).

La taille des cellules de phytoplancton est un critère très utile à la modélisation des cycles biogéochimiques, car les processus de croissance et de mortalité du phytoplancton dépendent fortement de la taille [START_REF] Marañón | Phytoplankton size structure[END_REF]. Schématiquement, on considère que les petites cellules phytoplanctoniques sont avantagées par rapport aux grandes pour l'acquisition de la lumière et des nutriments grâce à des ratios surface/volume avantageux. Inversement, la grande taille de certaines cellules leur offrirait une meilleure défense, au moins de façon temporaire, contre les brouteurs microzooplanctoniques. Ces principes généraux de croissance et mortalité déterminent la structure de taille des communautés phytoplanctoniques et expliquent la dominance globale du petit phytoplancton dans les eaux tropicales oligotrophes, à l'inverse des cellules plus grandes, qui tendent à dominer la biomasse dans les upwellings ou les eaux côtières riches en nutriments (Fig. A4).

Au sein du phytoplancton de petite et grande taille, les différentes lignées taxonomiques sont caractérisées par des traits écologiques particuliers (sensibilité à la qualité de la luminosité reçue, qualité/quantité des nutriments nécessaires à la croissance, mécanismes défensifs face aux prédateurs, pour n'en citer que quelques-uns [START_REF] Glibert | Margalef revisited: a new phytoplankton mandala incorporating twelve dimensions, including nutritional physiology[END_REF]. Par conséquent, les groupes de phytoplancton présentent des différences dans leurs niches écologiques [START_REF] Brun | Ecological niches of open ocean phytoplankton taxa[END_REF].

L'océan Austral abrite des communautés phytoplanctoniques généralement dominées par le picoet le nanophytoplancton eucaryote, auxquels se superposent ponctuellement des blooms de diatomées et colonies de Phaeocystis (haptophyte) (par exemple, Wright et al., 2010 ;[START_REF] Wolf | Protist community composition in the Pacific sector of the Southern Ocean during austral summer 2010[END_REF]. Les grandes diatomées, du fait de leur capacité à former des blooms très productifs ont été considérées comme le groupe phytoplanctonique le plus important pour la production primaire de l'océan Austral. Cependant, le petit phytoplancton semble contribuer davantage à la production primaire en dehors des blooms de diatomées, notamment en hiver (Fig. A5). De nombreuses incertitudes et controverses persistent sur la composition et la contribution respective du petit et grand phytoplancton à la production primaire dans l'océan Austral. Les mesures in situ sont ponctuelles et restreintes à certaines zones du fait de l'éloignement de cet océan. Par ailleurs, les observations satellite sont souvent rendues impossibles par la couverture nuageuse persistante, ce qui conduit à des données hivernales rares [START_REF] Losa | On modeling the Southern Ocean phytoplankton functional types[END_REF]. Parmi les taxons les plus importants du petit phytoplancton de l'océan Austral, les haptophytes, les petites diatomées, les cryptophytes et, dans une moindre mesure, les prasinophytes ont été décrits comme des acteurs clés des communautés phytoplanctoniques (Moline et al., 2004 ;Uitz et al., 2009 ;[START_REF] Georges | Protist community composition during early phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF][START_REF] Iida | Seasonal variability of phytoplankton biomass and composition in the major water masses of the Indian Ocean sector of the Southern Ocean[END_REF]. L'identification précise des genres représentatifs importants au sein de ces grands groupes taxonomiques, ainsi que des facteurs écologiques responsables de leur succès, est toutefois rare.

Les grandes cellules phytoplanctoniques sont principalement représentées par les diatomées, dont la croissance est favorisée par d'importantes concentrations en fer dissous, acide silicique et une stratification élevée de la colonne d'eau [START_REF] Arrigo | Photophysiology in two major Southern Ocean phytoplankton taxa: photosynthesis and growth of Phaeocystis antarctica and Fragilariopsis cylindrus under different irradiance levels[END_REF][START_REF] Mills | Photophysiology in two Southern Ocean phytoplankton taxa: photosynthesis of Phaeocystis antarctica (prymnesiophyceae) and Fragilariopsis cylindrus (Bacillariophyceae) under simulated mixed-layer irradiance[END_REF]. Parmi le petit phytoplancton, les haptophytes, dont Phaeocystis antarctica, dominent parfois la biomasse phytoplanctonique. Sa capacité à croître à faible luminosité lui permet de se développer tôt dans la saison ou sous la glace de mer [START_REF] Peperzak | Development of the diatom-Phaeocystis spring bloom in the Dutch coastal zone of the North Sea: the silicon depletion versus the daily irradiance threshold hypothesis[END_REF][START_REF] Hilst | Photosynthesis/irradiance relationships in the Ross Sea, Antarctica, and their control by phytoplankton assemblage composition and environmental factors[END_REF][START_REF] Boyd | Environmental control of openocean phytoplankton groups: Now and in the future[END_REF]. P. antarctica bénéficie également de constantes de demi-saturation en fer plus faibles que les diatomées (Alderkamp et al., 2012). En général, on observe une alternance entre forme coloniale et forme flagellée solitaire au fil des saisons [START_REF] Smith | The temporal dynamics of the flagellated and colonial stages of Phaeocystis antarctica in the Ross Sea[END_REF]. La formation de colonies de Phaeocystis nécessite de fortes concentrations en fer [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF]. La grande taille des colonies semble réduire les taux de broutage par rapport aux cellules solitaires, qui sont facilement consommées par le microzooplancton [START_REF] Caron | Microzooplankton herbivory in the Ross Sea, Antarctica[END_REF][START_REF] Tang | Colony size of Phaeocystis antarctica (Prymnesiophyceae) as influenced by zooplankton grazers[END_REF]. Par conséquent, les formes coloniales de Phaeocystis augmenteraient l'export de carbone par sédimentation de grands agrégats mucilagineux riches en carbone, tandis que les formes solitaires alimenteraient le réseau trophique microbien et seraient rapidement recyclées au sein de la couche de mélange [START_REF] Smith | The temporal dynamics of the flagellated and colonial stages of Phaeocystis antarctica in the Ross Sea[END_REF]. Les prasinophytes et les cryptophytes ont également été identifiés dans des études pigmentaires comme des contributeurs majeurs à la Chl a dans certains secteurs de l'océan Austral (Moline et al., 2004 ;[START_REF] Iida | Seasonal variability of phytoplankton biomass and composition in the major water masses of the Indian Ocean sector of the Southern Ocean[END_REF]. Les prasinophytes sont des algues vertes caractérisées par une préférence disproportionnée pour l'ammonium par rapport au nitrate (Litchman et Klausmeier, 2008) mais semblent avoir des exigences en fer plus élevées que les autres groupes de phytoplancton [START_REF] Quigg | The evolutionary inheritance of elemental stoichiometry in marine phytoplankton[END_REF]. Récemment, il a été suggéré que la bactérivorie pourrait constituer une ressource complémentaire importante pour les algues vertes dans des conditions de faible teneur en nutriments, même à de faibles concentrations bactériennes [START_REF] Mckie-Krisberg | Phagotrophy by the picoeukaryotic green alga Micromonas: implications for Arctic Oceans[END_REF][START_REF] Mckie-Krisberg | Physiological responses of three species of Antarctic mixotrophic phytoflagellates to changes in light and dissolved nutrients[END_REF][START_REF] Anderson | Mixotrophy in chlorophytes and haptophytes-effect of irradiance, macronutrient, micronutrient and vitamin limitation[END_REF]. L'importance des cryptophytes a été principalement signalée dans les eaux côtières de l'Antarctique occidental, où les blooms sont historiquement dominés par les diatomées. L'importance croissante des cryptophytes au détriment des diatomées de grande taille, en lien avec une forte stratification de la colonne d'eau et de fortes luminosités [START_REF] Ducklow | West Antarctic peninsula: an ice-dependent coastal marine ecosystem in transition[END_REF][START_REF] Mendes | New insights on the dominance of cryptophytes in Antarctic coastal waters: a case study in Gerlache Strait[END_REF] affecterait la composition des communautés de zooplancton et pourrait entraîner une diminution du flux de carbone vers l'océan profond (Moline et al., 2004).

En conclusion, peu d'informations sont actuellement disponibles sur la diversité du petit phytoplancton et ses déterminants écologiques, bien que certains modèles de production primaire de l'océan Austral estiment que le petit phytoplancton contribuerait davantage à la production primaire annuelle (2,5 Gt C an -1 ) que le microphytoplancton (0,9 Gt C an -1 , Uitz et al. 2010).

Le phytoplancton dans le cycle du carbone

Historiquement, la diversité du phytoplancton a profondément influencé le cycle du carbone. Par exemple, le succès écologique grandissant des grandes cellules phytoplanctoniques eucaryotes au Mésozoïque a augmenté l'export de carbone organique et de nutriments vers l'intérieur de l'océan [START_REF] Katz | Geochemical and biological consequences of phytoplankton evolution[END_REF]. Ainsi, la composition taxonomique et la taille des cellules phytoplanctoniques contrôlent potentiellement le flux d'export de carbone vers l'océan intérieur et influencent la concentration à long terme du CO2 dans l'atmosphère.

On considère usuellement que le carbone produit par le phytoplancton de petite ou de grande taille suit différentes voies : le réseau trophique microbien pour les petites cellules et l'intégration vers la chaîne alimentaire classique pour les plus grandes [START_REF] Legendre | Microbial food webs and the export of biogenic carbon in oceans[END_REF][START_REF] Laender | Carbon transfer in a herbivore-and microbial loop-dominated pelagic food webs in the southern Barents Sea during spring and summer[END_REF]. Dans le réseau microbien, la production autotrophe est dominée par de petites cellules et rapidement respirée dans la boucle microbienne en surface (par exemple, Michaels & Silver 1988), ce qui entraîne un faible export de carbone. Inversement, les grandes cellules phytoplanctoniques seraient associées à des chaînes alimentaires courtes et directes (par exemple diatomées-krillbaleines dans l'océan Austral ; [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]. On pense généralement que cette chaîne alimentaire classique alimente la pompe biologique à carbone par sédimentation directe de grandes cellules phytoplanctoniques, ainsi que par production de grandes pelotes fécales par les niveaux trophiques supérieurs (Michaels et Silver, 1988 ; Fig. A9). Cependant, ce paradigme classique selon lequel les grandes cellules contribuent de manière disproportionnée à l'export de carbone est de plus en plus débattu, avec des preuves indirectes et directes croissantes que le petit phytoplancton peut être un contributeur majeur à l'export de carbone, notamment via des mécanismes d'agrégation (Richardson and Jackson, 2007). Avec le réchauffement climatique et l'oligotrophisation des eaux de surface, l'abondance du petit phytoplancton va connaître une forte hausse et il paraît d'autant plus nécessaire d'explorer davantage les routes possibles d'export des petites cellules vers l'océan profond [START_REF] Bopp | Potential impact of climate change on marine export production[END_REF][START_REF] Sarmiento | Response of ocean ecosystems to climate warming[END_REF]).

Le phytoplancton et le cycle du carbone dans l'océan Austral

L'océan Austral comptabilise 43%±3% (42±5 Pg C) du puit océanique de carbone anthropogénique de 1861-2005 (Frölicher et al., 2014). L'océan Austral devrait prochainement connaître de profonds bouleversements (réchauffement accru, renforcement des vents, augmentation de l'acidification, de la stratification des eaux de surface, diminution des remontées d'eaux profondes, de la glace de mer, de la salinité et migration vers le sud des principaux fronts océaniques ; [START_REF] Deppeler | Southern Ocean phytoplankton in a changing climate[END_REF]. Tous ces changements soulèvent des incertitudes majeures concernant les assemblages phytoplanctoniques futurs, leur productivité et le devenir de la pompe biologique à carbone.

L'océan Austral est la plus grande zone HNLC (High nutrient Low Chlorophyll), où les macronutrients abondants (N, P, Si) ne sont pas utilisés par le phytoplancton de surface du fait d'une limitation par des micronutriments, principalement le fer (de Baar et al., 2005;Blain et al., 2007;[START_REF] Boyd | Mesoscale iron enrichment experiments 1993-2005: synthesis and future directions[END_REF]. Le fer est en effet un micronutriment essentiel pour le phytoplancton, nécessaire notamment au fonctionnement des chaînes de transport d'électrons utilisées lors de la photosynthèse (par exemple par le PSI, PSII, le complexe cytochrome b6-f-FeS; [START_REF] Behrenfeld | Photophysiological expressions of iron stress in phytoplankton[END_REF]. Par conséquent, la production primaire reste faible dans l'essentiel de l'océan Austral à l'exception de quelques zones où des blooms de larges cellules sont observés à proximité d'îles, dans des eaux profitant d'une fertilisation naturelle en fer.

Les expériences explorant l'effet d'une fertilisation artificielle ou naturelle en fer au cours des deux dernières décennies ont montré sans équivoque que cette dernière favorisait une croissance accrue du phytoplancton [START_REF] Boyd | Control of phytoplankton growth by iron supply and irradiance in the subantarctic Southern Ocean: experimental results from the SAZ Project[END_REF][START_REF] Coale | Southern Ocean Iron Enrichment Experiment: carbon cycling in high-and low-Si Waters[END_REF]. Au Sud du Front Polaire, dans les eaux riches en acide silicique, la fertilisation en fer favorisait la prolifération de diatomées de grande taille, tandis qu'au Nord du Front Polaire, un assemblage mixte de petites cellules était observé [START_REF] Boyd | A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization[END_REF][START_REF] Buesseler | Particle export during the Southern Ocean Iron Experiment (SOFeX)[END_REF][START_REF] Martin | Iron fertilization enhanced net community production but not downward particle flux during the Southern Ocean iron fertilization experiment LOHAFEX[END_REF], en lien avec une plus forte limitation en acide silicique des diatomées. L'augmentation de la biomasse phytoplanctonique était plus faible dans les assemblages dominés par les petites cellules, du fait de leur importante régulation par les prédateurs microzooplanctoniques [START_REF] Schulz | Remarkable structural resistance of a nanoflagellate-dominated plankton community to iron fertilization during the Southern Ocean experiment LOHAFEX[END_REF]. L'impact sur l'export de carbone des changements occasionnés par la fertilisation en fer était variable selon les expériences. L'augmentation de la production ne s'accompagnait pas pour chaque expérience d'une augmentation significative de l'export, sans lien systématique entre export mesuré et composition des assemblages phytoplanctoniques de surface (Blain et al., 2007;[START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF][START_REF] Smetacek | Deep carbon export from a Southern Ocean iron-fertilized diatom bloom[END_REF].

Contexte scientifique et objectifs de la thèse

Le plateau des Kerguelen est un plateau océanique antarctique s'étendant de 46°S à 64°S à l'isobathe des 3000 m. Il forme une barrière à l'écoulement vers l'est du Courant Circumpolaire Antarctique (ACC) (Fig. A11 ; [START_REF] Park | Large-scale circulation over and around the Northern Kerguelen Plateau[END_REF]. Dans cette région, le Front Polaire est une zone frontale majeure, marquant la limite entre des eaux subtropicales au Nord et les eaux antarctiques au sud. La position du Front Polaire présente de grandes variations saisonnières, se déplaçant vers le sud du printemps à l'été, poussé vers le sud par des eaux plus chaudes et plus douces amenées du nord-ouest par l'ACC (Pauthenet et al., 2018). Sur le plateau, la circulation entre les îles Kerguelen et Heard est plutôt stagnante, <5 cm 2 s -1 en moyenne [START_REF] Robinson | A tale of three islands: downstream natural iron fertilization in the Southern Ocean[END_REF].

Cette thèse s'inscrit dans le cadre du projet MOBYDICK (Marine Ecosystem Biodiversity and Dynamics of Carbon around Kerguelen ; https://mobydick.mio.osupytheas.fr/), qui visait à évaluer les liens entre la biodiversité pélagique marine, le transfert du carbone dans le réseau trophique et l'export de carbone vers l'océan intérieur au large des îles Kerguelen. Par rapport aux études précédentes, l'originalité de MOBYDICK réside dans l'étude simultanée de la biodiversité pélagique à tous les niveaux trophiques, et de chaque groupe fonctionnel, du picoplancton aux prédateurs supérieurs, dans des environnements contrastés sur et en dehors du plateau. De précédentes campagnes océanographiques dans la région ont montré que le plateau naturellement fertilisé en fer était dominé par le microphytoplancton (principalement des diatomées), qui contribuait à 80-90% de la production primaire totale, alors que la région HNLC était principalement (65 -74 %) dominée par les nano-flagellés, notamment Phaeocystis antarctica, et les petites diatomées (Uitz et al., 2009 ;[START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF][START_REF] Georges | Protist community composition during early phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF].

Avant MOBYDICK, aucun projet n'avait exploré en détails la structure des communautés de phytoplancton sur le plateau des Kerguelen en dehors de la période du bloom de diatomées.

Cependant, les données satellitaires et les prélèvements effectués juste avant le bloom semblaient indiquer que les petites cellules constituaient un élément important des communautés phytoplanctoniques en dehors du bloom [START_REF] Rembauville | Plankton assemblage estimated with BGC-Argo Floats in the Southern Ocean: implications for seasonal successions and particle export[END_REF][START_REF] Penna | Mesoscale variability of conditions favoring an iron-induced diatom bloom downstream of the Kerguelen Plateau[END_REF]. L'étude de la diversité et de l'importance du phytoplancton de petite taille dans la fixation du CO2 sur et autour des eaux naturellement fertilisées en fer du plateau après le bloom des diatomées constitue une étape importante dans la compréhension du fonctionnement des écosystèmes et du cycle du carbone dans cette région où la production de phytoplancton est fortement saisonnière.

Les deux principaux objectifs de cette thèse étaient les suivants :

1. Décrire la diversité du petit phytoplancton après le bloom de diatomées dans des écosystèmes habituellement caractérisés par des régimes de productivité contrastés sur et en dehors du plateau fertilisé en fer, 2. Évaluer la contribution de différents groupes de taille du phytoplancton à la fixation du CO2 après le bloom, en prenant en compte la variabilité intra-groupe au niveau cellulaire.

L'évaluation de la variabilité de la fixation du CO2 entre et au sein de différents groupes devrait aider à comprendre comment la diversité taxonomique et de taille du phytoplancton affecte le cycle du carbone dans l'océan Austral. La comparaison de ces résultats avec les données collectées en début et fin de bloom apportera des informations précieuses sur les facteurs écologiques déterminant l'équilibre entre le petit phytoplancton et les diatomées au fil des saisons, et sur la manière dont les communautés phytoplanctoniques affectent le fonctionnement de la pompe biologique à carbone.

Matériel et méthodes

Différentes approches ont été utilisées au cours de cette thèse pour étudier les liens entre diversité du phytoplancton et fixation du CO2. La composition taxonomique du phytoplancton de petite et grande taille a été étudiée en combinant différentes méthodes (métabarcoding, CARD-FISH et chemotaxonomie pigmentaire ; Tableau B1) afin d'obtenir une compréhension qualitative et quantitative de la diversité du phytoplancton dans la zone d'étude. Le rôle du phytoplancton de petite et grande taille dans la fixation du CO2 a été étudié grâce à des incubations des communautés phytoplanctoniques en présence de DI 13 C. L'incorporation du carbone marqué ( 13 C) dans le carbone organique particulaire (POC) total, ainsi qu'au niveau cellulaire, a ensuite été évaluée par spectrométrie de masse EA-IRMS (POC total) NanoSIMS (cellules < 20µm) et IMS1280 (cellules >20µm). Les instruments NanoSIMS et IMS1280 utilisent un faisceau d'ions primaires (Cs + ), pour éroder lentement la couche superficielle d'un échantillon plat. Les ions secondaires ainsi crées sont ensuite transportés dans un spectromètre de masse, permettent la création de cartes isotopiques, où le rapport entre le nombre d'ions rares (par exemple 13 C) et les ions naturels plus abondants (par exemple 12 C) peut être visualisé (Fig. B4).

L'utilisation d'outils complémentaires de biologie moléculaire, de chemotaxonomie pigmentaire et de spectrométrie de masse au niveau cellulaire nous a ainsi permis d'explorer de manière originale la diversité et l'importance dans la fixation du CO2 du petit et grand phytoplancton.

Résultats

1. La diversité du petit phytoplancton autour de Kerguelen Dans l'océan Austral, les blooms de diatomées ont attiré beaucoup d'attention, tandis que la diversité taxonomique des cellules phytoplanctoniques de petite taille a été très peu étudiée. Cette étude examine la diversité du petit phytoplancton, défini ici comme l'ensemble des cellules <20 μm (petites diatomées et petit phytoplancton non silicifié), sur deux zones contrastées : le plateau productif de Kerguelen et les eaux environnantes à faible productivité. Pour évaluer la diversité et la structure spatiale du phytoplancton après le bloom de diatomées, des échantillons de plancton ont été prélevés à des profondeurs distinctes (de 0 à 300 m) pour deux fractions de taille (<20 et 20 à 100 μm), puis analysés par metabarcoding Illumina de l'ADNr 18S. La succession saisonnière du phytoplancton a été décrite en combinant des données pigmentaires, de cytométrie en flux et environnementales de deux campagnes précédentes sur cette même zone, au début et lors du déclin du bloom de diatomées.

Dans la couche de mélange, le petit phytoplancton non silicifié représentait moins de 10% de la chlorophylle a (Chl a) en début et fin de bloom sur le plateau, mais il contribuait pour 53-70% de Chl a après le bloom. Cette augmentation relative du petit phytoplancton dans la biomasse phytoplanctonique après le bloom pourrait principalement être liée à l'épuisement de l'acide silicique sur le plateau durant le bloom, limitant la croissance de nombreuses espèces de diatomées.

Au sein du petit phytoplancton, Phaeocystis antarctica (sous sa forme de flagellé solitaire) était relativement abondant à toutes les stations échantillonnées après le bloom, mais la distribution spatiale des autres groupes présentait des différences marquées sur et en dehors du plateau. Des concentrations plus élevées en NH4 + sur le plateau pourraient favoriser la présence de Micromonas, tandis que les pélagophytes étaient principalement séquencés en dehors du plateau.

Un assemblage diversifié de petites diatomées était également observé au large du plateau, où les concentrations en acide silicique étaient encore élevées.

En profondeur, les diatomées contribuaient dans l'ensemble d'avantage au nombre de séquences que le petit phytoplancton non-silicifié. Cependant, P. antarctica représentait jusqu'à 25% du nombre de séquences à 300 m de profondeur en dehors du plateau dans la fraction de plus grande taille, ce qui suggère qu'il pourrait contribuer de façon significative, probablement sous forme d'agrégats, à l'export de carbone dans les eaux peu productives de cette région.

Contribution du petit phytoplancton à la fixation du CO2 autour de Kerguelen

Le phytoplancton est composé de micro-organismes aux tailles et appartenances phylogénétiques très variées. Cette diversité influence le métabolisme, et par là, la capacité de différents groupes de phytoplancton à fixer le CO2 lors de la photosynthèse, ainsi que le devenir dans la pompe biologique du carbone fixé en surface. Afin d'améliorer notre compréhension du fonctionnement de la pompe à carbone, il faut donc évaluer la variabilité dans la fixation du CO2 entre différents groupes, mais également au sein de chaque groupe de phytoplancton. Seules ces mesures complémentaires permettront de déterminer la contribution respective des différents groupes, et si tous les individus au sein de ces groupes participent de façon égale ou non à la fixation du carbone.

Pour ce faire, nous avons mesuré à l'échelle cellulaire la fixation du CO2 par différents groupes de phytoplancton autour du plateau de Kerguelen, naturellement fertilisé en fer (océan Austral). Cette zone était principalement connue pour ses importants blooms de diatomées au printemps-été, favorisant l'export de carbone vers l'océan profond.

Notre étude a montré qu'après le bloom, les petites cellules phytoplanctoniques (<20 μm), composées de taxons éloignés sur le plan phylogénétique (prymnesiophytes, prasinophytes et petites diatomées) présentaient des taux de croissance supérieurs (0,22 ± 0,09 division j -1 en dehors du plateau et 0,37 ± 0,13 division j -1 sur le plateau) à ceux des grandes diatomées (0,11 ± 0,14 sur le plateau et 0,09 ± 0,11 en dehors du plateau). Du fait des faibles taux de croissance des grandes diatomées, le petit phytoplancton (principalement non silicifié) pourrait représenter 41 à 61% de la fixation totale de CO2 sur le plateau de Kerguelen, bien plus que ce qui est habituellement mesuré dans les régions naturellement fertilisées en fer de l'océan Austral (Tableau 2). Sur le plateau, la contribution élevée du petit phytoplancton à la fixation de carbone était principalement attribuable à des petites cellules (<5µm) non-silicifiées. Autour du plateau, les petites diatomées (3,8 ± 1,5 μm) étaient également abondantes et actives (Fig. 4b). Beaucoup de diatomées de plus grande taille (>20 μm) étaient inactives (19 ± 13%) à ce moment de la saison, tandis que quelques cellules très actives seulement au sein de ce groupe assuraient la majeure partie de la fixation du CO2 (Fig. 4c). Il est possible que certains groupes de grandes diatomées possèdent des caractéristiques avantageuses qui leur permettent de continuer à croitre dans les conditions environnementales défavorables après le bloom. Ainsi, les diatomées pennées, possédant la ferritine, pourraient être avantagées dans les zones où le fer est limitant, tandis que Corethron inerme serait avantagé sur le plateau par sa capacité à se développer dans des environnements où les concentrations en acide silicique sont faibles.

L'analyse de la distribution verticale des pigments dans la colonne d'eau suggère que les diatomées participent le plus à la sédimentation directe de phytoplancton, mais souligne également qu'aux stations les plus productives, une importante part du petit phytoplancton pourrait être consommée en surface, puis exportée en profondeur sous forme de pelotes fécales. De façon générale, cette étude met en évidence la nécessité d'explorer davantage le rôle des petites cellules dans la fixation et l'export du CO2.

Discussion générale

Révéler le rôle fonctionnel des micro-organismes et les facteurs environnementaux qui contrôlent la fonction qu'ils assurent dans l'écosystème est l'une des tâches les plus difficiles de l'écologie microbienne [START_REF] Madsen | Identifying microorganisms responsible for ecologically significant biogeochemical processes[END_REF][START_REF] Gutierrez-Zamora | An appraisal of methods for linking environmental processes to specific microbial taxa[END_REF]. Les deux principaux objectifs de cette thèse -explorer la diversité du petit phytoplancton et sa contribution à la fixation du CO2 dans des environnements contrastés -s'inscrivent dans ce cadre conceptuel.

Trois questions ont été abordées dans cette thèse pour répondre à ces deux objectifs :

1. La composition du petit phytoplancton diffère-t-elle sur et en dehors de la zone naturellement fertilisée en fer après le bloom de diatomées ? 2. Quels sont les facteurs environnementaux qui déterminent la dominance des petites cellules et des grandes diatomées au fil des saisons ? 3. Quelle est la contribution du petit phytoplancton à la fixation du CO2 après le bloom et quelles en sont les implications pour la pompe biologique à carbone dans la région ?

1. Premier aperçu de la diversité du petit plancton autour de Kerguelen Cette thèse a clairement mis en évidence la contribution importante du petit phytoplancton -Phaeocystis antarctica -en particulier, à la biomasse et production phytoplanctonique sur et en dehors du plateau après le bloom de diatomées (article 1 ; Fig. 3, Fig. 6). Le succès de P. antarctica peut être lié au polymorphisme de ses différents stades de vie (cellules flagellées individualisées ou grandes colonies), qui constituent différentes stratégies d'adaptation à des contrôles environnementaux contrastés au fil des saisons. Dans les eaux fertilisées en fer, la capacité à former de grandes colonies permet à P. antarctica d'échapper en partie à la forte prédation par le microzooplancton [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF]. La formation des colonies est en effet déclenchée par la disponibilité du fer [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF], mais la taille des colonies peut augmenter de 30% en présence de prédateurs, ce qui tend à confirmer l'importance écologique de la formation de colonies comme mécanisme de défense [START_REF] Tang | Colony size of Phaeocystis antarctica (Prymnesiophyceae) as influenced by zooplankton grazers[END_REF]. Sous la forme de cellules flagellées individualisées, cette espèce peut s'acclimater à la limitation en fer en réduisant sa taille, son taux de croissance, sa concentration en chlorophylle a et sa production de carbone organique particulaire [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF]Koch et al., 2019). L'importance de Phaeocystis va d'ailleurs probablement continuer à augmenter avec les changements attendus dans l'océan Austral (Zhu et al., 2016 ;Koch et al., 2019). La distribution d'autres taxons de petit phytoplancton semblait dans notre étude influencée par des concentrations contrastées en silice, ammonium et fer sur et autour du plateau (article 1 ; fig. 5). Comme aucune étude n'a exploré en détail la structure taxonomique des communautés de petit phytoplancton tout au long de l'année, nous ne pouvons pas exclure que différents groupes de petit phytoplancton puissent prévaloir plus tard dans la saison. Ainsi, d'autres groupes de petit phytoplancton pourraient être aussi, voire même davantage compétitifs en automne et en hiver, notamment lorsque la luminosité est faible quand la couche de mélange est beaucoup plus profonde (jusqu'à 185 m) en hiver. En dehors du plateau, la position du Front Polaire semble également fortement influencer la composition du petit phytoplancton à petite échelle spatiale. La limitation en acide silicique au nord du Front Polaire semble limiter la croissance des diatomées de petite taille, très présentes au sud de ce front. D'autres facteurs, en lien possible avec la circulation océanique et la distribution biogéographique de certains groupes pourraient également influencer la composition taxonomique des prasinophytes de part et d'autre du Front Polaire. Etant donné que des espèces spécifiques de ce groupe, comme Micromonas, pourraient servir de "sentinelles" du réchauffement des océans [START_REF] Demory | Picoeukaryotes of the Micromonas genus: sentinels of a warming ocean[END_REF], il pourrait être intéressant d'explorer davantage la composition taxonomique détaillée des taxons de petit phytoplancton dans les zones frontales de l'océan Austral.

Saisonnalité et variabilité interannuelle des communautés phytoplanctoniques autour de

Kerguelen

En comparant les campagnes MOBYDICK, KEOPS I et II, il a été possible de décrire la succession saisonnière des communautés phytoplanctoniques sur le plateau, du bloom de diatomées à la dominance par les petites cellules non-silicifiées après le bloom (article 1, fig. 6).

Au début du bloom, la stabilisation de la colonne d'eau et les fortes concentrations en nutriments inorganiques majeurs et en fer (maximum de 0,6 nM, réduit à 0,05 nM en été) profite à la fois aux diatomées et au petit phytoplancton sur le plateau [START_REF] Mongin | Seasonality and scale of the Kerguelen plateau phytoplankton bloom: a remote sensing and modeling analysis of the influence of natural iron fertilization in the Southern Ocean[END_REF]. Cependant, le petit phytoplancton est efficacement brouté par le microzooplancton, ce qui empêche l'accumulation de sa biomasse en début de bloom. Bien au contraire, une forte diminution des pigments associés au petit phytoplancton (prymnesiophytes et prasinophytes) est observée sur le plateau en début de bloom (article 1 ; fig. 6), période durant laquelle la biomasse des ciliés était multipliée par 7 [START_REF] Christaki | Microzooplankton community associated with phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF], contrôlant fortement le petit phytoplancton. Pendant MOBYDICK, des expériences ont également montré que le taux de croissance net du phytoplancton était inférieur au taux de broutage par le microzooplancton après le bloom (article 3; Christaki et al., en révision).

Le microzooplancton était alors cependant fortement contrôlé par les copépodes (Fig. D2). Cela peut expliquer pourquoi les abondances de phytoplancton de petite taille restaient supérieures à 10 6 cellules L -1 malgré la forte prédation par le microzooplancton après le bloom (article 1 ; Fig. 6).

Au fil du bloom, l'épuisement en acide silicique (<2 μmol L -1 ) est probablement le facteur principal limitant la croissance des diatomées. De surcroît, les interactions biotiques, comme la prédation et le parasitisme, peuvent accélérer le déclin du bloom. Pendant MOBYDICK, une forte proportion de parasites Syndiniales (55-70% des séquences) a été séquencée dans la petite fraction de taille au cours des deux premières visites sur le plateau. Ces séquences pourraient correspondre à des spores de Syndiniales libérées après la mort de l'hôte, reflétant potentiellement la fin d'une infection parasitaire d'importance en fin de bloom (article 4 ; [START_REF] Sassenhagen | Protist interactions and community structure during early autumn in the Kerguelen Region (Southern Ocean)[END_REF].

Le bloom de diatomées est caractérisé par une grande variabilité interannuelle (article 1, figure 6a), en lien probable avec de fortes variations des apports d'acide silicique et de fer dans cette région caractérisée par des fronts dynamiques [START_REF] Mongin | Seasonality and scale of the Kerguelen plateau phytoplankton bloom: a remote sensing and modeling analysis of the influence of natural iron fertilization in the Southern Ocean[END_REF][START_REF] Robinson | A tale of three islands: downstream natural iron fertilization in the Southern Ocean[END_REF]. Dans ce contexte, la position du Front Polaire au nord ou au sud du plateau des Kerguelen pourrait être cruciale, car les eaux subantarctiques au nord du Front Polaire sont pauvres en acide silicique nécessaire aux diatomées. Le déplacement saisonnier du Front Polaire vers le sud du plateau est d'ores et déjà plus prononcé lorsque des anomalies de température de surface des eaux chaudes se produisent [START_REF] Moore | Location and dynamics of the Antarctic Polar Front from satellite sea surface temperature data[END_REF] et s'intensifiera à mesure que les eaux se réchaufferont et se stratifieront davantage en raison du changement climatique [START_REF] Moore | Location and dynamics of the Antarctic Polar Front from satellite sea surface temperature data[END_REF][START_REF] Deppeler | Southern Ocean phytoplankton in a changing climate[END_REF][START_REF] Freeman | The variable and changing Southern Ocean silicate front: insights from the CESM Large Ensemble[END_REF]. Cela pourrait favoriser les petites cellules de phytoplancton au détriment des grosses diatomées, à l'instar de la situation observée pendant MOBYDICK. Des changements dans la fréquence et l'ampleur des blooms de diatomées sur le plateau sont susceptibles d'avoir un impact sur l'ensemble de la chaîne alimentaire, provoquant un déclin des populations de krill et une augmentation des salpes gélatineuses (Moline et al., 2004), qui pourrait en conséquence avec des conséquences négatives sur les consommateurs de krill, comme les manchots et les phoques [START_REF] Atkinson | Long-term decline in krill stock and increase in salps within the Southern Ocean[END_REF].

3. Impact de la structure des communautés phytoplanctoniques de surface et du régime de productivité sur la pompe biologique à carbone En théorie, quand les petites cellules dominent le phytoplancton, l'export de carbone est faible, en raison du recyclage rapide de la majeure partie de la matière organique dans la zone euphotique [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]. Une réduction de l'export de carbone à 200 m a été observée après le bloom par rapport à l'export mesuré en fin de bloom (KEOPS1), mais l'export demeurait plus élevé après le bloom qu'en début de bloom (KEOPS2) (7, 25 et 4 mmol m-2 j-1, respectivement) (article 5 ; Christaki et al. 2020). Cela suggère que le petit phytoplancton contribue à l'export de carbone, par agrégation ou broutage par les niveaux trophiques supérieurs et export sous forme de pelotes fécales, comme le suggèrent plusieurs résultats de cette thèse. La forte proportion de Phaeocystis hors du plateau à 300 m dans la fraction de grande taille (article 1 ; fig. 3) indique que Phaeocystis pourrait être incorporé dans la neige marine sous forme d'agrégats. L'étude de la distribution verticale des pigments suggère en outre que les petits taxons non-silicifiés sont rapidement ingérés par les brouteurs, en particulier sur le plateau et à M1, où se trouvent des populations denses de salpes (article 2 ; fig. 2). Pendant MOBYDICK, les diatomées du genre Odontella, Eucampia et Chaetoceros étaient bien représentées dans les données de séquençage en dessous de 125m sur le plateau, ce qui suggère que les espèces productrices de spores et formes de résistance y sont efficacement exportées. En dehors du plateau, les grandes diatomées lourdement silicifiées comme Fragilariopsis et Thalassiosira étaient également courantes dans les résultats de séquençage à 300m (Paper 1 ; Fig. 7). Ainsi, même lorsque le petit phytoplancton contribue grandement à la biomasse du phytoplancton et à la production primaire en surface, certaines grandes diatomées semblent contribuer de façon disproportionnée à l'export de carbone.

La qualité de la matière organique exportée au cours des différentes saisons reflète la succession des assemblages de phyto-et zooplancton. Les agrégats de phytoplancton dominaient le flux descendant au début du bloom (Laurenceau-Cornec et al., 2015b), lorsque les brouteurs étaient encore dans une phase de développement. Le régime de productivité était alors élevé (BPC 344 mmol m -2 j -1 ) mais l'efficacité de l'export très faible (1 %), ce qui suggère un système de rétention caractérisé par une forte reminéralisation bactérienne et une accumulation de biomasse en surface (Laurenceau-Cornec et al., 2015b ;Christaki et al., 2020). En revanche, en fin de bloom les grosses pelotes fécales (>100 μm) dominaient le flux d'export, du fait du développement d'importantes communautés de mésozooplancton [START_REF] Ebersbach | Sinking particle properties from polyacrylamide gels during the KErguelen Ocean and Plateau compared Study (KEOPS): zooplankton control of carbon export in an area of persistent natural iron inputs in the Southern Ocean[END_REF]. À cette période, l'efficacité de l'export sur le plateau était maximale (26 %). Après le bloom (MOBYDICK), les particules < 50 μm représentaient la quasi-totalité de la biomasse du POC jusqu'à 300 m (Planchon, communication personnelle). Cette caractéristique suggère que les pelotes fécales des petits brouteurs (microzooplancton et petits copépodes) et des salpes jouent alors un rôle plus important l'export. L'efficacité de l'export après le bloom n'était plus que de 5 % (article 3 ;Christaki et al., 2020), ce qui tend à confirmer que l'efficacité de l'export est plus faible lorsque les organismes de petite taille dominent le phyto-et zooplancton.

Compte tenu de cette saisonnalité marquée, il pourrait être tentant d'associer des assemblages caractéristiques de phyto-et zooplancton à des régimes d'export spécifiques, puis d'extrapoler ces mesures à un cycle annuel. Cependant, l'utilisation de pièges à sédiments déployés sur une année a montré que les spores de Chaetoceros spp. et Thalassiosira antarctica étaient responsables de plus de 60% du flux annuel de POC lors de pulses d'export brefs (Rembauville, Blain, et al., 2015).

Ces évènements ponctuels majeurs soulignent la dimension idiosyncrasique des liens entre écologie pélagique et biogéochimie des océans et soulignent la difficulté pour des campagnes océanographiques ponctuelles d'utiliser des expériences de fertilisation naturelle ou artificielle en fer pour comprendre le fonctionnement de la pompe biologique à carbone. Compte tenu de l'éloignement et du rôle central de l'océan Austral dans le cycle global du carbone, cela souligne la nécessité de développer des dispositifs d'échantillonnage automatisés pour explorer la saisonnalité, la récurrence et les liens environnementaux entre les communautés phytoplanctoniques de surface et l'export qui leur est associé.

A. Introduction

Phytoplankton are photoautotrophic microorganisms that live in the euphotic layer of freshwaters or marine environments. During oxygenic photosynthesis, they use the light energy to oxidize inorganic carbon and convert it to energy-rich organic carbon molecules. As the main marine primary producer community, phytoplankton provide food, directly or indirectly, to virtually every other marine creature. At a global scale, they account for less than 1% of Earth's photosynthetic biomass but are responsible for more than 45% of the global annual net primary production (Field et al., 1998;[START_REF] Bar-On | The biomass distribution on Earth[END_REF]. Thus, they modulate actively atmospheric carbon dioxide and oxygen concentrations. Within the phytoplankton, functional groups (characterized by distinct traits associated to size or taxonomy) have variable impacts on carbon and other major biogeochemical cycles, such as nitrogen, phosphorus and silica [START_REF] Litchman | Global biogeochemical impacts of phytoplankton: a trait-based perspective[END_REF]. Phytoplankton are composed of a broad suite of taxa, from cyanobacteria to eukaryotes, that vary considerably in size (<1 to >100 µm). The diversity in size and metabolism of phytoplankton communities affects food web structure and biogeochemical cycling of major elements.

In the first part of this introduction, I will briefly provide an overview of the origin of the major phytoplanktonic lineages and discuss what is known on their physiology, metabolism and ecology of broad size-and taxonomic groups, focusing on phytoplankton of the Southern Ocean (SO). I will then discuss how phytoplankton size and taxonomy affect the marine carbon cycle with a focus on the SO. The SO is the most important sink of anthropogenic carbon dioxide and thus a key region in understanding the role of marine biogeochemical carbon cycling (Frölicher et al., 2014). It is the largest high nutrient, low chlorophyll (HNLC) area of the global ocean, where primary production is limited by iron availability despite high concentrations of macronutrients (Martin, 1990;de Baar et al., 2005). The "Iron hypothesis" proposed by Martin, (1990) suggested that relief from iron deficiency via increased dust deposition enhanced phytoplankton primary production in the SO during the Last Glacial Maximum (20 000 years ago), lowering atmospheric CO2 by as much as 100 ppm in comparison with preindustrial times (∼ 280 ppm).

Since the discovery of the essential role of iron in the productivity of the SO and potential effect in mitigation of CO2 emissions, many studies focused on the influence of artificial or natural iron fertilization on phytoplankton communities, CO2-fixation and carbon export (reviewed by [START_REF] Yoon | Reviews and syntheses: Ocean iron fertilization experiments -past, present, and future looking to a future Korean Iron Fertilization Experiment in the Southern Ocean (KIFES) project[END_REF]. In the SO, large phytoplankton blooms occur in spring and summer in naturally ironfertilized waters downstream of island plateaus, such as those of Kerguelen, Crozet and South Georgia. These blooms are usually characterized by different phytoplankton communities than surrounding low productive HNLC waters and enhanced carbon export to the deep ocean (Blain et al., 2007;[START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF][START_REF] Korb | Regional and seasonal differences in microplankton biomass, productivity, and structure across the Scotia Sea: implications for the export of biogenic carbon[END_REF]. Thus, these contrasted ecosystems on and off naturally-fertilized waters constitute unique natural laboratories to explore the influence of contrasted phytoplankton communities and productivity regimes on the marine carbon cycle.

Phytoplankton diversity

"Phytoplankton is a nineteenth century ecological construct for a biologically diverse group of pelagic photoautotrophs that share common metabolic functions but not evolutionary histories." [START_REF] Quigg | The evolutionary inheritance of elemental stoichiometry in marine phytoplankton[END_REF] 1

.1 Evolution and diversity of phytoplankton

The first marine photoautotrophs appeared about 3.8 Ga years ago in the Archaean period with the origin of photosynthesis in prokaryotic Cyanobacteria [START_REF] Katz | Geochemical and biological consequences of phytoplankton evolution[END_REF][START_REF] Nutman | Rapid emergence of life shown by discovery of 3,700-million-year-old microbial structures[END_REF] , which led to the rise of oxygen on Earth ~2.3 billion years ago [START_REF] Bekker | Dating the rise of atmospheric oxygen[END_REF]. Oxygenic photosynthesis is supposed to have evolved only once in an ancestral Cyanobacteria, but it subsequently spread via endosymbiosis to a wide variety of eukaryotic clades [START_REF] Delwiche | Tracing the thread of plastid diversity through the tapestry of life[END_REF]. The engulfment of a cyanobacteria by a heterotrophic eukaryote ~1 900 to 900 Myr ago marked the origin of eukaryotic phytoplankton [START_REF] Sánchez-Baracaldo | Early photosynthetic eukaryotes inhabited low-salinity habitats[END_REF]. This primary endosymbiosis gave rise to the Archaeplastida lineage, a monophyletic group that includes Glaucophyta, Rhodophyta (red algae), Chlorophyta (green algeae) and streptophyta (land plants) (Fig. A1). In turn, secondary endosymbioses involving archaeplastid lineages (red or green algae) spread photosynthesis to the haptophytes, cryptophytes, dinoflagellates, stramenopiles, euglenids (red lineage) and chlorarachniophyte rhizarians [START_REF] Gentil | Review: origin of complex algae by secondary endosymbiosis: a journey through time[END_REF]. In modern oceans, the red lineage (especially haptophytes, dinoflagellates and diatoms) is extremely successful and groups the most common bloom-forming species [START_REF] Falkowski | The Evolution of Modern Eukaryotic Phytoplankton[END_REF].

Fig. A1: Evolution and diversification of eukaryotic autotrophic lineages. Taxa that inherited their plastids from a secondary endosymbiosis with green algae or red algae are labeled in green and red, respectively (Source: [START_REF] Worden | Rethinking the marine carbon cycle: factoring in the multifarious lifestyles of microbes[END_REF] Phytoplankton has now representatives in 4 of the 6 super-groups of in the eukaryotic tree of life described by [START_REF] Adl | The new higher level classification of eukaryotes with emphasis on the taxonomy of protists[END_REF] (Fig. A2). Not et al., 2012) Historically, the diversity of eukaryotic phytoplankton has been assessed by microscope-based description of morphological features and approximately 4 000-25 000 phytoplankton morphospecies were described this way [START_REF] Sournia | Marine phytoplankton: how many species in the world ocean?[END_REF][START_REF] Falkowski | The Evolution of Modern Eukaryotic Phytoplankton[END_REF]. This number probably largely underestimates the real extent of phytoplankton diversity and molecular approaches keep unveiling new uncultured lineages (e.g. [START_REF] Moreira | The molecular ecology of microbial eukaryotes unveils a hidden world[END_REF][START_REF] Massana | Unveiling new microbial eukaryotes in the surface ocean[END_REF][START_REF] Massana | Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles[END_REF] and the existence of cryptic species [START_REF] Sáez | Pseudo-cryptic speciation in coccolithophores[END_REF][START_REF] Amato | Reproductive isolation among sympatric cryptic species in marine diatoms[END_REF]. Extensive molecular surveys of phytoplankton diversity based on sequencing of the 18S rRNA gene (V9 region) revealed that the actual numbers of phytoplankton taxa based on operational taxonomic units (OTUs, a proxy for species in molecular surveys) are consistently approximately 10-fold higher than morphology-based estimates [START_REF] Pierella Karlusich | Phytoplankton in the Tara Ocean[END_REF].

Ecological traits of small and large phytoplankton cells

As phytoplankton taxonomic diversity is large, phytoplankton taxa are often classified based on cell size. The cell size of phytoplankton spans over at least 9 orders of magnitude, from a cell volume around 0.1 µm 3 for the smallest cyanobacteria to more than 10 8 µm 3 for the largest diatoms (Fig. A3).

Fig. A3: A comparison of the size range of phytoplankton relative to macroscopic objects (Source: [START_REF] Finkel | Phytoplankton in a changing world: cell size and elemental stoichiometry[END_REF] The arbitrary size ranges of 0.2 to 2 μm, >2 to 20 μm, and >20 to 200 μm, are historically referred to as picophytoplankton, nanophytoplankton, and microphytoplankton, respectively. In this thesis, I will use the definition from [START_REF] Richardson | Mechanisms and pathways of small-phytoplankton export from the surface ocean[END_REF] and consider cells <20 µm to be small and cells >20 µm to be large.

The distinction between small and large cells is particularly useful as phytoplankton growth and loss process are strongly size dependent (reviewed in [START_REF] Marañón | Phytoplankton size structure[END_REF]. Small cells are in theory advantaged over larger ones in nutrients and light uptake. They are characterized by higher surface to volume ratio, resulting in increased diffusion capability of nutrients through the cell membrane (e.g Chisholm, 1992;[START_REF] Raven | The twelfth Tansley Lecture. Small is beautiful: the picophytoplankton[END_REF]. The amount of light absorbed per pigment unit also decreases with increasing cell size, as high intracellular pigment concentration creates self-shading among the different pigment molecules (the "package effect"; [START_REF] Finkel | Light absorption and size scaling of light-limited metabolism in marine diatoms[END_REF]. Moreover, a large cell size increases the cell's sinking velocity (mechanism also known as Stokes' law), which implies a reduction of their residence time in the euphotic layer unless they have a way to regulate their position in the water column (e.g. active swimming, gas vacuoles). These advantages of being small are particularly relevant in strongly stratified water columns, where nutrients input from deep waters are scarce and large cells are less likely to return to the photic layer in the absence of water mixing.

While being small is advantageous in terms of resource and light acquisition, large cell size is assumed to lower, or at least offset the predation pressure. The main reason is that the generation time of predators increases with body size more rapidly than the generation time of phytoplankton.

Small phytoplankton are typically consumed by microzooplankton like dinoflagellates and ciliates, which have generation times similar to those of phytoplankton (in the order of hours to days) and exert a strong control on their biomass. In contrast, large cells may accumulate biomass temporarily by their ability to escape high predation rates [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF][START_REF] Irigoien | Phytoplankton blooms: a 'loophole' in microzooplankton grazing impact?[END_REF]. This theoretical approach of allometric relationship between prey size and its grazing mortality is however oversimplified as it appears that heterotrophic protists with short generation times (in particular dinoflagellates) are important consumers of large cells such as diatoms [START_REF] Sherr | Capacity of herbivorous protists to control initiation and development of mass phytoplankton blooms[END_REF]. Consequently, although some studies have found that small phytoplankton suffer higher grazing losses than larger cells [START_REF] Latasa | Pigment specific growth and grazing rates of phytoplankton in the central equatorial Pacific[END_REF][START_REF] Landry | Biological response to iron fertilization in the eastern equatorial Pacific (IronEx II). III. Dynamics of phytoplankton growth and microzooplankton grazing[END_REF][START_REF] Strom | Microzooplankton grazing in the coastal Gulf of Alaska: Variations in top-down control of phytoplankton[END_REF], others have reported similar grazing pressure on phytoplankton groups of markedly different mean cell size [START_REF] Latasa | Estimating the carbon flux through main phytoplankton groups in the northwestern Mediterranean[END_REF][START_REF] Gutiérrez-Rodríguez | Growth and grazing rate dynamics of major phytoplankton groups in an oligotrophic coastal site[END_REF][START_REF] Chang | Scaling of growth rate and mortality with size and its consequence on size spectra of natural microphytoplankton assemblages in the East China Sea[END_REF].

These general growth and loss patterns play a role in determining the size structure of phytoplankton communities in contrasting marine environments and explain the overall dominance of small phytoplankton in oligotrophic tropical waters, whereas larger cells dominate the chlorophyll a biomass in upwellings or nutrient-rich coastal waters (Fig. A4). [START_REF] Roy | The global distribution of phytoplankton size spectrum and size classes from their light-absorption spectra derived from satellite data[END_REF] Within small and large phytoplankton, specific taxonomic lineages are characterised by particular ecological traits (e.g. light and nutrient quality/quantity requirements or grazer avoidance, to cite only a few [START_REF] Glibert | Margalef revisited: a new phytoplankton mandala incorporating twelve dimensions, including nutritional physiology[END_REF]. Consequently, phytoplankton groups feature differences in their ecological niches [START_REF] Brun | Ecological niches of open ocean phytoplankton taxa[END_REF].

In the next paragraph, I will briefly review the specific ecological characteristics determining the fitness and success of important "small" and "large" phytoplankton groups that thrive in the ocean, with a focus on emblematic groups of the SO.

Phytoplankton diversity in the Southern Ocean

The SO harbours phytoplankton communities typically dominated by diatoms and haptophytes, superimposed upon a background of pico-and nanophytoplankton (e.g. Wright et al., 2010;[START_REF] Wolf | Protist community composition in the Pacific sector of the Southern Ocean during austral summer 2010[END_REF]. For decades, large diatoms have been considered as the most important phytoplankton group for primary production in the SO, because of their ability to form large blooms.

Estimates from remote sensing studies considered that diatoms could account for 89% of the primary production of the SO [START_REF] Rousseaux | Interannual variation in phytoplankton primary production at a global scale[END_REF]. This important role of diatoms in SO primary production constitutes the foundation of the emblematic "food chain of the giants" (diatomskrill-whales) [START_REF] Smetacek | Are declining Antarctic krill stocks a result of global warming or of the decimation of the whales? In Impacts of global warming on Polar ecosystems[END_REF]. However, large disagreements still exist between the models regarding the distribution and contribution to production of major phytoplankton groups of the SO (Fig. A5). In situ measurements to calibrate models are sparse in space and time in this remote ocean and satellite observations require cloud-free scenes which leads to sparse winter data in the often cloud-covered SO [START_REF] Losa | On modeling the Southern Ocean phytoplankton functional types[END_REF]. [START_REF] Alvain | Seasonal distribution and succession of dominant phytoplankton groups in the global ocean: a satellite view[END_REF], the Darwin-MITgcm version of [START_REF] Dutkiewicz | Capturing optically important constituents and properties in a marine biogeochemical and ecosystem model[END_REF] and [START_REF] Losa | On modeling the Southern Ocean phytoplankton functional types[END_REF] . "SCL" represents Synechococcus like prokaryotic phytoplankton. Simulated haptophytes include coccolithophores and Phaeocystis. The dominance between diatoms and small phytoplankton (haptophytes, Phaeo and small eukaryotes) varies greatly between models. However, in all models, the dominance of small phytoplankton over diatoms is observed during winter months (July).

Among the most important groups of small phytoplankton of the SO, haptophytes, small diatoms, cryptophytes and to a lesser extent prasinophytes have been described as key players of phytoplankton communities (Moline et al., 2004;Uitz et al., 2009;[START_REF] Georges | Protist community composition during early phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF][START_REF] Iida | Seasonal variability of phytoplankton biomass and composition in the major water masses of the Indian Ocean sector of the Southern Ocean[END_REF]. Precise identification of important representative genera within these broad taxonomic groups, as well as the ecological drivers of their success is, however, scarce.

Diatoms

Diatoms (Bacillariophyceae), are photoautotrophic stramenopiles, characterized by a silica cell wall, the frustule. [START_REF] Sarthou | Growth physiology and fate of diatoms in the ocean: a review[END_REF] reported that diatoms may contribute up to 40% of oceanic primary productivity. This group exerts a major influence upon the biogeochemical cycles of carbon, silicon, nitrate, and iron in the open ocean (Tréguer et al., 2018). Diatoms tend to have significantly higher maximum uptake rates of nutrients than any other group and relatively high maximum growth rates [START_REF] Litchman | The role of functional traits and trade-offs in structuring phytoplankton communities: scaling from cellular to ecosystem level[END_REF]Litchman and Klausmeier, 2008). Large diatoms are able to effectively utilize nutrient pulses by storing nutrients in disproportionately large vacuoles compared to smaller diatoms [START_REF] Sicko-Goad | Estim ation of intracellular carbon and silica content of diatoms from natural assemblages using morphometric techniques1[END_REF][START_REF] Raven | The role of vacuoles[END_REF].

In the SO, iron and silicic acid availability are critical for diatoms to sustain high relative growth rates and biomass. They are able to form large seasonal blooms in iron-enriched regions in summer in high macronutrient regions such as the Ross Sea, the Kerguelen Plateau and South Georgia (Korb and Whitehouse, 2004;Blain et al., 2007). Diatom dominance also seems to be favored by high water column stratification as they are characterized by higher tolerance to photoinhibition as compared to P. antarctica populations in high light conditions [START_REF] Arrigo | Photophysiology in two major Southern Ocean phytoplankton taxa: photosynthesis and growth of Phaeocystis antarctica and Fragilariopsis cylindrus under different irradiance levels[END_REF][START_REF] Mills | Photophysiology in two Southern Ocean phytoplankton taxa: photosynthesis of Phaeocystis antarctica (prymnesiophyceae) and Fragilariopsis cylindrus (Bacillariophyceae) under simulated mixed-layer irradiance[END_REF].

In addition to iron availability, the supply of silicate, required for diatoms to build their silica frustules, is also considered to be an important factor controlling the dominance of diatoms in the SO. The silicate concentrations show a decreasing northward gradient, in particular on either side of the Antarctic Polar Front, with low silicate concentrations (< 5 µM) in the sub-Antarctic waters north of the Polar Front and high silicate concentrations (> 60 µM) to the south of the Polar Front [START_REF] Tréguer | The Southern Ocean silica cycle[END_REF]; Fig. A6). As a result, diatoms typically dominate phytoplankton communities in summer South of the Polar Front in iron-enriched, stratified waters like marginal ice zones [START_REF] Wang | Impact of sea ice on the marine iron cycle and phytoplankton productivity[END_REF] or over naturally iron-fertilized areas near islands (Korb and Whitehouse, 2004;Blain et al., 2007). (nitrate, phosphate and silicic acid in particular). This favors the growth of diatoms and intense biogenic silica production, which triggers abundant export of biogenic silica to deep waters, ultimately depositing opal on abyssal sediments (mostly south of the PF). Unused nutrients are exported to the rest of the world ocean through the Sub-Antarctic Mode Water (SAMW), the Antarctic Intermediate Water (AAIW), and the Antarctic Bottom Water (AABW). (Source [START_REF] Tréguer | The Southern Ocean silica cycle[END_REF] Iron availability can greatly influence diatoms' size, chain length and taxonomic composition.

Typically, adding iron to iron-limited surface waters particularly stimulate growth of large and chainforming diatoms [START_REF] Boyd | Mesoscale iron enrichment experiments 1993-2005: synthesis and future directions[END_REF]. Inversely, most diatoms decrease their cell volumes by as much as 50% when grown under iron-limited conditions [START_REF] Sunda | Interrelated influence of iron, light and cell size on marine phytoplankton growth[END_REF][START_REF] Leynaert | Effect of iron deficiency on diatom cell size and silicic acid uptake kinetics[END_REF] and tend to be present as solitary cells or form very short chains [START_REF] Hoffmann | Iron, silicate, and light co-limitation of three Southern Ocean diatom species[END_REF].

Different diatom taxonomic compositions were observed on and off naturally iron-fertilized areas [START_REF] Armand | Late summer diatom biomass and community structure on and around the naturally iron-fertilised Kerguelen Plateau in the Southern Ocean[END_REF][START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF]. This likely results from species-specific responses to iron availability and some diatom families like pennate diatoms, Pseudo-nitzschia in particular, consistently thrive when iron is non-limiting (de Baar et al., 2005;Marchetti et al., 2009).

Haptophytes

Haptophytes constitute a widespread lineage, dominating the Chl a-normalized phytoplankton standing stock in modern oceans and possibly accounting for 30-50% of total Chl a biomass in the global ocean [START_REF] Liu | Extreme diversity in noncalcifying haptophytes explains a major pigment paradox in open oceans[END_REF]. Their success in diverse environments (open-ocean, shelf, upwelling, coastal, littoral, brackish and freshwater environments) has been attributed to their ability to complement photosynthesis with other particulate (bacteria mostly) and/or dissolved organic food sources [START_REF] Kawachi | The haptonema as a food-capturing device: observations on Chrysochromulina hirta (Prymnesiophyceae)[END_REF][START_REF] Nygaard | Bacterivory in algae: a survival strategy during nutrient limitation[END_REF][START_REF] Liu | Extreme diversity in noncalcifying haptophytes explains a major pigment paradox in open oceans[END_REF].

Molecular evidence support the division of the Haptophyta into two main classes, the Pavlovophyceae and the Prymnesiophyceae [START_REF] Edvardsen | Phylogenetic reconstructions of the Haptophyta inferred from 18S ribosomal DNA sequences and available morphological data[END_REF]. The vast majority of the known diversity of haptophytes occurs in the Prymnesiophyceae, which comprises two orders of non-calcifying (Phaeocystales and Prymnesiales) and one calcifying taxa (coccolithophores containing four orders: Isochrysidales, Coccolithales, Syracosphaerales and Zygodiscales; Fig. A7). Haptophytes include famous bloom-forming taxa, such as the famous calcifying species (coccolithophore) Emiliania huxleyi, as well as Chrysochromulina, Prymnesium and Phaeocystis [START_REF] Gjøsaeter | A longterm perspective on the Chrysochromulina bloom on the Norwegian Skagerrak coast 1988: a catastrophe or an innocent incident?[END_REF][START_REF] Tyrrell | Emiliania huxleyi: bloom observations and the conditions that induce them[END_REF][START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF][START_REF] Granéli | The ecophysiology and bloom dynamics of Prymnesium spp[END_REF].

Phaeocystis is an ubiquitous marine genus that contains three bloom-forming species with typical geographic distributions: P. pouchetii dominating in the Arctic Ocean, P. globosa in temperate coastal seas and P. antarctica in the SO, respectively [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF]. The divergence of the "cold-water forms" (P. pouchetii and P. antarctica) from the "warm-water forms" (P. globosa) was estimated to happen about 30 Mya and the divergence of P. antarctica and P. pouchetii to be about 15 Mya [START_REF] Lange | Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes[END_REF].

Ecological success of bloom-forming Phaeocystis was attributed to its polymorphic life cycle alternating freeliving cells (3-9 µm in diameter) and gelatinous colonies usually reaching several milimeters [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF]; Fig. A8).

In the SO, P. antarctica colonies regularly form huge blooms in the seasonal ice zone and coastal Antarctic waters, that can lead to rapid carbon export through the formation of fast sinking aggregates [START_REF] Ditullio | Rapid and early export of Phaeocystis antarctica blooms in the Ross Sea, Antarctica[END_REF]. Nutrients such as nitrate and phosphate appear to have little influence on P. antarctica growth, as they remain at relatively high concentrations over the annual cycle. Its ability to grow at low irradiance allows it to bloom early in the season or under the sea ice [START_REF] Peperzak | Development of the diatom-Phaeocystis spring bloom in the Dutch coastal zone of the North Sea: the silicon depletion versus the daily irradiance threshold hypothesis[END_REF][START_REF] Hilst | Photosynthesis/irradiance relationships in the Ross Sea, Antarctica, and their control by phytoplankton assemblage composition and environmental factors[END_REF][START_REF] Boyd | Environmental control of openocean phytoplankton groups: Now and in the future[END_REF]. P. antarctica also benefits from lower iron half-saturation constants for growth than diatoms (Alderkamp et al., 2012). However, iron availability triggers Phaeocystis colony formation which can result in the bloom in the Ross Sea for example [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF]. Typically, a switch from colony to solitary forms is observed throughout the season as a consequence of more severe micronutrient limitation for the colonies relative to solitary cells [START_REF] Smith | The temporal dynamics of the flagellated and colonial stages of Phaeocystis antarctica in the Ross Sea[END_REF]. The large size of colonies appear to lower the grazing rates in comparison to solitary cells, which are easily consumed by microzooplankton [START_REF] Caron | Microzooplankton herbivory in the Ross Sea, Antarctica[END_REF][START_REF] Tang | Colony size of Phaeocystis antarctica (Prymnesiophyceae) as influenced by zooplankton grazers[END_REF]. Consequently, colonial forms of Phaeocystis are assumed to enhance carbon export by direct sinking of large ungrazed mucilaginous aggregates rich in carbon, whereas solitary forms are assumed to fuel the microbial network and be quickly recycled within the mixed layer [START_REF] Smith | The temporal dynamics of the flagellated and colonial stages of Phaeocystis antarctica in the Ross Sea[END_REF].

Other phytoplanktonic groups of the SO Prasinophytes and cryptophytes have been identified in pigment studies as major contributors to Chl a in some sectors of the SO (Moline et al., 2004;Iida and Odate, 2014 and references therein).

Prasinophytes form a paraphyletic group belonging to division Chlorophyta ("Green algae"), represented by flagellated small cells characterized by chlorophylls a and b as the major photosynthetic pigments (reviewed in [START_REF] Leliaert | Phylogeny and molecular evolution of the green algae[END_REF]. Mamiellophyceae is the largest lineage Phaeocystis colonies in a Ross Sea bloom. (Source: [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF] of prasinophytes, including some of the smallest eukaryotes known (e.g., Ostreococcus, Micromonas; < 2 µm).

Prasinophytes are characterized by intermediate values of nutrient uptake and growth parameters, except for ammonium, for which they have a disproportionately high preference over nitrate (Litchman and Klausmeier, 2008). This high preference for ammonium may be linked to the higher availability of ammonium under lower pO2 conditions 1.5 billion years ago, when prasinophytes likely appeared (Litchman and Klausmeier, 2008). Furthermore, at this time, iron concentrations in the water were more elevated than at the present time, which may explain their higher iron requirements than phytoplankton cells from the red lineage [START_REF] Quigg | The evolutionary inheritance of elemental stoichiometry in marine phytoplankton[END_REF]. These two characteristics could partly explain why they dominate the eukaryotic picoplankton in coastal eutrophic areas especially, reaching 10 5 cells ml -1 (O' Kelly et al., 2003;[START_REF] Guillou | Diversity of picoplanktonic prasinophytes assessed by direct nuclear SSU rDNA sequencing of environmental samples and novel isolates retrieved from oceanic and coastal marine ecosystems[END_REF][START_REF] Falkowski | The Evolution of Modern Eukaryotic Phytoplankton[END_REF]. Recently, it has been suggested that bacterivory could be a significant complementary resource acquisition strategy for green algae under low nutrient conditions, even at low bacterial concentrations [START_REF] Mckie-Krisberg | Phagotrophy by the picoeukaryotic green alga Micromonas: implications for Arctic Oceans[END_REF][START_REF] Mckie-Krisberg | Physiological responses of three species of Antarctic mixotrophic phytoflagellates to changes in light and dissolved nutrients[END_REF][START_REF] Anderson | Mixotrophy in chlorophytes and haptophytes-effect of irradiance, macronutrient, micronutrient and vitamin limitation[END_REF]. In the SO, pigment analyses have reported the presence of prasinophytes mostly north in the sub-Antarctic zone north of the Polar Front [START_REF] Wright | Analysis of phytoplankton of the Australian sector of the Southern Ocean: comparisons of microscopy and size frequency data with interpretations of pigment HPLC data using the CHEMTAX matrix factorisation program[END_REF][START_REF] Iida | Seasonal variability of phytoplankton biomass and composition in the major water masses of the Indian Ocean sector of the Southern Ocean[END_REF]Wolf et al., 2014;[START_REF] Van Leeuwe | Phytoplankton and pigment patterns across frontal zones in the Atlantic sector of the Southern Ocean[END_REF]. However, classical pigment and microscopy analyses are not adapted to reveal the taxonomic identity of these prasinophytes as these tiny cells lack easily identifiable morphological features. Recently, a genetic study demonstrated that Micromonas was present in the SO and that Antarctic Micromonas may be connected to Arctic Micromonas through deep-sea currents [START_REF] Simmons | Intron invasions trace algal speciation and reveal nearly identical Arctic and Antarctic Micromonas populations[END_REF]. Micromonas also represented a major fraction of the sequences of the pico-and nanoplankton community within an artificially Fe-fertilized patch [START_REF] Thiele | Stable composition of the nano-and picoplankton community during the ocean iron fertilization experiment LOHAFEX[END_REF].

The importance of cryptophytes has been mostly reported in coastal waters of West Antarctica, where blooms are usually dominated by diatoms. Some studies have noted the increasing importance of cryptophytes that can prevail over diatoms in association with highly illuminated conditions in shallow upper mixed layers and strong water column stratification [START_REF] Ducklow | West Antarctic peninsula: an ice-dependent coastal marine ecosystem in transition[END_REF][START_REF] Mendes | New insights on the dominance of cryptophytes in Antarctic coastal waters: a case study in Gerlache Strait[END_REF]. Such a recurrent transition from diatoms to cryptophytes represents a fundamental decrease in the size spectrum of the phytoplankton community, which can impact grazing efficiencies of different zooplankton species, enhancing microbial activity in the region and, consequently, promoting changes in carbon fluxes within the water column (Moline et al., 2004).

Concluding, even though models of primary production in the SO estimates that small phytoplankton contributes more to annual primary production (2.5 Gt C yr -1 ) than microphytoplankton (0.9 Gt C yr -1 , Uitz et al. 2010), little information is currently available on small phytoplankton diversity and ecological drivers in the SO. This knowledge gap partly originates from the timing of most cruises exploring phytoplankton community composition during spring and summer diatom blooms, a period where small cells represent a small fraction of the biomass.

Phytoplankton in the carbon cycle

Historically, the evolution and diversity of phytoplankton deeply influenced the carbon cycle. For instance, the radiation of large-celled eukaryotic marine phytoplankton enhanced the export of organic carbon and nutrients to the ocean interior through the Mesozoic [START_REF] Katz | Geochemical and biological consequences of phytoplankton evolution[END_REF]. This established stronger lateral, vertical and temporal variations of nutrient levels that further advantaged eukaryotic phytoplankton relative to cyanobacteria, because of their larger nutrient storage capacity and complex behavioral and trophic strategies [START_REF] Worden | Rethinking the marine carbon cycle: factoring in the multifarious lifestyles of microbes[END_REF]. Therefore, taxonomic composition and size-structure of phytoplankton communities potentially control carbon export flux and influence the long-term concentration of CO2 in the atmosphere.

The Biological Carbon Pump (BCP)

Currently, phytoplankton in the global ocean is responsible for the photosynthetic fixation of around 45-50.10 15 g C annually, which represents almost half of global net primary production on Earth (Field et al., 1998). Some 20% of phytoplankton net primary production (5-10 Gt C) is exported toward the ocean's interior, either in the form of sinking particles or as dissolved material (Falkowski et al., 1998;Palmer and Totterdell, 2001). The vertical transfer processes include passive sinking of organic particles, physical mixing of particulate and dissolved organic carbon (POC and DOC, respectively), and active transport by zooplankton migration. The mineralization of the organic matter increases with depth the concentration of dissolved inorganic carbon. The net effect of this phytoplankton-fueled biological pump, is the transport of CO2 from the atmosphere to the deep ocean, where it may be sequestered over the timescales of deep-ocean circulation (10 2 -10 3 years).

A small fraction (<1%) of the organic matter transported toward the deep ocean escapes mineralization and is buried in the ocean sediments, where it is retained over timescales of >10 6 years. It is predicted that if the biological pump stops pumping carbon down to the ocean interior, atmospheric levels of CO2 would in time rise by another 100-200 ppm thereby accelerating global warming (Field et al., 1998;[START_REF] Ito | Preformed phosphate, soft tissue pump and atmospheric CO<SUB>2</SUB>[END_REF]. Thus, phytoplankton play a role in the regulation of the atmospheric content of CO2 and therefore affect climate variability.

Several studies have demonstrated that regional differences exist in the strength, overall export efficiency and depth-dependent export efficiency of the biological pump [START_REF] Henson | Global patterns in efficiency of particulate organic carbon export and transfer to the deep ocean[END_REF].

These differences are driven by the structure of pelagic communities, which ultimately determine the magnitude of the biological sequestration of carbon in the deep ocean and sediments [START_REF] Turner | Zooplankton fecal pellets, marine snow, phytodetritus and the ocean's biological pump[END_REF].

Impact of phytoplankton community size structure on the BCP

Size-structure of phytoplankton communities is often considered as an important determinant of the magnitude of the BCP. Classically, it is considered that the carbon produced by small or large phytoplankton follow different pathways: the microbial food web for small cells and classical food chain for larger ones [START_REF] Legendre | Microbial food webs and the export of biogenic carbon in oceans[END_REF][START_REF] Laender | Carbon transfer in a herbivore-and microbial loop-dominated pelagic food webs in the southern Barents Sea during spring and summer[END_REF]. In the microbial food web, autotrophic production is dominated by small cells and rapidly respired within the microbial loop in the euphotic layer (e.g. Michaels & Silver 1988), resulting in little export. In contrast, the dominance of larger phytoplanktonic cells is thought to be associated with short, direct food chains (e.g. "diatoms-krill-whales" in the SO; [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]. This classical food chain is generally thought to fuel the BCP through direct sinking of large phytoplankton cells and the production of large fast-sinking fecal pellets by higher trophic levels, with a high carbon content (Michaels and Silver, 1988) (Fig. A9).

Fig. A9: The two-level food web of Michaels & Silver (1988), which was a modification of the basic food web (black arrows) with the size of prey ingested by zooplankton expanded (green arrows). In this scheme, only large phytoplankton can directly sink and their export pathway through grazing is more direct (only one step, than for small phytoplankton). (Source: [START_REF] Richardson | Mechanisms and pathways of small-phytoplankton export from the surface ocean[END_REF] These general ecological and biogeochemical features associated with small and large phytoplankton marine communities (Table A1) have motivated the development of marine ecosystem and biogeochemistry models integrating different phytoplankton size classes (Poulin and Franks, 2010;Ward et al., 2012).

Table A1: General ecological and biogeochemical properties of plankton communities in which phytoplankton are dominated by small vs large cells [START_REF] Marañón | Phytoplankton size structure[END_REF] However, the classical view that large cells contribute disproportionately to export is increasingly debated with growing indirect and direct evidences that small phytoplankton can be a major contributor to C-export. The debate started in 2007 when Richardson and Jackson advanced that the relative direct and indirect contribution of picoplankton to export were proportional to their total

Microphytoplankton Nanophytoplankton Picophytoplankton

Heterotrophic nanoflagellates

Microzooplankton Macrozooplankton net primary production, despite their small size (Richardson and Jackson, 2007). Their conclusion, based on inverse modelling and network analyses, underlined the importance of aggregation that made small cells available to mesozooplankton grazers and allowed them after transformation in fecal pellets to reach relatively high sinking rates. Indeed, direct observations from sinking aggregates confirmed that small cells could make substantial contributions to export production via the production of fecal pellets by mesozooplankton or salps grazers (e.g. [START_REF] Sutherland | Filtration of submicrometer particles by pelagic tunicates[END_REF][START_REF] Wilson | Autotrophic picoplankton in mesozooplankton guts: evidence of aggregate feeding in the mesopelagic zone and export of small phytoplankton[END_REF][START_REF] Ebersbach | Particle flux characterisation and sedimentation patterns of protistan plankton during the iron fertilisation experiment LOHAFEX in the Southern Ocean[END_REF]. Interestingly, intact Prochlorococcus cells at concentrations of 10 2 -10 4 cells mL -1 were reported as deep as 800 m in the Pacific [START_REF] Jiao | Presence of Prochlorococcus in the aphotic waters of the western Pacific Ocean[END_REF]. These observations suggest that small phytoplankton could play an important role in Cexport. Overall, direct observations based on modern underwater particle imaging combined with field POC flux data estimated that higher mass fluxes were measured for large phytoplanktondominated communities than in systems dominated by small phytoplankton. However, small phytoplankton-dominated communities were characterized by lower remineralization rates, resulting in higher export efficiencied than in systems dominated by larger cells (Guidi et al., 2009).

Predicted increase in small phytoplankton dominated communities

Ongoing climate warming is expected to increase ocean stratification, and hence reduce nutrient input from deep waters to the upper mixed layer, with an overall negative effect on phytoplankton primary production [START_REF] Bopp | Potential impact of climate change on marine export production[END_REF][START_REF] Sarmiento | Response of ocean ecosystems to climate warming[END_REF]. Such a change is already taking place and oceanic areas exhibiting the lowest chlorophyll concentrations (<0.07 mg L -1 ) are expanding at annual rates varying between 0.8% and 4.3% [START_REF] Polovina | Ocean's least productive waters are expanding[END_REF]. For the reasons explained in part 1.2., this scenario is thought to favor small phytoplankton at the expense of diatoms [START_REF] Bopp | Response of diatoms distribution to global warming and potential implications: a global model study[END_REF][START_REF] Li | Smallest algae thrive as the Arctic Ocean freshens[END_REF].

Resulting changes in phytoplankton community structure are expected to have a cascading effect on structure of marine food webs and biogeochemical cycling of carbon [START_REF] Finkel | Phytoplankton in a changing world: cell size and elemental stoichiometry[END_REF]. Using a coupled oceanic biogeochemical model and climate model, a large decrease in export production as a result of this shift, by as much as 25% at in case of a four-fold increase of CO2 concentrations was predicted [START_REF] Bopp | Response of diatoms distribution to global warming and potential implications: a global model study[END_REF]. However, treatment of plankton communities is relatively simple in these models, with 1-3 phytoplankton functional types and typically one zooplankton group [START_REF] Bopp | Multiple stressors of ocean ecosystems in the 21st century: projections with CMIP5 models[END_REF]. The models including both large and small phytoplankton, assume a higher export efficiency (by a factor of 3 or more) for the large phytoplankton [START_REF] Aumont | Globalizing results from ocean in situ iron fertilization studies[END_REF].

Consequently, it is not surprising that these models predicted that a shift towards smaller phytoplankton cells is expected to decrease C-export. Other models combining inverse modeling and ecological network analysis predicted that a shift from large to small phytoplankton-dominated communities would alter the pathways of carbon flow, but result in minimal changes in carbon export [START_REF] Vernet | Models of plankton community changes during a warm water anomaly in Arctic waters show altered trophic pathways with minimal changes in carbon export[END_REF]. In this inverse model, high export in nanoflagellates dominated community was possible, if a link between the classical and microbial food webs develops through the consumption of microzooplankton and detritus by copepods. More integrative in situ studies are needed to study the effects of a shift from large to small phytoplankton communities and settle this debate.

Southern Ocean phytoplankton and the C-cycle

The SO (south of 30°S), occupies 30% of global surface ocean area and is estimated to account for 43%±3% (42±5 Pg C) of anthropogenic CO2 and 75%±22% of heat uptake by the ocean over 1861[START_REF] Madsen | Identifying microorganisms responsible for ecologically significant biogeochemical processes[END_REF](Frölicher et al., 2014)). The SO holds a special place in view of its possible impact on the biogeochemistry of the world ocean. The circulation of Antarctic Intermediate Water, formed in the Polar Frontal Zone, redistributes major nutrients to lower latitudes and controls the dynamics of primary producers over large regions of the world ocean, directly influencing the biological pump at the global scale (see Fig. A8).

Over the last century, large decadal variations in the SO carbon sink have raised several concerns regarding to the future capacity of the SO carbon sink [START_REF] Quéré | Saturation of the Southern Ocean CO2 sink due to recent climate change[END_REF]Landschützer et al., 2015;[START_REF] Sitch | Recent trends and drivers of regional sources and sinks of carbon dioxide[END_REF]. Climate of the SO is predicted to experience increased warming, strengthening wind, acidification, shallowing mixed layer depths, increasing light, changes in upwelling and nutrient replenishment, declining sea ice, reduced salinity, and the southward migration of major fronts [START_REF] Deppeler | Southern Ocean phytoplankton in a changing climate[END_REF]. All these changes raise major uncertainties concerning the response of phytoplankton communities and the impact of SO productivity on the BCP.

Control of Southern Ocean productivity

The Southern Ocean paradox

The SO is the largest upwelling region of the globe. Remarkably, major nutrients (N, P, Si) brought to surface waters by the upwelling of deep waters on the Antarctic shelf are not fully used and the SO is the largest high-nutrient, low-chlorophyll (HNLC) region of the world. The fact that macronutrients were not used up was termed the "Antarctic paradox" since the 1930s [START_REF] Hart | On the phytoplankton of the South-west Atlantic and the Bellingshausen Sea[END_REF].

Several hypotheses have been ventured to explain this paradox: growth limitation due to unfavorable light (because of deep mixing and light limitation in winter), trace element deficiency, and high mortality due to intense grazing pressure by micro-and microzooplankton, limiting the accumulation of phytoplankton biomass. [START_REF] Smetacek | Seasonal and regional variation in the pelagial and its relationship to the life history cycle of krill[END_REF] described the Antarctic ecosystem as a "retention type system", in which relatively high abundances of zooplankton are sustained in an ecosystem despite low phytoplankton biomass. The recycling of the bulk of zooplankton faeces in the surface layer would provide the essential nutrients to maintain phytoplankton growth [START_REF] González | Distribution and abundance of minipellets around the Antarctic peninsula. Implications for protistan feeding behaviour[END_REF]. The relative importance of these mutually inclusive explanations is still under debate, however there is strong evidence that the key factor limiting phytoplankton production in vast areas of the SO is iron availability (de Baar et al., 2005;Blain et al., 2007;[START_REF] Boyd | Mesoscale iron enrichment experiments 1993-2005: synthesis and future directions[END_REF].

The Iron hypothesis

The Iron hypothesis formulated by Martin in 1990 suggested that the higher productivity observed in the SO during glacial periods to HNLC waters has been debated as a solution to mitigate the effects of anthropogenic CO2 by stimulating phytoplankton production (e.g. [START_REF] Chisholm | Stirring times in the Southern Ocean[END_REF][START_REF] Buesseler | Will ocean fertilization work?[END_REF]. These events marked the begin of the "iron age" in oceanography (de Baar et al., 2005;[START_REF] Stoll | 30 years of the iron hypothesis of ice ages[END_REF], with many cruises and experiments trying to disentangle the interplay between Fe-fertilization, plankton community structure, food webs and their effects on the BCP.

Impact of Fe-fertilization on phytoplankton communities and C-export

Artificial iron fertilization experiments in HNLC waters conducted over the past two decades have unequivocally led to enhanced phytoplankton growth in iron limited regions [START_REF] Boyd | Control of phytoplankton growth by iron supply and irradiance in the subantarctic Southern Ocean: experimental results from the SAZ Project[END_REF][START_REF] Coale | Southern Ocean Iron Enrichment Experiment: carbon cycling in high-and low-Si Waters[END_REF] Experiment; [START_REF] Martin | Iron fertilization enhanced net community production but not downward particle flux during the Southern Ocean iron fertilization experiment LOHAFEX[END_REF] have been carried out so far. During SOIREE (February 1999) and EisenEx (November 2000), increased growth rates, as well as a floristic change from piconanoflagellates to large chain-forming diatoms was observed over the first days of fertilization, with no measurable change in the downward POC flux with the 234 Th method [START_REF] Boyd | A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization[END_REF][START_REF] Assmy | Mechanisms determining species dominance in a phytoplankton bloom induced by the iron fertilization experiment EisenEx in the Southern Ocean[END_REF]. The short observational periods (13 and 27 days), as well as other intrinsic limits and artefacts of the small-scale fertilization technique, may have prevented a clear assessment of carbon export and explain why no increase in the C-export was observed during these experiments.

During EIFEX, Fe-fertilization lead to a massive diatom bloom and it was estimated that at least half the bloom biomass sank far below a depth of 1,000 metres [START_REF] Smetacek | Deep carbon export from a Southern Ocean iron-fertilized diatom bloom[END_REF]. SoFEX (genus Pseudo nitzschia), benefited the most from the fertilization in the low-silicate "North Patch", whereas in the high-silicate "South Patch", up to 65% of the phytoplankton in the bloom were diatoms [START_REF] Buesseler | Particle export during the Southern Ocean Iron Experiment (SOFeX)[END_REF]. Increased C-export was observed in both patches. Although northern production and biomass were dominated by nonsilicious phytoplankton, the communities driving export production shifted toward diatoms in the north, but remained diatom-dominated in the south [START_REF] Coale | Southern Ocean Iron Enrichment Experiment: carbon cycling in high-and low-Si Waters[END_REF]. SOLAS-SAGE and LOHAFEX also explored the effect of Fefertilization in Si-limited waters. During SOLAS-SAGE, a modest increase in picophytoplankton biomass was observed, but without an increase in particulate organic carbon (POC) at the surface or in C-export. Phytoplankton growth was thought to have been kept in check by the resident microzooplankton grazers with the increase in POC being recycled through the microbial food web [START_REF] Harvey | The SOLAS air-sea gas exchange experiment (SAGE) 2004[END_REF]. LOHAFEX was a three-weeks experiment in the productive south-west Atlantic sector of the Southern Ocean depleted in Si in late summer 2009. Chlorophyll levels tripled and the biomass of autotrophic pico-and nanoflagellates doubled [START_REF] Martin | Iron fertilization enhanced net community production but not downward particle flux during the Southern Ocean iron fertilization experiment LOHAFEX[END_REF]. The biomass levels of almost all other groups of the phyto-and microzooplankton (heterotrophic nanoflagellates, dinoflagellates and ciliates) remained remarkably stable throughout the experiment both inside and outside the fertilized patch [START_REF] Schulz | Remarkable structural resistance of a nanoflagellate-dominated plankton community to iron fertilization during the Southern Ocean experiment LOHAFEX[END_REF]. Fe-fertilization during LOHAFEX didn't enhance downward export of particles, but many small flagellates and coccoid cells were found in the sediment traps, and it was suggested that these small cells probably contributed the majority of downward POC flux [START_REF] Ebersbach | Particle flux characterisation and sedimentation patterns of protistan plankton during the iron fertilisation experiment LOHAFEX in the Southern Ocean[END_REF].

Other studies analyzed the effect of natural iron fertilization near land mass (Crozet and Kerguelen

Plateau, South Georgia) on phytoplankton communities and C-export. CROZEX established that Phaeocystis represented 78% of phytoplankton biomass in Fe-fertilized subantarctic waters north of the Crozet Plateau, whereas HNLC waters South of the plateau were characterized by large diatoms [START_REF] Poulton | Phytoplankton community composition around the Crozet Plateau, with emphasis on diatoms and Phaeocystis[END_REF]. Primary production was 3 to 5 times higher and C-export two to three times larger in naturally iron-fertilized waters by the Crozet plateau than in the adjacent HNLC area [START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF]. KEOPS 1 and 2 studied respectively the decline (February 2005) and onset (November 2011) of a large diatom bloom over the Kerguelen Plateau. Phytoplankton communities on the Plateau were largely dominated by diatoms, whereas small phytoplankton (flagellates and small diatoms) were abundant off-plateau (Uitz et al., 2009;[START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF].

POC export over the plateau was approximately twice larger than in HNLC waters off-plateau during both cruises (Blain et al., 2007;Planchon et al., 2015). Sinking material contained a mixture of phytodetrital aggregates and fecal pellets at bloom onset and mainly fecal pellets at bloom decline, highlighting temporal variations in ecosystem structure and export pathways throughout the season (Laurenceau-Cornec et al., 2015a).

Near South Georgia, diatoms were dominant in and out of iron replete waters, but diatoms were larger and primary production rates were three times higher at stations situated within the main bloom compared to those outside of the bloom (Korb et al., 2008). C-export was more than five times greater in Fe-fertilized areas, with a greater contribution of fecal pellets [START_REF] Manno | The contribution of zooplankton faecal pellets to deep-carbon transport in the Scotia Sea (Southern Ocean)[END_REF].

In conclusion, these experiments showed unequivocally that Fe-fertilization enhanced primary production rates. However, a shift to diatom-dominated phytoplankton community was only observed in Si-replete ecosystems. Increased C-export in Fe-fertilized waters was not observed during all experiments, but could involve both diatoms as well as small flagellates dominated surface assemblages (Blain et al., 2007;[START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF][START_REF] Smetacek | Deep carbon export from a Southern Ocean iron-fertilized diatom bloom[END_REF].

Inverse relationship between primary production and export

In most of the global ocean, export efficiency is positively correlated to primary production [START_REF] Laws | Simple equations to estimate ratios of new or export production to total production from satellite-derived estimates of sea surface temperature and primary production[END_REF]. However, in the SO, the use in models of this global relationship has been shown to overestimate two to fourfold the export production in comparison with in situ export rates (Maiti et al., 2013). Maiti et al. (2013) concluded that one of the main reasons for this overestimation was that ecosystems of the SO were characterized by decreasing export efficiency with increasing production which is contrary to existing export models.

Numerous in situ measurements confirmed this inverse relationship between primary production and export efficiency (e.g. [START_REF] Froneman | Size-fractionated phytoplankton biomass, production and biogenic carbon flux in the eastern Atlantic sector of the Southern Ocean in late austral summer 1997-1998[END_REF][START_REF] Cavan | Attenuation of particulate organic carbon flux in the Scotia Sea, Southern Ocean, is controlled by zooplankton fecal pellets[END_REF]Le Moigne et al., 2016). To mention only data collected in our study area, export efficiency at 100 m was on average lower (28%) above the plateau, than in surrounding HNLC waters (58%) [START_REF] Savoye | 234 Th-based export fluxes during a natural iron fertilization experiment in the Southern Ocean (KEOPS)[END_REF] during the bloom decline (KEOPS1) although primary production was more than 3 times higher on the plateau (Uitz et al., 2009). At bloom onset (KEOPS1), export efficiency was 37% at HNLC reference station and only 1-2% on the plateau (Planchon et al., 2015).

Several studies have investigated the possible causes for this inverse relationship, suggesting that bacteria and zooplankton may play an important role in the regulation of export efficiency through grazing, remineralization of organic matter, and production/export of fecal pellets [START_REF] Cavan | Attenuation of particulate organic carbon flux in the Scotia Sea, Southern Ocean, is controlled by zooplankton fecal pellets[END_REF]Le Moigne et al., 2016). Around Kerguelen, it was also suggested that direct export of heavysilicified, grazing resistant diatoms found intact in sediment traps may favor the high export efficiency observed in HNLC waters (Laurenceau-Cornec et al., 2015a).

As phytoplankton communities in low productive waters are mostly dominated by small cells, further exploration of the taxonomic and functional diversity of small phytoplankton, as well as direct estimates of their C-fixation with in situ measurements are needed to shed light on the drivers of the high export efficiency observed in HNLC waters of the SO. waters brought from the northwest by the ACC (Pauthenet et al., 2018). The circulation over the plateau between the Kerguelen and Heard islands is rather stagnant, <5 cm 2 s on average [START_REF] Robinson | A tale of three islands: downstream natural iron fertilization in the Southern Ocean[END_REF].

Scientific context and PhD objectives

KEOPS and MOBYDICK cuises

This thesis is part of the MOBYDICK project (Marine Ecosystem Biodiversity and Dynamics of Carbon around Kerguelen; https://mobydick.mio.osupytheas.fr/), which aimed at evaluating the links between marine pelagic biodiversity and the carbon sequestration and transfer along the food KEOPS1 cruise further demonstrated that the iron-enriched bloom region was dominated by microphytoplankton (mainly diatoms), which contributed 80-90% to the total primary production, whereas the HNLC area was mainly (65%) dominated by nano-flagellates and small diatoms (Uitz et al., 2009). The importance of small phytoplankton was also highlighted during KEOPS2 at the HNLC reference station, where autotrophic pico-and nanoeukaryotes represented 74% of the plankton biomass [START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF]. High throughput sequencing of planktonic communities further identified P. antarctica as the dominant small phytoplankton taxa [START_REF] Georges | Protist community composition during early phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF].

On-plateau, it was suggested, that diatoms growth were co-limited by iron and silicic acid during the bloom decline (Mosseri et al., 2008). Diatom community composition and succession

Mechanism of iron and nutrients supply over the Kerguelen Plateau (Source: [START_REF] Boyd | Mesoscale iron enrichment experiments 1993-2005: synthesis and future directions[END_REF] Waters surrounding the Kerguelen area are enriched with macronutrients and trace elements from the crustal interface with the plateau. The interactions between geostrophic current disturbances due to the presence of the island, the tidal activity over the plateau, and strong winds generate internal waves, eddies, jets, Ekman pumping, and a complex hydrodynamical environment [START_REF] Park | Large-scale circulation over and around the Northern Kerguelen Plateau[END_REF]Gille et al., 2014). These characteristics enhance the vertical mixing of the deep waters above the plateau, characterized by high iron and nutrient concentrations, and stimulate higher productivities and C-export above the Plateau in comparison to HNLC surrounding waters (Blain et al., 2007) Box 1: Mechanism of iron fertilization over the Kerguelen Plateau throughout the season were studied in details. On-plateau, diatoms evolved from a chain-forming Chaetoceros bloom to a remnant Eucampia antarctica assemblage, whereby off-plateau, heavy silicified Fragilariopsis kerguelensis dominated throughout the season [START_REF] Armand | Late summer diatom biomass and community structure on and around the naturally iron-fertilised Kerguelen Plateau in the Southern Ocean[END_REF][START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF].

PhD objectives

Before MOBYDICK, no project explored thoroughly the structure of phytoplankton communities on the Kerguelen Plateau outside of the bloom period. However, satellite data and sampling just before the bloom seemed to indicate that small cells were an important component of phytoplankton communities outside of the bloom period [START_REF] Rembauville | Plankton assemblage estimated with BGC-Argo Floats in the Southern Ocean: implications for seasonal successions and particle export[END_REF][START_REF] Penna | Mesoscale variability of conditions favoring an iron-induced diatom bloom downstream of the Kerguelen Plateau[END_REF].

Investigating small phytoplankton diversity and importance in CO2-fixation on and around naturally iron-fertilized waters of the SO when the diatom bloom is over will constitute a milestone in understanding ecosystem functioning and C-cycling in this region where phytoplankton production is highly seasonal.

The two main objectives of this thesis were:

1. To describe the diversity of small phytoplankton after the diatom bloom in ecosystems usually characterized by different productivity regimes on and off the naturally iron-fertilized plateau of Kerguelen.

2. To evaluate the contribution of different taxonomic and size groups of phytoplankton to CO2fixation after the bloom, considering intra-group variability at the single cell level.

Assessing CO2-fixation variability between and within different phytoplankton groups should help understanding how phytoplankton taxonomic and size diversity affects the C-cycle within these ecosystems. Comparing these results to the description of phytoplankton communities and productivity regimes at bloom onset and decline will bring valuable insights into the ecological drivers determining the balance between small phytoplankton and diatoms throughout the season and how this affects the functioning of the biological carbon pump.

B. Material and methods

One of the most exciting challenge for microbial ecologists consist in linking identity of the microorganisms to functions. Over the past two decades, the development of molecular tools such as genomics has completely changed our qualitative understanding of microbial taxonomic and functional diversity in the marine environment [START_REF] Moran | The global ocean microbiome[END_REF][START_REF] Sunagawa | Structure and function of the global ocean microbiome[END_REF]. In parallel, new methodologies, like NanoSIMS have been used to directly quantify the role specific microbial populations in biogeochemical processes (e.g. [START_REF] Orphan | Methaneconsuming archaea revealed by directly coupled isotopic and phylogenetic analysis[END_REF][START_REF] Musat | Detecting metabolic activities in single cells, with emphasis on nanoSIMS[END_REF][START_REF] Nuñez | NanoSIMS for biological applications: current practices and analyses[END_REF].

Different approaches have been used during this thesis to study phytoplankton diversity and link this diversity to CO2-fixation. Small and large phytoplankton community composition have been studied by combining different methods (metabarcoding, CARD-FISH and chemotaxonomy) to gain a qualitative and quantitative understanding of phytoplankton's diversity in the study area. The role of small and large phytoplankton in CO2-fixation at the single cell-level has then been assessed

with NanoSIMS and large geometry SIMS. In this chapter, I will briefly review the principles, interests and limits of these methods.

Description of small phytoplankton community composition

We combined molecular methods (metabarcoding and CARD-FISH) and chemotaxonomy to investigate the diversity, abundance and structure of phytoplankton communities, focusing on small phytoplankton taxa (Table B1). [START_REF] Bachy | Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study[END_REF].

Process

In our study, 10L of seawater was filtered successively on 20 µm and 0.2 µm to collect large and small planktonic organisms, respectively. After extracting the DNA present on the filter, an identitygene marker, in our case the short variable regions V4 or V9 of the RNA of the small subunit of the ribosome (18S rRNA gene), was amplified by polymerase chain reaction (PCR). The 18S rRNA gene comprises both conserved and hypervariable regions. Primers targeting conserved regions enable to amplify almost all eukaryotes, while sequencing of hypervariable regions enable to assign the taxonomic affiliation of the different organisms within the sample. We used general eukaryotic primers biased against metazoan to mainly target unicellular eukaryotes (Bower et al., 2004).

Informatic processing of the sequences obtained from the pool of amplicons makes it possible to describe the molecular diversity present in the environmental sample (Fig. B1).

Limits

The first applications of metabarcoding attempted to provide quantitative data (based on the assumption that reads obtained from a sequencing run correlate with biomass in the original sample) [START_REF] Symondson | Special issue on molecular detection of trophic interactions: unpicking the tangled bank[END_REF]. However, the quantitative relationship between the biomass and sequences produced is not accurate in all cases [START_REF] Lamb | How quantitative is metabarcoding: a meta-analytical approach[END_REF]. Each step of the metabarcoding method, from sampling to bio-informatic data analysis is indeed associated with potential biases that can distort the community picture (Table B2).

Collect and filter water

Amplify 18S V4 Taxonomy assignment

Table B2: Overview of the factors that can create quantitative biases at each step of the metabarcoding process. Adapted and completed from [START_REF] Lamb | How quantitative is metabarcoding: a meta-analytical approach[END_REF]. These biases may lead to over-or underestimation of specific taxa. Indeed, amplification rates may differ between species as a result from the difficulty to find universal primers matching with the same efficiency a wide range of divergent lineages [START_REF] Elbrecht | Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass-sequence relationships with an innovative metabarcoding protocol[END_REF]. For example, diplonemid flagellates have been revealed as very diverse and abundant using the v9 region, while they are barely detected with conventional v4 approaches [START_REF] Flegontova | Extreme diversity of diplonemid eukaryotes in the Ocean[END_REF]. Moreover, the number of rRNA gene copies per cell varies among eukaryotes from one to several thousand [START_REF] Gong | Estimation of 18S gene copy number in marine eukaryotic plankton using a next-generation sequencing approach[END_REF]. Specific taxonomic groups, dinoflagellates for example, have a high number of gene copies [START_REF] Wisecaver | Dinoflagellate genome evolution[END_REF] and are likely overrepresented in molecular data.

Stage of protocol Potential biases

The number of copies also depends on cell size and while picoeukaryotes have one or a few copies per cell [START_REF] Rodríguez-Martínez | Distribution of the uncultured protist MAST-4 in the Indian Ocean, Drake Passage and Mediterranean Sea assessed by real-time quantitative PCR[END_REF], larger eukaryotes show variations up to several orders of magnitude among taxa [START_REF] Biard | Biogeography and diversity of Collodaria (Radiolaria) in the global ocean[END_REF] leading to a higher representation of large organisms in sequencing datasets. To avoid contamination by larger organisms with high rRNA gene content, we used size-fractionated sequencing to access the diversity of small and large protists. A prefiltration on 100 µm was chosen to remove large metazoans and focus on protist communities.

We didn't use a prefiltration on 200 µm -considered as a common threshold to define mesoplankton-as this threshold is too high and results in high contamination by large metazoans [START_REF] Liu | DNA metabarcoding reveals that 200μm-size-fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes[END_REF]. The 20 µm size-fractionation removed most of the larger cells and allowed to have a more in-depth sequencing of the small eukaryotes that we focused on in this study.

However, size fractionation only allowed us to infer that the sequence-linked organism was probably smaller than the prefilter pore-size (<20 µm), but it is possible that some of the sequences obtained from small size-fraction samples derive from larger organisms, which could have broken during the prefiltration step. Alternatively, certain life-stages of larger organisms (e.g., swarmers, resting spores) could be small enough to pass through the prefilter. These concerns highlight the interest of completing the molecular description of phytoplankton communities by microscopic observations to assess the reliability of the data in case sequencing results are unexpected.

In addition to the methodological concerns linked with organisms biology described in the above paragraph, PCR artifacts, sequencing errors and PCR chimeras (sequences formed from two or more biological sequences joined together) are known to artificially inflate diversity estimates [START_REF] Kunin | Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates[END_REF][START_REF] Lee | Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing[END_REF]. Discriminating between natural amplicons and technical artifacts is a challenge that has to be addressed by the informatic pipeline for accurate interpretation of diversity estimates. Customarily, the analysis of metabarcoding data begins with the construction of molecular operational taxonomic units (OTUs), i.e. clusters of reads that differ by less than a fixed sequence dissimilarity threshold, most commonly 3% or 1% [START_REF] Caron | Defining DNA-based operational taxonomic units for microbial-eukaryote ecology[END_REF]. The taxonomic composition of a sample then lists the number of reads of each OTU, which are often treated alike 'species' in diversity analysis. Using dissimilarity thresholds allows to reduce the artificial inflation of diversity resulting from the technical artifacts mentioned above. Nevertheless, these thresholds are arbitrary and the resulting sequences do not always correspond to exact biological sequences. In an attempt to overcome this issue, new methods try to infer the biological sequences in the sample prior to the introduction of amplification and sequencing errors, and distinguish sequence variants differing by as little as one nucleotide [START_REF] Quince | Removing noise from pyrosequenced amplicons[END_REF]Callahan et al., 2016). It is the case of the DADA2 pipeline, an open-source R package implementing a full amplicon workflow for Illumina sequencing denoising: filtering, dereplication, sample inference, chimera identification, and merging of paired-end reads (Callahan et al., 2016). To discriminate biological sequences from sequencing errors, DADA2 builds an error model on the basis of the expectation that biological sequences are more likely to be repeatedly observed than are errorcontaining sequences. Sequencing quality is also considered to determine the likelihood of a new amplicon sequence variant (ASV) to result from a sequencing error. Although metabarcoding methods can't be considered as quantitative for the reasons exposed in the next paragraph, ASV have been shown to be more accurate than conventional OTU methods in representing a sample's diversity and they constitute consistent labels that can be used to compare Illumina datasets using the same marker-gene [START_REF] Callahan | Exact sequence variants should replace operational taxonomic units in marker-gene data analysis[END_REF][START_REF] Caruso | Performance of microbiome sequence inference methods in environments with varying biomass[END_REF]. In spite of these advantages, DADA2 can also introduce spurious ASVs, but these spurious ASVs account for a low proportion (<4%) of read numbers [START_REF] Prodan | Comparing bioinformatic pipelines for microbial 16S rRNA amplicon sequencing[END_REF]. In this case, ASVs can be grouped based on similarity or abundance thresholds [START_REF] Needham | Ecological dynamics and cooccurrence among marine phytoplankton, bacteria and myoviruses shows microdiversity matters[END_REF].

The last major limitation of sequencing comes from the under-representation of uncultured organisms in databases, resulting in a consequent number of unidentified organisms, for which the taxonomic identification is restricted to a low taxonomic resolution (class or order). Although being the most comprehensive database, using NCBI's GenBank is not recommended to classify 18S rRNA gene sequences due to large amount of wrongly annotated sequences [START_REF] Geisen | A user guide to environmental protistology: primers, metabarcoding, sequencing, and analyses[END_REF].

At least two curated databases are dedicated to the 18S rRNA gene, Silva [START_REF] Pruesse | SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[END_REF] and PR 2 (Guillou et al., 2013). We decided to use PR 2 as it implements more specialized databases designed for specific taxonomic groups, e.g. foraminifera [START_REF] Morard | PFR2: a curated database of planktonic foraminifera 18S ribosomal DNA as a resource for studies of plankton ecology, biogeography and evolution[END_REF] or dinoflagellates [START_REF] Mordret | dinoref: a curated dinoflagellate (Dinophyceae) reference database for the 18S rRNA gene[END_REF].

CARD-FISH

Considering the biases described above, marker-gene sequencing needs to be completed with quantitative methods. The most straightforward quantitative monitoring technique available for the specific detection of active small phytoplanktonic cells consists in whole-cell fluorescence in situ hybridization (FISH) of the ribosomal RNA (rRNA). Fluorescently labelled groups can subsequently be visualized under an epifluorescence microscope [START_REF] Delong | Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells[END_REF]. FISH can be used to detect, identify, and enumerate active microorganisms without requiring culture. The probe can be selected to bind organisms at different taxonomic levels, e.g. group-specific to species-specific, and/or multiple probes can be used to locate multiple taxonomic groups. Fluorochromes are attached to the probe, so that when the probe binds to its target, the cells belonging to the target taxa will become fluorescent when observed at a particular wave length with an epifluorescence microscope (Fig. B2). Standard FISH is not always sufficient to detect pico-sized cells with low rRNA copy numbers, and as such, CARD-FISH (Catalysed Reporter Deposition-FISH), also known as Tyramide Signal Amplification CARD-FISH, is preferably used when studying the composition of marine plankton communities [START_REF] Pernthaler | Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria[END_REF].

Most probes designed for phytoplankton target broad taxonomic groups, however species-specific probes also exist (Not et al., 2004). Developing probes for finer taxonomic levels is possible, but is time consuming and requires many cultures of closely related taxonomic groups to test the sensitivity and specificity of the probes developed.

In our study, we used results from metabarcoding of the small size fraction to choose the primers that would target most organisms sequenced within Prymnesiophyceae, Pelagophyceae, Mamiellophyceae (Table B3). In silico validity of these probes was recently re-assessed and showed that they were indeed specific to their target groups, with some exceptions such as PRAS04 that do not hybridize with 17 targeted taxa [START_REF] Riou | Specificity re-evaluation of oligonucleotide probes for the detection of marine picoplankton by Tyramide Signal Amplification-Fluorescent In Situ Hybridization[END_REF]. Abundances obtained in this study should thus be considered as minima.

Chemotaxonomy

Chemotaxonomy was used to complete CARD-FISH quantitative approach and evaluate the contribution of a few small phytoplankton broad taxonomic groups to the chlorophyll a (Chl a) pool.

Chl a is the most common index to estimate the biomass of phytoplankton. In some cases, the biomass of Chl a can in turn be used as a proxy of phytoplankton carbon biomass using a conversion factor C:Chl a. Classically, ratios ranging from 35 to 60 (mean value=50) are used.

However, these ratios can be highly variable (e.g. from 6 to 333) depending on the season, light, temperature, depth and nutrient availability [START_REF] Geider | Light and temperature dependence of the carbon to chlorophyll a ratio in microalgae and cyanobacteria: implications for physiology and growth of phytoplankton[END_REF][START_REF] Harrison | Effects of nutrient and light limitation on the biochemical composition of phytoplankton[END_REF][START_REF] Jakobsen | Carbon-to-chlorophyll ratio for phytoplankton in temperate coastal waters: seasonal patterns and relationship to nutrients[END_REF]. Consequently, the measure of Chl a, often used as a proxy of phytoplankton biomass, remains particularly challenging to convert to carbon.

In addition to the use of Chl a as a general proxy for phytoplankton biomass, other pigments, characteristic of specific broad taxonomic groups can be used to estimate the contribution of these groups to Chl a. Pigment analyses by high-performance liquid chromatography (HPLC) are widely applied to study algal distributions on a large scale (e.g. [START_REF] Roy | Phytoplankton pigments: characterization, chemotaxonomy and applications in oceanography[END_REF]. The development of softwares designed to convert pigment patterns into algal groups (CHEMical TAXonomy:

CHEMTAX) has further promoted the application of HPLC analyses (Mackey et al., 1996).

CHEMTAX is a tool that derives the quantitative contributions of phytoplankton taxonomic groups to the total chlorophyll pool by using the likely pigment:Chl a ratios of each group. Expected composition of taxonomic groups in the sample is needed to choose which groups must be included in the pigment:Chl a ratio matrix. Then, pigment:Chl a ratios are ideally obtained directly from local cultures, or based on published literature data. Indeed, in addition to geographic variations, pigment:Chl a ratios of the different groups can vary according to species, light, depth or nutrients, so that they cannot be known beforehand. To avoid introducing strong biases in the results due to the initial pigment ratio matrix used for predicting the contribution of the different groups to Chl a, it is highly recommended to modify iteratively the initial ratios to successively reduce the amount of unexplained Chl a of the produced model [START_REF] Coupel | Pigment signatures of phytoplankton communities in the Beaufort Sea[END_REF].

In our study, samples were first clustered according to their similarity in pigment:Chl a ratios, as CHEMTAX requires to run the analysis on homogeneous pigment:Chl a ratios to reach an optimal solution. The initial ratio matrix was based on the literature available on pigment composition of Southern Ocean phytoplankton (Wright et al., 2010;van Leeuwe et al., 2014). The ratio matrix was then randomized a minimum of 60 times for each cluster of samples to achieve root mean square of the residuals <0.1, considered as the threshold for checking model accuracy. The average contribution of each taxonomic group to Chl a was obtained by averaging the six best runs.

Contribution of specific groups of small phytoplankton to CO2-fixation

Once the diversity of small phytoplankton and its contribution to biomass were evaluated, we investigated the contribution of specific phytoplankton groups to CO2-fixation. To understand how phytoplankton size structure and taxonomic diversity affected CO2-fixation, it was necessary to combined the measurements of CO2-fixation at different levels: at the community, specific sizeranges, taxa, and single cell scale. The latter was essential to assess if all cells contributed equally to CO2-fixation or if the function depended mainly on a few individual cells.

Incubation with carbon isotopes: principles and limitations

The first experiments measuring phytoplankton CO2-fixation using isotopic tracers ( 14 C or This method is relatively easy to implement but faces methodological and conceptual limitations.

Methodological concerns can be regrouped in the so-called "bottle effect", a term describing how the incubation bottle could affect metabolic processes of the community sampled. These effects are more pronounced over long incubations into small volumes (<1L). They arise from different factors, like contamination with trace metals from the bottle walls, loss of turbulence, changes in the community composition or damage to organisms due to incubation conditions (e.g. [START_REF] Gieskes | Current 14 C methods for measuring primary production: gross underestimates in oceanic waters[END_REF][START_REF] Fitzwater | Metal contamination and its effect on primary production measurements1[END_REF][START_REF] Berg | Dimension effects of enclosures on ecological processes in pelagic systems[END_REF][START_REF] Calvo-Díaz | Decrease in the autotrophic-to-heterotrophic biomass ratio of picoplankton in oligotrophic marine waters due to bottle enclosure[END_REF]. Additionally to bottle effects problems, it remained unclear from the very beginning if the 14 C method was measuring net or gross primary production or an intermediate between net and gross [START_REF] Steemann Nielsen | The interaction of photosynthesis and respiration and its importance for the determination of 14 C-discrimination in photosynthesis[END_REF][START_REF] Marra | Net and gross productivity: weighing in with 14 C[END_REF]. No consensus has been reached over the years. It is established that 13 C or 14 C-based estimates of photosynthesis would considerably underestimate gross production [START_REF] Grande | Primary production in the North Pacific gyre: a comparison of rates determined by the 14 C, O2 concentration and 18 O methods[END_REF]. This conclusion is unsurprising and based on the assumption that phytoplankton respires recently fixed, i.e. labelled carbon, so that the measure at the end of the incubation already incorporates part of the respiratory loss of labelled carbon. For Marra (2009), 14 C uptake is a proxy of net production since phytoplankton would respire labelled carbon as they incorporate it. Nevertheless, [START_REF] Pei | Does the 14 C method estimate net photosynthesis? Implications from batch and continuous culture studies of marine phytoplankton[END_REF] showed that this assumption wasn't always true and that some species would respire preferentially old unlabelled carbon. In this case, using labelled carbon fixation would overestimate net production. Despite these shortcomings, labelling techniques with 13 C and 14 C remain useful key methods to evaluate phytoplankton CO2-fixation.

In this study, plankton communities were incubated mimicking in situ temperature and light conditions in 13 C-enriched water from dawn to dusk (Fig. B3). Bulk CO2-fixation was measured by conventional mass spectrometry (EA-IRMS). Detailed description of the incubations and calculations performed are available in Paper 2.

Group specific CO2-fixation

Bulk measurements don't provide any information on the functional groups responsible for CO2fixation. Regarding the major diversity of natural phytoplankton communities, measuring the carbon uptake of different phytoplankton groups is paramount to explain ecosystem functioning.

To establish the relative contribution of different groups to CO2-fixation, the most common approach consists in measuring CO2-fixation of different size groups. This is most commonly achieved by successive filtration of natural communities incubated with labelled carbon on arbitrary mesh sizes (for example 20 µm, 5 µm, 2 µm) (e.g. [START_REF] Teira | Variability of chlorophyll and primary production in the Eastern North Atlantic Subtropical Gyre: potential factors affecting phytoplankton activity[END_REF][START_REF] Poulton | Phytoplankton carbon fixation, chlorophyll-biomass and diagnostic pigments in the Atlantic Ocean[END_REF][START_REF] Pérez | Vertical distribution of phytoplankton biomass, production and growth in the Atlantic subtropical gyres[END_REF][START_REF] Tilstone | Micro-phytoplankton photosynthesis, primary production and potential export production in the Atlantic Ocean[END_REF], or by flow cytometry sorting of populations characterized by homogeneous fluorescence and forward scatter, a proxy of cell size (Li, 1994;Jardillier et al., 2010;Rii et al., 2016). Flow cytometry enables the rapid counting and sorting of small autotrophic prokaryotes and pico-and nano-eukaryotes. Radiocarbon ( 14 C) incorporated in sorted cells can subsequently be quantified using a liquid scintillation cocktail (e.g. Li, 1994). Studies exploring contribution of different size groups to primary production showed that their contribution wasn't proportional to their abundance, reflecting different cell-specific CO2-fixation rates of specific size or phylogenetic groups (Li, 1994;Jardillier et al., 2010;[START_REF] Hartmann | Efficient CO2 fixation by surface Prochlorococcus in the Atlantic Ocean[END_REF]Rii et al., 2016). These differences in cell-specific CO2-fixation rates could be linked with taxonomic affiliations or size-specific nutrient requirements of the different groups (Jardillier et al., 2010). Cell biomass could be an important determinant of CO2-fixation rates as biomass-specific CO2-fixation rates were equivalent in several studies, independently of taxonomic affiliation or nutrient concentration [START_REF] Grob | Cell-specific CO2 fixation rates of two distinct groups of plastidic protists in the Atlantic Ocean remain unchanged after nutrient addition[END_REF]. This isometric relationship between biomass and CO2-fixation rates goes against the assumption that rates of metabolism decrease universally with increasing organism size, also known as the Kleiber's law [START_REF] Kleiber | Body size and metabolic rate[END_REF]. For phytoplankton, different studies have found contradictory results

regarding the universality of this rule (e.g. Huete-Ortega et al., 2012;[START_REF] Ward | The size dependence of phytoplankton growth rates: a trade-off between nutrient uptake and metabolism[END_REF]Zaoli et al., 2019), underlying the need to further explore this question in different natural environments.

Studies combining flow cytometry sorting of radiolabelled phytoplankton groups paved the way in linking phytoplankton diversity to CO2-fixation. However, they measured average CO2-fixation on a group of cells and did not inform on the actual dispersion of CO2-fixation by individual cells around that average. As microbial communities are characterized by high genetic, metabolic and phenotypic heterogeneity of single cells [START_REF] Ackermann | Microbial individuality in the natural environment[END_REF], understanding how the group average is constructed can help to understand whether all individual cells contribute equally or not to CO2fixation.

Cell-specific CO2-fixation

Recently, secondary ion mass spectrometry (large-geometry SIMS and NanoSIMS) has emerged in marine science as one of the most valuable approaches to quantitavely link microbial identity of single cells to function (reviewed by [START_REF] Mayali | NanoSIMS: microscale quantification of biogeochemical activity with largescale impacts[END_REF]. SIMS can provide spatially resolved information about uptake and transfer between organisms of isotopically-labelled compounds in environmental samples. SIMS instruments use a primary ion beam (Cs + in our case), that rasters across a flat and dry sample, slowly eroding the surface layer of that sample and creating secondary ions that are then transported into a mass spectrometer. Images of the detected ions allow the creation of isotope maps, where the ratio between counts of rare ions (e.g., 13 C) and more abundant natural ions (e.g., 12 C) can be visualized (Fig. B4). In simple words, SIMS combines the qualities of a microscope with those of a mass spectrometer and reveals elemental and isotopic compositions at a spatial resolution of 1 μm (SIMS) or even 50 nm (NanoSIMS) [START_REF] Musat | Detecting metabolic activities in single cells, with emphasis on nanoSIMS[END_REF].

As far as phytoplankton is concerned, SIMS was used to investigate virus-host interactions [START_REF] Sheik | Viral infection of Phaeocystis globosa impedes release of chitinous star-like structures: quantification using single cell approaches: single cell view on virally infected P. globosa[END_REF][START_REF] Pasulka | Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods[END_REF], transfers between phytoplankton and heterotrophic bacteria (de- et al., 2016;[START_REF] Arandia-Gorostidi | Elevated temperature increases carbon and nitrogen fluxes between phytoplankton and heterotrophic bacteria through physical attachment[END_REF], 2017), symbioses between nitrogen N2-fixing prokaryotes and photosynthetic eukaryotes [START_REF] Thompson | Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga[END_REF] and contribution of specific size and taxonomic groups to CO2-or N-fixation (e.g. Ploug et al., 2010;[START_REF] Bonnet | Diazotroph derived nitrogen supports diatom growth in the South West Pacific: a quantitative study using nanoSIMS[END_REF]Berthelot et al., 2019;Olofsson et al., 2019). All NanoSIMS studies highlighted great variability in carbon or nitrate metabolism between and within phytoplankton groups, confirming that single cell studies can shed light on how community composition affects biogeochemical process. 12C -, 13 C -, 28 Si). In this example, plankton communities were incubated with H 13 CO3 -from dawn to dusk. The resulting ratio image of 13 C/ 12 C was used to quantify CO2fixation of individual cells sorted by flow cytometry. 28 Si was used to identify the silicified frustule of diatoms. Modified from [START_REF] Musat | Detecting metabolic activities in single cells, with emphasis on nanoSIMS[END_REF].

Bashan

In our study, large diatoms collected on 20 µm mesh-size filter were measured with large-geometry SIMS and cells <20 µm with NanoSIMS. Cells <20 µm were sorted by flow cytometry into 3 different size-groups. 28 Si was used to identify diatoms. Cell C-content was computed from cell biovolume and the conversion factor to carbon from Verity et al., (1992). Finally, the group CO2-fixation was calculated by multiplying mean CO2-fixation of single cells by cell abundance of the group obtained by flow cytometry. We deduced the contribution of different size groups sorted by flow cytometry to total CO2-fixation by comparing the estimates of each groups' CO2-fixation to bulk CO2-fixation measured by conventional mass spectrometry (EA-IRMS). This method enabled to compare CO2fixation rates of different size groups, of small non-silicifed cells and small and large diatoms.

Using complementary tools of molecular biology, chemotaxonomy and SIMS at the single cell level in this thesis allowed us exploring in an original way the diversity and importance in the CO2-fixation of small phytoplankton in comparison to their larger counterparts.

C. Results

In this section, I will present the results obtained as part of my thesis project. The first chapter addresses the diversity, ecological drivers and importance of small phytoplankton in the structuring of phytoplankton communities during and after the diatom bloom. The second chapter focuses on the contribution of small phytoplankton to CO2-fixation after the bloom and its contribution to the functioning of the biological carbon pump in the area.

Chapters begin with a short introduction detailing the objectives of each study. Published or submitted papers offer an in-depth presentation of the context, questions, methods and main findings associated with each chapter. Finally, diverse perspectives for further research are suggested. On that occasion, unpublished results from analysis or methodological developments attempted during this thesis will be discussed.

Small phytoplankton diversity around Kerguelen

The naturally iron-fertilized plateau of Kerguelen has been extensively studied for the occurrence of large diatom blooms during spring and summer (see KEOPS1 and KEOPS2 publications; e.g [START_REF] Armand | Late summer diatom biomass and community structure on and around the naturally iron-fertilised Kerguelen Plateau in the Southern Ocean[END_REF][START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF]. Off-plateau, previous studies showed that unidentified pico-and nanoflagellates were the most abundant cells throughout the year (Kopczyńska et al., 1998). On-plateau, the intensity of diatom blooms is highly variable from one year to the other [START_REF] Robinson | A tale of three islands: downstream natural iron fertilization in the Southern Ocean[END_REF] and nano-sized phytoplankton probably dominate phytoplankton communities in the absence of a bloom [START_REF] Rembauville | Plankton assemblage estimated with BGC-Argo Floats in the Southern Ocean: implications for seasonal successions and particle export[END_REF][START_REF] Penna | Mesoscale variability of conditions favoring an iron-induced diatom bloom downstream of the Kerguelen Plateau[END_REF]. However, small phytoplankton communities on-plateau have never been specifically targeted before this study, and no information was available on their taxonomic composition. Off-plateau, previous studies were mainly based on microscopy observations, not adapted to identify small cells with little morphological characteristics [START_REF] Fiala | Seasonal and interannual variability of size-fractionated phytoplankton biomass and community structure at station Kerfix, off the Kerguelen Islands, Antarctica[END_REF]Kopczyńska et al., 1998). These studies highlighted the importance of naked flagellates, most likely prasinophytes, cryptophytes and prymnesiophytes in winter, where the <10 µm size fraction contributed 80% of total Chl a biomass [START_REF] Fiala | Seasonal and interannual variability of size-fractionated phytoplankton biomass and community structure at station Kerfix, off the Kerguelen Islands, Antarctica[END_REF]. Only one metabarcoding study highlighted the importance of Phaeocystis antarctica off-plateau [START_REF] Georges | Protist community composition during early phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF].

This knowledge gap on the composition of small phytoplankton at a fine taxonomic resolution concerns not only the Kerguelen area, but the SO more generally and to date, few studies specifically investigated the composition of small phytoplankton in the SO with modern molecular tools and little information is available on small phytoplankton biogeography and ecological drivers in the SO [START_REF] Díez | Distribution of eukaryotic picoplankton assemblages across hydrographic fronts in the Southern Ocean, studied by denaturing gradient gel electrophoresis[END_REF][START_REF] Thiele | Stable composition of the nano-and picoplankton community during the ocean iron fertilization experiment LOHAFEX[END_REF][START_REF] Wolf | Protist community composition in the Pacific sector of the Southern Ocean during austral summer 2010[END_REF][START_REF] Clarke | Eukaryote plankton assemblages in the southern Kerguelen axis region: ecological drivers differ between size fractions[END_REF].

The objectives of this study were to:

1. Examine the diversity of small and large protists after the bloom, with an emphasis on small phytoplankton, through 18S rDNA metabarcoding of small (<20µm) and large (20-100 µm) planktonic communities 2. Determine if small phytoplankton communities were different on and off the naturally ironfertilized plateau and if so, which environmental drivers could explain the spatial distribution of specific taxa 3. Explore the contribution of small phytoplankton to total chlorophyll throughout and after the diatom bloom. For this, a general overview of the contribution to chlorophyll biomass of broad taxonomic groups over the bloom was assessed by comparing pigment analysis (CHEMTAX) during MOBYDICK with two previous cruises in the area at bloom onset (KEOPS2) and decline (KEOPS1).

Paper 1: Marked spatiotemporal variations in small phytoplankton structure in contrasted waters of the Southern Ocean (Kerguelen area)

Diatom pictograms (Fig. 7) were modified from K. Leblanc diatom pictures available on https://plankton.mio.osupytheas.fr/mobydick-2018/ Analysis of all samples of the small size fraction showed that on and off-plateau community structures were not different (Permanova, P=0.33). Cluster analysis confirmed that samples of the < 20 µm size fraction were grouped by depth rather than by location (Fig. S5 A,B). Surface samples formed two different clusters (clusters 1 and 3). Cluster 3 corresponded to the first visits at M2 and M3, and was characterized by a high proportion of parasitic Syndiniales reads (Dino-Group 1, clade 1 and 4). In contrast, cluster 1 was dominated by autotrophs, mostly by Phaeocystis, and other small autotrophs such as Pelagomonas and the diatom Fragilariopsis at M4 (Fig. S5 B). Cluster 2 grouped all samples from 125 m and the 300 m samples of M2-3 and M1. Community composition of the samples within this cluster showed higher dispersion than in other clusters (Tukey HSD, P<0.05) and constituted a transition between deep samples (cluster 4) and surface samples of clusters 1 and 3 (Fig. S5 A). As a consequence of this heterogeneity, this cluster showed up to 50% dissimilarity between samples whereas other clusters were less than 40% dissimilar (Fig S5 B). In cluster 2, reads of Dinophyceae, Radiolaria and diatoms, in particular Chaetoceros, were abundant, which resulted in samples from 300 m depth at M1 and M2-3 also being included in this cluster. The remaining 300 m samples (all but M1 and M2-3) were grouped in cluster 4 and characterized by Radiolaria, mostly belonging to cyst-forming order Chaunacanthida (19-68% of the reads).

M1 M2_1 M2_2 M2_3 M3_1 M3_3 M4_1 M4_2 M1 M2_1 M2_2 M2_3 M3_1 M3_3 M4_1 M4_2 M1 M2_1 M2_2 M2_3 M3_1 M3_3 M4_1 M4_2 M1 M2_1 M2_2 M2_3 M3_1 M3_3 M4_1 M4_2
0 0 0 0 0 10 0 1 0 0 1 1 00 0 0 0 0 1 0 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 1 0 0 8 5 0 0 4 2 0 0 0 0 0 0 0 0 0 0 1 00 2 1 0 1 1 0 0 1 1 1 0 10 11 1 0 9 1 0 0 1 0 0 0 0 0 0 0 0 00 0 1 0 0 2 3 0 1 2 1 0 1 2 1 0 2 1 0 0 1 0 0 0 0 0 0 1 0 00 0 0 0 1 1 0 0 1 0 1 0 2 4 0 0 4 4 0 0 6 0 0 0 0 0 0 0 1 00 0 0 0 0 3 2 0 3 3 0 0 1 6 0 0 8 2 0 0 4 0 0 0 0 0 0 1 1 00 0 0 0 0 2 3 0 5 1 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 0 0 3 1 11 0 1 0 0 2 2 1 1 1 2 0 1 1 2 1 1
0 0 1 0 0 0 0 0 0 0 0 0 11 0 7 0 0 0 2 1 0 0 1 0 0 0 7 1 0 1 0 0 0 1 0 1 2 0 0 2 4 11 4 1 0 2 2 1 1 2 1 1 0 2 1 1 0 1 3 1 0 2 0 1 0 0 0 0 1 1 00 2 1 0 3 2 2
Table S2: Seasonal drivers and key players of the balance between diatoms and small phytoplankton.

Diatom silicate limitation

Grazing pressure on small phytoplankton (ciliates biomass ng C l -1 ) a Balance between diatoms and pico-and nanoflagellates

Perspectives

Complementary approaches are needed to explore phytoplankton diversity

In this study, we used 18S rDNA metabarcoding of <20 µm cells to provide a detailed description of small phytoplankton species composition and CHEMTAX to gain a more quantitative understanding of the contribution of broad taxonomic groups to the chlorophyll pool. For the moment, size-fractionated metabarcoding studies constitute the best method to explore small phytoplankton diversity, especially for non-silicified small cells lacking characteristic morphological features. For example, the presence of Micromonas was documented for the first time in the SO only recently, thanks to metagenomic analyses [START_REF] Simmons | Intron invasions trace algal speciation and reveal nearly identical Arctic and Antarctic Micromonas populations[END_REF]. However, molecular description should be considered as a complementary approach, and not an alternative, to conventional methods such as microscopy observations.

During MOBYDICK, light microscopy counts of phytoplankton and microzooplankton were also performed ( Paper 3,Christaki et al.,submitted;Lafond et al.,submitted). In our study,

Fragilariopsis, Chaetoceros, Pseudo-nitzschia and Dactyliosolen were enriched in off-plateau samples of the two size fractions (Fig. 4). Contamination by large broken cells in the small size fraction could not be excluded. However, comparison with microscopy observations confirmed that the size range of Fragilariopsis kerguelensis (10-93 µm), Chaetoceros atlanticus (7-30 µm) and

Dactyliosolen antarctica (14-72 µm) overlapped the two size fractions and that small specimens were common off-plateau (Lafond et al., submitted). For Pseudo-nitzschia sp., specimens were larger than 20 µm (22-150 µm), but very thin (2-6 µm), which could explain their presence in the small size fraction (Lafond et al., submitted). Microscopy also informed on the occurrence of diverse life forms. Chaetoceros spores/winter forms were frequently observed during microscopic observations of bottlenet samples, used to sample deep stocks (below 100m) (Leblanc, personal communication). This corroborates the high contribution of Chaetoceros reads (up to 31%) observed in this study in the small size fraction at 125 m. Although sequencing results somehow differed with microscopic counts, the two techniques highlighted the same dominant diatom genera, when biomass -and not abundances-were considered (Appendix C1).

In other cases, the discrepancy observed with microscopy highlighted problematic taxonomic affiliations. Maximum likelihood phylogenetic trees were constructed to refine or correct taxonomic assignments that were incongruent with current knowledge of diatom taxa in the SO. The only taxonomic change we made with some confidence was the assignment of Hemiaulus sequences to the microscopically observed genus Dactyliosolen (Appendix C2). It is necessary to complete existing taxonomic databases by isolating and sequencing organisms, such as Cylindrotheca, that were frequently observed with microscopy and were rare, or even missing in sequencing results (Appendix C.1). Phylogenetic trees were also constructed based on the v4 region of the 18S rRNA gene to check visually the taxonomic resolution and coherence of ASVs within lineages containing a relatively high number of unassigned sequences (e.g up to 25% of unassigned "Dinophyceae" reads). However, in most cases, phylogenetic trees could not help to resolve higher taxonomic affiliation considering the lack of reference sequences from the SO in databases and the limited variability of the sequenced region (Paper 3, Christaki et al., submitted).

From small phytoplankton diversity to functional significance

In addition to refining the identification of the taxa sequenced, another challenge would be to determine their functional significance. In this study, ASVs affiliated to divisions Chlorophyta, Cryptophyta, Haptophyta, Ochrophyta, and class marine ochrophytes (MOCH), as well as Tripos (Dinophyceae), were characterized as photoautotrophs. This classification likely includes ambiguities, such as the classification of certain mixotrophs/heterotrophs in the phytoplankton category. For example, MOCH lineages contain non-pigmented as well as pigmented cells [START_REF] Massana | Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles[END_REF]. To overcome this issue, it was initially planned to establish the composition of autotrophic eukaryotes thanks to the sequencing of the 18S rDNA of pigmented cells sorted by flow cytometry. Stringency in the definition of cytometric populations to avoid contamination with heterotrophic cells skewed the results towards large autotrophic cells, mainly diatoms. As a result, it was decided to work on the diversity of small phytoplankton based on size-fractionated sequencing (0.2-20 µm) of the whole eukaryotic community.

In addition to their role as primary producers, many small phytoflagellates are mixotrophic and major predators of prokaryotes (reviewed by Stoecker et al., 2017). Mixotrophy present a competitive edge over obligate phototrophic cells in nutrient acquisition [START_REF] Zubkov | High bacterivory by the smallest phytoplankton in the North Atlantic Ocean[END_REF].

For example, incubation experiments showed in the equatorial Pacific, that iron flux through mixotrophic flagellates could amount to 35-58% of the total iron uptake by the entire autotrophic community [START_REF] Maranger | Iron acquisition by photosynthetic marine phytoplankton from ingested bacteria[END_REF]. It would be interesting to run similar experiments in HNLC waters of the SO to see if mixotrophic small phytoplankton can alleviate iron limitation by ingesting bacteria. In our dataset, Micromonas contribution to number of reads strongly increased on-plateau over the repeated visits (Fig. S5b). This species has been reported as mixotrophic in the Arctic (McKie-Krisberg and Sanders, 2014). During MOBYDICK, uptake of fluorescently labeled bacteria by small phytoplankton was insignificant (Christaki et al., 2020). However, the fluorescently labeled bacteria may have been too large (appr. 0.8 µm) to be efficiently grazed by abundant pico-sized phytoplankton such as Micromonas, which have been shown to prefer small-sized prey (<0.5 µm) [START_REF] Mckie-Krisberg | Phagotrophy by the picoeukaryotic green alga Micromonas: implications for Arctic Oceans[END_REF].

Mixotrophy is predicted to enhance the transfer of biomass to larger organisms at higher trophic levels and increase the efficiency of the BCP through the production of larger, faster-sinking, and carbon-rich organic detritus, it could be interesting to further explore the trophic status of specific species of small phytoplankton identified in this study as key players in the area.

Describing phytoplankton community succession in remote areas

CHEMTAX analysis over the different cruises established the seasonal succession of phytoplankton communities over the bloom and metabarcoding precised who were the key players of small phytoplankton communities after the bloom. More size-fractionated metabarcoding studies are needed to further explore the spatial and temporal distribution of small phytoplankton taxa in the SO at the species level. This would enable to explore how specific taxonomic groups or species, such as pelagophytes or Micromonas respond to nutrient availability throughout the season (iron and ammonium particularly). However, exploring possible seasonal succession within small phytoplankton taxa throughout the season is for the moment impossible due to limited access to this remote ocean. Recently, high-resolution seasonal observations of the prokaryotic community were conducted using a moored remote autonomous sampler (RAS) deployed over 4 months [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF]. In the frame of this project, protist communities were also sequenced. Some promising patterns emerged from the analysis, that could shed light on dynamic interactions between phytoplankton and their grazers or parasites (Appendix C3). However, the fixative usedglutaraldehyde or mercuric chloride (HgCl2)-profoundly affected the picture and diversity of the community sequenced. Preliminary tests showed that glutaraldehyde fixed samples showed 70% similarity with unfixed controls for eukaryotic communities, while similarity dropped to 40% when the samples were preserved with HgCl2 (Liu, personal communication). Moreover, sequencing results of diatoms communities were too incongruent with microscopy counts to be confidently interpreted (Liu, personal communication). We tested a kit specifically designed to counterbalance the DNA damages due to the use of fixatives (QIAamp DNA FFPE Tissue Kit, Qiagen, Germany), but sequencing results after extraction using this kit still differed considerably between fixed and unfixed samples (Appendix C4). These major technical obstacles and the difficulty to sample this stormy remote ocean during winter months explain why composition of small phytoplankton communities of the SO remain virtually unexplored during autumn and winter, the seasons when the ecosystems most presumably rely on them.

Abundances Biomass <20µm >20µm

Sequencing Microscopy Sequencing Microscopy

0% 100% M2-2 10m M2-2 10m M2-2 10m M2-2 10m M2-2 70m M2-2 70m M2-2 60m M2-2 60m
100% 0% M4-1 10m M4-1 10m M4-1 10m M4-1 10m M4-1 70m M4-1 70m M4-1 60m M4-1 60m a.
b.

CO2-fixation by small phytoplankton around Kerguelen

Introduction

The high abundance and important contribution to Chl a of small phytoplankton after the bloom (Paper 1) suggested they could play an important, yet totally overlooked, role in CO2-fixation onplateau. Indeed, previous studies on the Kerguelen Plateau occurred during the bloom onset and decline and established that large diatoms accounted for 80-90% to the total primary production (of 1 g C m -2 d -1 ) on-plateau, whereas off-plateau production was much lower (0.30 C m -2 d -1 ) and mainly (65%) achieved by small diatoms and nanoflagellates (Uitz et al., 2009). Diatom-dominated phytoplankton communities on-plateau were characterized by low export efficiency, in contrast to ecosystems dominated by small cells, where export efficiency at 100 m reached 58% [START_REF] Savoye | 234 Th-based export fluxes during a natural iron fertilization experiment in the Southern Ocean (KEOPS)[END_REF]Christaki et al., 2014;Planchon et al., 2015).

Moreover, we established in Paper 1 that small phytoplankton communities differed on-and offplateau, as a result of contrasted post-bloom conditions. Small non-silicified cells, as well as small and large diatoms are associated with unique physiological requirements and ecological characteristics, that are likely to impact CO2-fixation and C-export efficiency [START_REF] Litchman | Global biogeochemical impacts of phytoplankton: a trait-based perspective[END_REF]Tréguer et al., 2018). The seasonal succession and differences in phytoplankton community composition described in Paper 1 are thus expected to have consequences on production and export regime around Kerguelen.

The objectives of this study were to:

1. Evaluate the variability in CO2-fixation rates between and within different groups of phytoplankton on-and off-plateau. For this, CO2-fixation rates were measured at the single cell level using NanoSIMS for small cells (non-silicified cells and diatoms <20 µm) and large-geometry SIMS for larger diatoms (>20 µm).

2. Establish the contribution of small phytoplankton to community CO2-fixation 3. Make hypothesis on the fate of phytoplankton, by looking at grazing indicators and changes in the contribution of broad taxonomic groups to Chl a with depth.

Introduction

Carbon fixation (CO2-fixation) by marine phytoplankton accounts for about half the Earth's primary production [1][2][3]. Some 20% of phytoplankton's net primary production (5-10 Gt C) is exported to the deep ocean via the biological pump [4,5]. The magnitude and nature of the carbon exported to the deep ocean is impacted by the size-structure of phytoplankton communities [6,7]. High carbon export (Cexport) out of the photic zone is classically linked to the dominance of large phytoplankton (herein defined as >20 µm cells) because of their high sinking velocity or repackaging into dense fecal pellets produced by large grazers [8][9][10]. Alternatively, it has been suggested that small phytoplankton contribution to export was proportional to their total net primary production through aggregation into larger sinking particles, or, as for their larger counterpart, export as fecal pellets produced by higher trophic levels [11].

Determining the contribution of diverse phytoplankton size-groups to CO2-fixation is thus the first step to characterize the functioning of the carbon pump. This is routinely achieved by measuring size-fractionated CO2-fixation rates in natural communities using isotopic tracers ( 14 C or 13 C labelled substrates) that can be used to model marine production [12,13]. Defining a general size-scaling relationship for CO2-fixation from pico-flagellates to large diatoms based on accurate measurements represents thus a major challenge for models relying on the use of theoretical size-scaling relationships for phytoplankton growth [14,15]. However, size-based models may be biased due to metabolic variability within a size-class grouping diverse phylogenetic taxa [16,17]. Including phylogenetic features can further refine phytoplankton production models, but increase drastically their complexity [START_REF] Lancelot | Modeling phytoplankton blooms and carbon export production in the Southern Ocean: dominant controls by light and iron in the Atlantic sector in Austral spring 1992[END_REF][START_REF] Wang | Incorporating Phaeocystis into a Southern Ocean ecosystem model[END_REF][START_REF] Worthen | A coupled ocean-ecosystem model of the Ross Sea. Part 1: Interannual variability of primary production and phytoplankton community structure[END_REF]. Consequently, it is crucial to determine the degree of complexity required (e.g. size, species, population) and define the descriptors (e.g. biomass, abundances) needed to improve models. To do so, in situ measurements are required to appreciate the variability of CO2-fixation rates between and within different phytoplankton groups. Diverse studies have previously revealed that the contribution to biogeochemical cycles is not necessarily proportional to microbial group abundance or biomass. For example, flow cytometry sorting of different small-sized microbial autotrophs has revealed that although picoeukaryotes are far less abundant than cyanobacteria, their contribution to CO2-fixation is similar or even greater, relatively [START_REF] Li | Primary production of prochlorophytes, cyanobacteria, and eucaryotic ultraphytoplankton: Measurements from flow cytometric sorting[END_REF][START_REF] Jardillier | Significant CO2 fixation by small prymnesiophytes in the subtropical and tropical northeast Atlantic Ocean[END_REF][START_REF] Rii | Diversity and productivity of photosynthetic picoeukaryotes in biogeochemically distinct regions of the South East Pacific Ocean: Picophytoplankton diversity and productivity in the S. Pacific[END_REF]. More recently, secondary ion mass spectrometry (SIMS) has allowed measurements at the singlecell scale at a resolution of appr. 50 nm (NanosSIMS) or 1 μm (large-geometry SIMS). A pioneer lacustrine study revealed that rare phototrophic bacterial taxa (0.3% of the total cell number) could contribute to more than 70% of the total carbon uptake [START_REF] Musat | A single-cell view on the ecophysiology of anaerobic phototrophic bacteria[END_REF]. Subsequent studies also revealed higher marine phytoplankton contribution to C-or N-fixation than expected from their relative abundance or biomass for diverse microbial groups. This has been observed for example for diazotrophs-associated diatoms [START_REF] Foster | Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses[END_REF], chain-forming diatoms [START_REF] Olofsson | Nitrate and ammonium fluxes to diatoms and dinoflagellates at a single cell level in mixed field communities in the sea[END_REF], or specific pico-phytoplankton groups [START_REF] Berthelot | NanoSIMS single cell analyses reveal the contrasting nitrogen sources for small phytoplankton[END_REF]. This approach also unveiled a high microbial intra-group heterogeneity in C-or N-uptake, likely affecting the group's adaptation potential to changing environments [START_REF] Musat | A single-cell view on the ecophysiology of anaerobic phototrophic bacteria[END_REF][START_REF] Foster | Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses[END_REF][START_REF] Berthelot | NanoSIMS single cell analyses reveal the contrasting nitrogen sources for small phytoplankton[END_REF][START_REF] Ploug | Carbon and nitrogen fluxes associated with the cyanobacterium Aphanizomenon sp. in the Baltic Sea[END_REF][START_REF] Olofsson | High single-cell diversity in carbon and nitrogen assimilations by a chain-forming diatom across a century[END_REF]. The reasons for this high heterogeneity are unclear but could result from intra-group genetic diversity, intra-group differences in gene expression, or cell life history [START_REF] Musat | A single-cell view on the ecophysiology of anaerobic phototrophic bacteria[END_REF]. Recently, it has been suggested that intra-specific variability in C-or N-uptake is correlated to differences in biovolumes from one cell to the other [START_REF] Zaoli | Generalized size scaling of metabolic rates based on single-cell measurements with freshwater phytoplankton[END_REF].

The Southern Ocean (SO) is an ideal study area to explore phytoplankton CO2-fixation, as it contributes up to 40% (42 ± 5 Pg C) of the oceanic uptake of anthropogenic CO2 [START_REF] Frölicher | Dominance of the Southern Ocean in anthropogenic carbon and heat uptake in CMIP5 models[END_REF][START_REF] Landschützer | The reinvigoration of the Southern Ocean carbon sink[END_REF]. Most of the SO is composed of high-nutrient, low-chlorophyll (HNLC) areas, where primary production is limited by iron despite high macronutrients concentrations [START_REF] Martin | Glacial-interglacial CO2 change: The Iron Hypothesis[END_REF][START_REF] De Baar | Importance of iron for plankton blooms and carbon dioxide drawdown in the Southern Ocean[END_REF][START_REF] De Baar | Synthesis of iron fertilization experiments: From the Iron Age in the Age of Enlightenment[END_REF]. In these low productive environments, phytoplankton communities and primary production are typically dominated by small cells (<20 µm) [START_REF] Weber | Contributions of the net, nano-and picoplankton to the phytoplankton standing crop and primary productivity in the Southern Ocean[END_REF][START_REF] Froneman | Size-fractionated primary production in the South Atlantic and Atlantic Sectors of the Southern Ocean[END_REF]. However, large diatoms have attracted most attention because of the enhanced production and C-export observed during diatom blooms in discrete, naturally iron-fertilized regions of the SO such as Kerguelen, Crozet, or South Georgia during spring and summer [START_REF] Blain | Effect of natural iron fertilization on carbon sequestration in the Southern Ocean[END_REF][START_REF] Pollard | The Crozet Natural Iron Bloom and Export Experiment (CROZEX). Deep Sea Res Part II[END_REF][START_REF] Korb | Magnitude and maintenance of the phytoplankton bloom at South Georgia: a naturally iron-replete environment[END_REF]. This study is part of the MOBYDICK cruise (Marine Ecosystem Biodiversity and Dynamics of Carbon around Kerguelen: an integrated view) that aimed at understanding the link between biodiversity and carbon fluxes on and off the naturally iron-fertilized Kerguelen plateau. Off-plateau, phytoplankton biomass and production are dominated throughout the year by small size-groups [12,[START_REF] Kopczyńska | Annual and interannual variability in phytoplankton at a permanent station off Kerguelen Islands, Southern Ocean[END_REF]. On-plateau, spring blooms of chain-forming and large diatoms typically end in February because of silicic acid and iron co-limitation [START_REF] Mosseri | Impact of iron on silicon utilization by diatoms in the Southern Ocean: A case study of Si/N cycle decoupling in a naturally iron-enriched area[END_REF]. MOBYDICK was the first study in this area that took place after the diatom bloom (March 2018).

Our main objective was to describe the diversity and assess the role of small phytoplankton in CO2fixation in post-bloom conditions. Surface CO2-fixation and division rates of phytoplankton at the single cell level were measured, focusing on small phytoplankton (non-silicified and small diatoms), which have been overlooked so far in this Oceanic region. Changes in the contribution of broad taxonomic groups to chlorophyll a (Chl a) with depth were used to discuss how small phytoplankton could potentially contribute to C-export.

Materials and methods

Sampling location

Four different sites were visited on and off the Kerguelen Plateau during the MOBYDICK cruise (Table 1). Station M2, located on the iron-fertilized plateau, was sampled three times at 9 to 10-day intervals.

Three off-plateau stations were also sampled (M1, M3, and M4). Station M4 was sampled twice at 2week intervals, and M1 and M3 was sampled only once. M2, M1 and M4 were located south of the polar front in Antarctic waters. M3 was located south-west of the plateau in subantarctic waters (Fig. 1).

Samples were collected with a rosette equipped with Niskin bottles and a CTD probe (SeaBird 911-plus).

Three casts were done at all stations for: i) nutrient concentration measurements as well as phytoplankton community composition based on pigment analyses; ii) microbial eukaryote community composition through a metabarcoding approach: and iii) CO2-fixation measurements using stable isotope ( 13 C) tracer experiments.

Water sampling for nutrients and pigment analysis

Samples for dissolved inorganic nutrients measurements (silicic acid, nitrate, phosphate and ammonium) and pigment analysis were taken at all stations at 9-10 depths (10,[START_REF] Foster | Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses[END_REF][START_REF] Strom | Production of pheopigments by marine protozoa: results of laboratory experiments analysed by HPLC[END_REF][START_REF] Christaki | Microbial food web dynamics during spring phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF]100,125,150,175,200 and 250 m). Ammonium was measured by fluorometry [START_REF] Holmes | A simple and precise method for measuring ammonium in marine and freshwater ecosystems[END_REF]. Other nutrients were analyzed colorimetrically as described in Aminot and Kérouel [START_REF] Aminot | Dosage automatique des nutriments dans les eaux marines: méthodes en flux continu[END_REF].

For pigment analysis, 2.3 L of seawater were collected and filtered onto Whatman GF/F filters. Filters were then flash-frozen in liquid nitrogen and stored at -80°C. Pigment determination was done using High Performance Liquid Chromatography (HPLC), following the method of Ras et al. [START_REF] Ras | Spatial variability of phytoplankton pigment distributions in the Subtropical South Pacific Ocean: comparison between in situ and predicted data[END_REF]. HPLC data from all sampling points in the first 250 m were considered for CHEMTAX analysis [START_REF] Mackey | CHEMTAX-a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton[END_REF]. The contribution to Chl a of seven different taxonomic groups (chlorophytes, prasinophytes, cyanobacteria (Synechococcus spp.), cryptophytes, diatoms, autotrophic dinoflagellates (with peridinin) and haptophytes (Phaeocystis like) was determined based on their characteristic pigment profiles. Samples were first clustered based on their pigment:Chl a ratios to form homogeneous bins. Then, pigment:Chl a ratios were adjusted for each bin using a 60 randomized ratio matrix varying by up to 35% of the initial ratio matrix to avoid any bias linked to the ratios chosen from the literature [START_REF] Wright | Phytoplankton community structure and stocks in the Southern Ocean (30-80 E) determined by CHEMTAX analysis of HPLC pigment signatures[END_REF][START_REF] Van Leeuwe | The pigment composition of Phaeocystis antarctica (Haptophyceae) under various conditions of light, temperature, salinity, and iron[END_REF]. The contribution of the different groups to Chl a was determined by averaging the six best runs.

Phaeopigments (the sum of phaeophytin-a and phaeophorbide-a) are degraded Chl a products.

Phaeophytin-a is traditionally thought to result from grazing, while phaeophorbide-a may arise from both phytoplankton senescence and grazing [START_REF] Szymczak-Żyla | The influence of microorganisms on chlorophyll a degradation in the marine environment[END_REF]. In this study, it was considered that phaeopigments were mostly likely associated with grazing activity [START_REF] Strom | Production of pheopigments by marine protozoa: results of laboratory experiments analysed by HPLC[END_REF], since chlorophyllide-a, a degradation pigment associated with cell senescence [START_REF] Wright | Phytoplankton community structure and stocks in the Southern Ocean (30-80 E) determined by CHEMTAX analysis of HPLC pigment signatures[END_REF], was only detected at very low concentration (<0.004 µg L -1 ) in two samples. The ratio phaeopigments:Chl a was determined from the surface down to 250 m. A ratio <1

indicates that phytoplankton material is mostly fresh, whereas a ratio >1 indicates mostly degraded material [START_REF] Roca-Martí | High particulate organic carbon export during the decline of a vast diatom bloom in the Atlantic sector of the Southern Ocean[END_REF].

Water sampling for metabarcoding of phytoplankton communities

Water samples were collected at 15 m at all stations to describe small and large phytoplankton communities with metabarcoding of 18S rDNA. After pre-filtering through 100 μm mesh to remove most of the metazoans, 10 L of seawater were successively filtered through 20 μm (20-100 µm size-fraction) and 0.2 μm (0.2-20 µm size-fraction). Filters were stored at -80°C until processing. DNA was extracted following PowerSoil DNA Isolation Kit (QIAGEN, Germany) standard manufacturer's protocol. The 18S rDNA V4 region was amplified using EK-565F (5'-GCAGTTAAAAAGCTCGTAGT) and UNonMet (5'-TTTAAGTTTCAGCCTTGCG) primers [START_REF] Bower | Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues[END_REF]. Pooled samples were sequenced on an Illumina MiSeq lane

(2 x 300bp) at the company Genewiz (South Plainfield, NJ, USA). Quality filtering of the reads, identification of amplicon sequencing variants (ASV) and taxonomic affiliation based on the PR2 database [START_REF] Guillou | The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy[END_REF] were done in the R-package DADA2 [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF]. ASVs affiliated to divisions Chlorophyta, Cryptophyta, Haptophyta, Ochrophyta, and class marine ochrophytes (MOCH) were filtered to describe phytoplankton communities. Relative abundances of ASVs were normalized to the total number of sequences affiliated to autotrophic phylogenetic taxa to build heatmaps of small (0.2-20 µm) and large (20-100 µm) phytoplankton taxa with package ampvis2 [START_REF] Andersen | ampvis2: an R package to analyse and visualise 16S rRNA amplicon data[END_REF]. Raw sequencing files in fastq format, as well as ASVs, taxonomy and metadata tables are available on MOBYDICK's public database at http://www.obs-vlfr.fr/ proof/ftpfree/mobydick/db/DATA/PAR_2251/.

Water sampling for stable isotope experiments

Seawater samples were collected at each site at least one hour before sunrise from surface waters ( 10or 15 m depth) to evaluate phytoplankton CO2-fixation. Five HCl-cleaned polycarbonate 12.5 L carboys were filled with 12 L of seawater prefiltered on a 100 µm mesh (Fig. S1). Three carboys were spiked with 12 mL of NaH 13 CO3 solution (99% 13 C, Cambridge Isotope Laboratories, Inc.), targeting an enrichment of 10% in DI 13 C. Two carboys were left unspiked as negative control at T0 and Tfinal. Four carboys (one control and three carboys enriched with DI 13 C) were incubated on-deck from dawn to dusk (Table S1). In situ temperature was reproduced in the incubator by a constant flow of sub-surface seawater. In situ light intensity of the sampling depth was mimicked using blue light screens attenuating direct sunlight by approximately 50%. Among the three 13 C enriched carboys, one was left in the dark. Incubations were stopped after sunset by adding paraformaldehyde (PFA; 1% final concentration w/v). After one hour of fixation in the dark, several sub-samples were taken:

(1) To calculate the bulk CO2-fixation of the community, triplicates from each carboy of 1.5 L were filtered onto precombusted (450°C, 4 h) GF/F filters, rinsed three times with 20 mL of filtered seawater (0.2 µm pore size membranes) and stored in pre-combusted dark glass tubes at -80°C. Back in the laboratory, these filters were dried at 60°C overnight, pelletized into tin capsules and analyzed by an elemental analyzer coupled to a continuous flow isotope-ratio mass spectrometer (EA-IRMS).

(2) To measure the CO2-fixation at the single cell level (large geometry SIMS and NanoSIMS analysis), large (>20µm) and small (<20µm) cells were collected in duplicates for each treatment by successive filtration of 2 L on 20 µm pore size nylon filter (Millipore, USA) and 0.65 µm pore size PVDF filter (Durapore, Germany) and stored at -80°C.

(3) To evaluate potential effects of the incubation conditions on plankton community composition, 2 x 5 mL of water were sampled at T0 and Tfinal from each carboy for cytometry analysis of pico-, and nanophytoplankton abundances.

(4) To determine more precisely the abundance of different taxonomic groups using FISH, 300 mL of water from the T0 carboy were filtered onto 0.4 µm polycarbonate filters (Nuclepore Track-Etch Membrane, Whatman, USA) and further dehydrated successively with 50%, 70%, and 100% ethanol for 3 minutes each [START_REF] Not | Application of fluorescent in situ hybridization coupled with tyramide signal amplification (FISH CARD) to assess eukaryotic picoplankton composition[END_REF] (Supplementary material).

Preparation of samples for secondary ion mass spectrometry (SIMS)

Single cells CO2-fixation analysis was performed with SIMS. For large cells, the 20 µm nylon filters were placed in 3 mL of 0.01 µm filtered seawater and gently vortexed to detach the cells. The solution was then pipetted onto a 0.2 μm polycarbonate membrane directly connected to a low-vacuum pump (< 0.5 atmosphere) in order to concentrate the cells on a spot of about 2 mm 2 on the filter. No samples were prepared for SIMS analysis of large cells at station M3 since almost no cells were collected on the 20 µm nylon filter during the sampling.

To detach and collect small cells (<20 µm), the 0.65 µm pore size PVDF filters were cut into small pieces, placed in a 3 mL solution composed of 0.01 µm filtered seawater and Pluronic (0.01% w/v final conc., Sigma-Aldrich), and sonicated twice for 1 minute. Small autotrophs were then sorted using BD FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA; UNICELL facility). Three different populations were gated at each station based on diverse combinations of red (690/50 nm, for chlorophyll a detection)

and orange (585/42 nm, for phycoerythrin detection characteristic of Synechococcus) fluorescence, forward scatter (FSC, related to cell size) and side scatter (SSC, related to cell structure). Synechococcus cells were used as standard to ensure homogeneity in the gating of cells of pico-size from one station to another. Synechococcus spp. abundances were low at all stations (45-400 cells mL -1 ), so that they were sorted together with pico-eukaryotes (pigmented eukaryote cells in the same size range as Synechococcus) in a population hereafter called Pico (Fig. S2). Small pigmented nano-eukaryotes were sorted into two groups (Nano1 and Nano2) according to their red fluorescence and forward scatter. Sorted cells were directly collected onto a 0.6 μm polycarbonate membrane (DTTP01300, Millipore) in the sorting chamber using a low-vacuum in order to maximize cell density on the filter [START_REF] Berthelot | NanoSIMS single cell analyses reveal the contrasting nitrogen sources for small phytoplankton[END_REF]. All filters were stored at -20 °C until analysis. Abundances of the populations sorted were determined in triplicates at the beginning and end of the incubations using a CytoFLEX (Beckman Coulter, Singapore) at a high flow rate (60 μl min -1 ) for 3 minutes. Homogeneity in the gating of the populations between the two flow cytometers used was ensured using Synechococcus as standard.

SIMS analyses (Large geometry SIMS and NanoSIMS)

Pieces of the filters prepared for SIMS analyses were placed on double-sided conductive adhesive copper tape and mounted on plots adapted to SIMS samples holders. They were then metalized by sputter deposition of a gold film (20-50 nm thickness).

The 13 C-fixation of large diatoms was measured using a large geometry SIMS (IMS1280, Cameca, Gennevilliers, France) at the Centre de Recherches Pétrographiques et Géochimiques (CRPG, CNRS-Univ. Lorraine, Nancy, France). Areas of interest (120 µm * 120 µm) were pre-sputtered with a primary 10 nA Cs + beam for 5 min to remove the silica frustules of most diatoms and access their cellular content.

Analyses were conducted on a 100 * 100 µm field using a 50-100 pA Cs + beam with a spatial resolution of approx. 1.5 µm for 80 cycles. Secondary ion images (512 * 512 pixels) were recorded for 12 C 14 N -(2 s per cycle), 13 C 14 N -(4 s per cycle) and 28 Si (2 s per cycle) at a mass resolution of 12 000 (M/ΔM).

The 13 C-fixation of small pigmented cells sorted by flow cytometry in three populations (Pico, Nano1 and Nano2) was measured using a NanoSIMS 50 (Cameca, Gennevilliers, France) at the Museum National d'Histoire Naturelle (MNHN, Paris, France). NanoSIMS analyses were conducted on a field size of 40 * 40 µm (255 * 255 pixels) with a primary Cs + ion beam of 1.2 pA with a lateral resolution of 60-120 nm for 1000 µs px -1 . A larger field (42 * 42 µm) was pre-sputtered with a high primary ion beam current (300 pA) for 2 to 2.5 min. Secondary ions 12 C, 13 C, 12 C 14 N -, 13 C 14 N -and 28 Si were collected on at least 20 planes.

For large geometry SIMS and NanoSIMS images, regions of interest corresponding to single cells were manually defined using Limage software (Larry Nittler, Carnegie Institution of Washington) based on the total 12 C 14 N -ion counts. 28 Si was further used to correct the shape of diatoms based on their silica frustule.

The equivalent spherical diameter (ESD) was measured on NanoSIMS images and used to estimate biovolumes of small non-silicified cells. For small diatoms, the biovolume was calculated after Sun and

Liu [START_REF] Sun | Geometric models for calculating cell biovolume and surface area for phytoplankton[END_REF], taking measures on NanoSIMS images for each silicified cell. The average ESD of Pico, Nano1 and Nano2 cells were 1.6 0.3, 2.5 0.4, and 4.8 1.6 µm, respectively.

A total of 344 cells were analyzed with large geometry SIMS and 2194 with NanoSIMS (1162 Pico, 944

Nano1, and 211 Nano2: Table S2). In addition, 774 non-enriched cells from the control carboys were analyzed to determine natural 13 C isotopic content of phytoplankton cells.

CO2-fixation calculations (EA-IRMS, large geometry SIMS and NanoSIMS)

Bulk CO2-fixation rates measured by EA-IRMS (µmol C L -1 d -1 ) were calculated as follows:

Bulk CO2-fixation = A sample POC -A control POC A enriched DIC -A natural DIC xPOC !"#$%&
Where A is the 13 C isotopic fractional abundance (in atom%) of the community labeled with For each cell analyzed with NanoSIMS, 13 C 14 N -and 12 C 14 N -ions were counted and specific fractional abundance (ACell) were calculated where

" '&%% = C () & * (+ ('&%%) C () & * (+ ('&%%) + C (.

& * (+ ('&%%)

× 100

To assess the metabolic activity of individual cells, C-based cell-specific division rates (d -1 ) were calculated as in Berthelot et al. [27], assuming that DIC was the only carbon source used for growth:

+ --./01 2033 -/4025652 1575/589 :.;0/(1 *( ) = 38= .

/ !"# */ #$%&'$( / !"# */ #)((
with " '01230% being the mean 13 C cell fractional abundance in non-enriched populations. Cells whose fractional abundance enrichment " '&%% -" '01230% was less than two times the standard deviation associated with the Poisson distribution parameterized by λ = " '&%% * & '45&%% , with & '45&%% being the CN - ion counts of the cell, were considered as inactive [START_REF] Berthelot | NanoSIMS single cell analyses reveal the contrasting nitrogen sources for small phytoplankton[END_REF].

Contribution of the different population sorted by flow cytometry was calculated by multiplying the mean cell-specific CO2-fixation by the abundance of the population.

For this, the C-based turnover of the cellular C-content was calculated as follows:

+ --./01 ;>:9870: 86 ;ℎ0 2033>3.: + -289;09; = " '&%% -" '01230% " 67' -" '01230%

Cell-specific CO2-fixation (fmol C cell -1 d -1 ) were obtained by multiplying the C-based turnover of the cellular C-content by the carbon content of the cell, calculated after Verity et al. [START_REF] Verity | Relationships between cell volume and the carbon and nitrogen content of marine photosynthetic nanoplankton[END_REF]:

+ -2033 289;09; = 0.433 × C58783>D0 8.:;)

Statistical analysis

All statistical analyses were conducted in R [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF]. Differences in CO2-fixation rates between groups and stations were assessed using the Kruskal Wallis test, followed by pairwise the Mann-Whitney test with

Bonferroni correction for multiple comparisons with ggpubr package. Interquartile range (IQR) was used as a measure of statistical dispersion within groups. The package fitdistrplus was used to select the best probability distribution fitting the division rates observed for small and large cells.

Results

Study area

Chl a was low at all stations and visits (0.18-0.31 and 0.28-0.58 mg m -3 on-and off-plateau, respectively).

Nutrient concentrations were contrasted on-and off-plateau (Table 1). Plateau station M2 was depleted in silicic acid (<2 µmol L -1 ), but silicic acid concentrations and Chl a doubled at the last visit (M2-3) after a storm on the 10 th March. Ammonium concentrations were higher at M2 than at off-plateau stations. Offplateau stations sampled in HNLC waters presented higher nitrate, silicic acid, and phosphate concentrations than on-plateau (Table 1). Stations M1 and M4, south of the polar front, were characterized by lower temperature and higher silicic acid concentrations than M3, located in subantarctic waters north of the polar front (Fig. 1).

Composition of phytoplankton communities

Haptophytes and diatoms contributed the most to Chl a at surface at all stations (36-70% and 18-40%, respectively: Fig. 2a,b). Chl a concentration strongly decreased between 75 and 125 m depending on the station. Down to 250 m, Chl a concentrations were low (0.01 µg Chl a L -1 ) and diatoms accounted for 77-96% of total Chl a.

Vertical distribution of haptophyte's pigments and Phaeo/Chl a ratio differed between stations (Fig. 2a,b). At M2 and M1 stations, haptophyte pigments were abundant at surface but decreased rapidly with depth and almost disappeared below 75m. These stations were also characterized by Phaeo/Chl a ratios >1 below 175m, indicating that pigments found below this depth were mostly degraded. At off-plateau stations M3 and M4, haptophyte pigments signature extended deeper. Phaeo/Chl a ratio was approximately equal to 1 at 250m, reflecting a similar contribution of fresh and degraded pigments at this depth (Fig. 2b).

Sequencing data revealed that Phaeocystis antarctica (haptophyte) was the most abundant phytoplankton taxa in the small size fraction on-and off-plateau (up to 76% of the reads: Fig. 3). Other common non-silicified phytoplankton taxa of the small size fraction included chlorophytes Prasinoderma (Prasinococcales family, 34% of the reads at M3) and Micromonas (Mamiellaceae family, 3-13% of the reads at M2). CARD-FISH counts confirmed the importance of haptophytes (2-5µm in size) on-and offplateau (735 -4950 cells mL -1 ), and of prasinophytes (<2µm in size) on-plateau (Fig. S3). Members of the Pelagophyceae family were common at off-plateau stations, in particular Pelagococcus (23% of the reads at M3) and Pelagomonas (5-10% at M4 and M1, respectively). Diatoms contributed for 10-45% of the total number of reads in the small size fraction, with a higher contribution of raphid pennates (Fragilariopsis and unidentified raphid pennates) off-than on-plateau (8-30% and 4-6% of reads number respectively: Fig. 3).

Diatoms were the dominant phytoplankton class of the large size fraction (>20 µm in size; 55-97% of the reads). Off-plateau, diatom communities were composed of pennate (Fragilariopsis, Pseudo-nitzschia)

and centric diatoms (Thalassiosira, Chaetoceros, Proboscia and Rhizosolenia). On-plateau, large diatom communities were dominated by centric diatoms (Eucampia during the first visit and Corethron for the two last visits). Phaeocystis was abundant in read numbers in the large size fraction at M1 and M3-1 ( 40and 21% of the reads respectively).

C-based division rates of small and large cells

Over 98% of the small cells measured with NanoSIMS were actively taking up carbon, at the exception of M1 where slightly less cells were active (92%: Fig. 4a). Division rates were significantly higher on-than off-plateau (mean from 0.33-0.38 and 0.18-0.26 division d -1 , respectively: Fig. 4a). However, Nano2 cells were characterized by lower division rates than Pico and Nano1 on-and off-plateau (Fig. 4b). Nano2 also presented higher variability and higher Interquartile Range (IQR) of the division rates than the two other small cells groups. Interestingly, Nano2 was mostly composed of small diatoms off-plateau (70%), while non-silicified cells were the major contributors of this group on-plateau (81%). Division rates of small diatoms were significantly lower than those of non-silicified cells on-plateau (Mann-Whitney, P<10 -9 ), but they were not different off-plateau (Fig. S4). Division rates of small cells (Pico, Nano1 and Nano2) followed a symmetrical logistic distribution, very similar to the normal distribution (Fig. S5 a,b).

The mean division rates of larger diatoms (>20 µm in size) were relatively low and showed great variability (0.11 ± 0.14 and 0.09 ± 0.11 division d -1 ). Mean division rates of large diatoms on-plateau were 0.17, 0.05 and 0.12 division d -1 during the first, second and third visit at M2, respectively, while they ranged between 0.08 to 0.10 division d -1 at off-plateau stations (Fig. 4c). The proportion of inactive diatoms varied from zero to 27% and 14 to 39 % on-and off-plateau, respectively. However, some active outliers (<7% of the diatoms measured with large-geometry SIMS) showed high division rates reaching 0.72 and 0.51 division d -1 on-and off-plateau, respectively (Fig. 4c). As a consequence, the distribution best fitted to large diatoms' division rates was a log-normal distribution skewed towards low values (Fig. S5c).

CO2-fixation by small phytoplankton

For small cells, the amount of carbon fixed at the single cell level (C-fix) scaled allometrically with cell volume (V) according to a power law C-fix= a.V α , where the scaling exponent α=0.81 and 0.75 on-and off-plateau respectively (Fig. 5). This relationship explained 66% of the variance observed in CO2-fixation of individual cells on-plateau and 54% off-plateau, where a few inactive cells departed from this relation.

Consequently, mean daily CO2-fixation rates at each station were highest for Nano2, intermediate for Nano1 and the lowest for Pico-cells (Table S3). However, when normalized to cell volume, the volumespecific CO2-fixation rates were decreasing with size (Fig. S6).

Estimated contribution of the different small phytoplankton's size-groups to total CO2-fixation was important on-and off-plateau (41-61% and 43-70% on and off plateau respectively; Fig. 6a). Nano1 was the most important contributor within small autotrophs to CO2-fixation at all stations (17-34%) except M2-1 where Pico contribution was higher (21%). Total community CO2-fixation off-plateau varied between 0.20 and 0.44 µmol C L -1 (Fig. 6b). The CO2-fixation on-plateau was slightly higher during the first two visits (0.37-0.48 µmol C L -1 ) and doubled at the third visit (0.92 µmol C L -1 ; Fig. 6b). The doubling of the CO2-fixation at the last visit at M2 was associated with the doubling of Chl a concentration as well as increases in abundances of the three small phytoplankton size-groups (Fig. S3; Table S2). Estimation of the contribution to CO2-fixation of large diatoms by extrapolation of their CO2-fixation rates was not possible because of: (i) the low number of large diatoms analyzed; and (ii) the variability observed in their division rates, with mean division rates very sensitive to the presence of active outliers.

Discussion

We report here, for the first time, that small phytoplankton (mainly non-silicified) could represent 41 to 61% of the total CO2-fixation on the Kerguelen Plateau, a naturally iron-fertilized area previously characterized by the dominance of chain-forming and large diatoms. Previous estimates of small phytoplankton contribution to CO2-fixation in other naturally iron-fertilized regions of the SO were usually much lower (Table 2). This high contribution on-and off-plateau was achieved by different communities of small phytoplankton, mostly represented by non-silicified pico and nano-eukaryotes on-plateau, whereas small diatoms (3.8 1.5 µm equivalent spherical diameter; Fig. S7) were also abundant and active off-plateau (Fig. 4b). Complementary SIMS analysis revealed that many larger diatoms (>20 µm)

were inactive at this time of the season and that most of the CO2-fixation within this group was achieved by a few cells only (Fig. 4c).

Drivers of small phytoplankton importance in CO2-fixation in contrasted areas

In this study, small phytoplankton communities and CO2-fixation rates were different on and off-plateau (Fig. 4a,b), but small cells' contribution to CO2-fixation was comparable in these two areas (41-70%; Fig. 6a). In HNLC waters, high contribution of small phytoplankton to CO2-fixation is a commonly observed phenomenon (Table 2), attributed to the advantage of a reduced size in iron acquisition [START_REF] Chisholm | Phytoplankton Size[END_REF][START_REF] Marchetti | Diatom elemental and morphological changes in response to iron limitation: a brief review with potential paleoceanographic applications[END_REF][START_REF] Alderkamp | The effect of iron limitation on the photophysiology of Phaeocystis antarctica (prymnesiophyceae) and Fragilariopsis cylindrus (bacillariophyceae) under dynamic irradiance[END_REF]. In our study, smaller cells showed higher volume-specific CO2-fixation rates than their larger counterparts, in line with their theoretical advantage of high surface/volume ratio for nutrient and light uptake. This theoretical allometric relationship has not always been verified, as some studies have suggested that CO2-fixation could also scale isometrically with cell volume, and that larger cells could be as, or even more competitive, than smaller ones depending on the environmental conditions [START_REF] Marañón | Inter-specific scaling of phytoplankton production and cell size in the field[END_REF][START_REF] Huete-Ortega | Isometric size-scaling of metabolic rate and the size abundance distribution of phytoplankton[END_REF].

During MOBYDICK, the allometric size-scaling relationship explained over half of the variability observed in CO2-fixation of small phytoplankton cells ranging over four orders of magnitude (66% on-and 54% offplateau: Fig. 5). Other sources of variability in CO2-fixation may come from taxa-specific physiology adapted to on-and off-plateau conditions. For example, pelagophytes and small pennate diatoms were mostly present off-plateau and Micromonas on-plateau (Fig. 3). The lower C-based division rates observed at off-plateau stations (Fig. 4a) likely resulted from higher iron limitation, whereas iron is continuously supplied to surface waters by internal waves on-plateau [START_REF] Blain | Effect of natural iron fertilization on carbon sequestration in the Southern Ocean[END_REF]. Higher competitiveness with respect to iron acquisition may favor pelagophytes and pennate diatoms off-plateau. [START_REF] Hogle | Pervasive iron limitation at subsurface chlorophyll maxima of the California Current[END_REF] observed over-expression of genes involved in iron metabolism in a metatranscriptomic study, suggesting pelagophytes were advantaged in HNLC waters [START_REF] Hogle | Pervasive iron limitation at subsurface chlorophyll maxima of the California Current[END_REF]. As for pennate diatoms, they possess the iron storage protein ferritin, which enables them to store iron on the long term and to be very efficient in using pulsed iron inputs [START_REF] Marchetti | Ferritin is used for iron storage in bloom-forming marine pennate diatoms[END_REF][START_REF] Lampe | Different iron storage strategies among bloom-forming diatoms[END_REF]. On-plateau, higher ammonium and lower silicic acid concentrations were observed than off-plateau. The relatively high ammonium concentrations could have benefited to the growing Micromonas population (Fig. 3 & S3), since prasinophytes preference for ammonium could be 10-fold superior to other phytoplankton groups [START_REF] Litchman | Trait-based community ecology of phytoplankton[END_REF]. In contrast, silicic acid limitation could have limited small diatom's growth on-plateau in comparison to small non-silicified cells (Fig. S4) and explain why fewer small diatoms were observed on-than off-plateau (Fig. 4b). Finally, some of the variability observed in CO2-fixation of small cells may originate from physiological heterogeneity within a species. For example, P. antarctica which was the most abundant taxa on-and off-plateau is characterized by highly variable responses to iron limitation, even within clonal populations (i.e. size reduction, decrease of Chl a concentration; [START_REF] Luxem | Intraspecific variability in Phaeocystis antarctica's response to iron and light stress[END_REF]).

Currently, little information is available on in situ division rates of small phytoplankton taxa in the SO, most of them been obtained from Phaeocystis cultures (Table S4). Despite the variability observed at the single cell level, mean division rates observed in our study on-and off-plateau were in the same range as the ones observed for P. antarctica in Fe-replete and Fe-limited cultures. Therefore, we suggest that the division rates measured in this study in natural communities composed of diverse phylogenetical groups could serve as a baseline to model small phytoplankton growth in HNLC (mean of 0.22 ± 0.09 division d -1 ) and naturally-iron fertilized areas (0.37 ± 0.13 division d -1 ).

During MOBYDICK, many large diatoms (>20 µm) were not actively growing, while few cells showed high CO2-fixation (Fig. 4c). Most likely, division rates of large diatoms change considerably throughout the season in relation with silicic acid and iron availability. Silicic acid concentrations on-plateau can be as high as 19 µmol L -1 in early spring at the onset of the bloom [START_REF] Closset | Seasonal evolution of net and regenerated silica production around a natural Fe-fertilized area in the Southern Ocean estimated with Si isotopic approaches[END_REF]. After the bloom, silicic acid concentrations were <2 µmol L -1 during the first two visits at M2, a level which is considered as an empirical threshold to support diatoms' dominance over flagellates [START_REF] Egge | Silicate as regulating nutrient in phytoplankton competition[END_REF]. The high proportion of inactive large diatoms observed with SIMS was in line with microscopic observations of surface samples during MOBYDICK showing 33 ± 7 % of empty/broken frustules (Lafond et al., submitted). It is worthy to note that highly heterogeneous division rates have been observed in culture studies within large diatoms (Table S4) in Fe-limited cultures (e.g. daily division rates from 0.03 to 0.43 d -1 ) and also within specific genera in Fe-replete conditions (e.g. daily division rates from 0.16 to 0.64 d -1 for Fragilariopsis sp.). These intriguing results relative to the highly heterogeneous division rates of larger diatoms observed in our study highlight the need to further explore species-specific changes in CO2-fixation rates at the single cell level in response to contrasted environmental conditions.

Indications on the fate of small phytoplankton

Currently, export fluxes in the SO cannot be predicted based on global primary production and food web structure. Several studies conducted in the SO have revealed an inverse relationship between primary production and carbon export efficiency [START_REF] Lam | High biomass, low export regimes in the Southern Ocean[END_REF][START_REF] Maiti | An inverse relationship between production and export efficiency in the Southern Ocean[END_REF][START_REF] Moigne | What causes the inverse relationship between primary production and export efficiency in the Southern Ocean?[END_REF]. This decoupling between the carbon produced in the surface layer and the carbon export efficiency below 200m has also been documented on the Kerguelen Plateau, where high productivity regime during early spring was associated with low carbon export efficiency (1-2%), and moderate productivity in summer showed high export efficiency (26%; [START_REF] Christaki | Microbial food web dynamics during spring phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF][START_REF] Christaki | Seasonal microbial food web dynamics in contrasting Southern Ocean productivity regimes[END_REF]). In low productive HNLC waters of the Kerguelen Plateau, high carbon export efficiencies were observed in spring and summer (35% and 44% respectively; [START_REF] Christaki | Microbial food web dynamics during spring phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF][START_REF] Planchon | Carbon export in the naturally iron-fertilized Kerguelen area of the Southern Ocean based on the 234 Th approach[END_REF]). Although the factors driving this inverse relationship between primary productivity and export efficiency are not fully understood, micro-and macrozooplankton-mediated grazing seem to be an efficient alternative pathway to export carbon in low productivity waters [START_REF] Moigne | What causes the inverse relationship between primary production and export efficiency in the Southern Ocean?[END_REF][START_REF] Cassar | The relation of mixed-layer net community production to phytoplankton community composition in the Southern Ocean[END_REF]. Counter to the classical view that only large phytoplankton are exported due to their high sinking velocity [8], there is growing evidence that the relative contribution of small phytoplankton to total C-export is proportional to its contribution to total primary productivity, when indirect export pathways (such as grazing through the production of fecal pellets by higher trophic levels) were also considered [11]. Considering the important contribution of actively growing small cells to CO2-fixation in the surface layer in our study, their possible export pathways -in particular indirectly via grazingdeserve some attention. Interesting observations relative to grazing could shed light on the vertical pigment distribution observed during MOBYDICK where pigments of small non-silicified groups (haptophytes and prasinophytes mostly) were almost absent below 100 m.

Grazing measurements showed that microzooplankton grazed actively on phytoplankton at all stations with grazing rates exceeding phytoplankton growth rates (Christaki et al., submitted). Consequently, an important part of the carbon fixed by small phytoplankton at the surface may have been channeled to higher trophic levels via microzooplankton grazing. Phaeo/Chl a ratio showed that grazing activity was intensified at stations M2 and M1 (Fig. 2a). These stations were characterized by higher productivity in the months before sampling, promoting the development of dense salps populations (Salpa thompsoni), making up 41-42 % of total micronekton biomass at these two stations while they were almost absent at M3 and M4 (Schenke et al.,submitted). Salps are major grazers of small phytoplankton in the SO [START_REF] Moline | Alteration of the food web along the Antarctic Peninsula in response to a regional warming trend[END_REF] and produce easily fragmented fecal pellets in the upper mesopelagic layer [START_REF] Iversen | Sinkers or floaters? Contribution from salp pellets to the export flux during a large bloom event in the Southern Ocean[END_REF], which could explain the pronounced Phaeo/Chl a ratio below the mixed layer at M2 and M1. Finally, molecular analysis of plankton communities at 300m revealed that 25% of the sequences recovered in HNLC waters in the >20 µm size fraction belonged to P. antarctica, confirming the contribution of small phytoplankton to carbon export through fecal pellet export and/or aggregation in low productive waters [START_REF] Irion | Marked spatiotemporal variations in small phytoplankton structure in contrasted waters of the Southern Ocean (Kerguelen area)[END_REF]. Our observations underline that grazing and aggregation may be important pathways of small phytoplankton export in both productive and HNLC waters.

Concluding, this study has shown for the first time the importance of actively growing small (silicified and non-silicified) phytoplankton cells in iron-fertilized and HNLC waters of the SO during post-bloom conditions. Single cell analysis revealed higher homogeneity in CO2-fixation within small phytoplankton composed of diverse phylogenetically distant taxa (prymnesiophytes, prasinophytes and small diatoms) than within large diatoms which were likely limited by silicic acid and iron in post-bloom conditions.

Considering the high inter-annual variability and limited duration (approx. 4 months) of diatom blooms, our data highlight the need to reassess the role of small phytoplankton in the SO. Further investigation of the indirect contribution of small phytoplankton to C-export via grazing is also needed as it may be an efficient export pathway especially in HNLC waters characterized by sparse productivity pulses. Data of phytoplankton division and CO2-fixation rates published here will also be useful for modelling parameterization of phytoplankton size-group contribution to the C-cycle in the SO. 2018) [START_REF] Pauthenet | Seasonal meandering of the Polar Front upstream of the Kerguelen Plateau[END_REF]. The best distribution was selected from a predefined family of distributions (normal, logistic, exponential, log-normal, uniform, gamma) based on the lowest AIC. Division rates of small cells were best fitted by a logistic distribution on and off-plateau with mean division rates µ=0. 37 and µ=0.23 on-and off-plateau respectively. Division rates of large diatoms fitted a lognormal distribution characterized by skewed distributions to the left (low division rates) with mean division rates µ=0.09. Table S3: Mean amount of carbon fixed per cell, biomass and unit of carbon fixed per unit of biomass for each station and size-group. Biomass was calculated by converting the cell biovolume to carbon content according to Verity et al. (1992) [4].

Size-group Station Mean C-fixed (pgC cell -1 )

Mean biomass (pgC cell

Perspectives

Linking identity to CO2-fixation activity

In this study, we managed to compare the variability in CO2-fixation rates between and within three different phytoplankton groups: small non-silicified cells, as well as small and large diatoms. More precise taxonomic identification of non-silicified cells or diatoms at the genus level were only inferred in parallel from metabarcoding or FISH data. Two methodological developments could help linking identity with function more directly.

The most direct method to link metabolic activity to phylogeny at the single-cell level is to couple FISH labelling of specific phylogenetic groups with NanoSIMS analysis [START_REF] Behrens | Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS[END_REF][START_REF] Li | Simultaneous analysis of microbial identity and function using NanoSIMS[END_REF]Musat et al., 2008). In this procedure, labeled cells are discriminated from other cells by recording their coordinates with fluorescence microscopy, or marking their location with a laser microdissection microscope. This ensures that the same fields of interest are imaged by fluorescence microscopy and during NanoSIMS analysis (FISH-NanoSIMS) (e.g. [START_REF] Mcglynn | Single cell activity reveals direct electron transfer in methanotrophic consortia[END_REF]. To avoid this two-step process (epifluorescence microscopy -NanoSIMS), direct visualization of hybridized cells during NanoSIMS analysis is also possible by using iodine-and fluorine-, or halogen labeled probes (EL-FISH-NanoSIMS, HISH-SIMS) [START_REF] Behrens | Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS[END_REF][START_REF] Li | Simultaneous analysis of microbial identity and function using NanoSIMS[END_REF]. Considering the low sample throughput (5-10 images per day) and high measurement costs (500 euros per day) of NanoSIMS analysis, these two techniques are adapted only for abundant organisms, which explains why only prokaryotes have been studied this way. For rare taxa, a method developed recently consists in using the flow cytometry signal of the target group from pure cultures, and using this signal to sort populations in natural communities, before CARD-FISH and NanoSIMS analysis [START_REF] Zimmermann | Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS[END_REF]. A flow-FISH procedure to allow the sorting of particular microbial groups by flow cytometry was set up in 2017 by U. Christaki and L.

Jardillier. This procedure is adequate for subsequent NanoSIMS analysis as it allowed to recover 90% of the target cells after CARD-FISH labelling, didn't damage the cells and did not seem to affect CO2-fixation rates measured after incubation with NaH 14 CO3 (Jardillier, personal communication). Chemical manipulation of the cells during CARD-FISH was however shown to decrease profoundly the 13 C fraction in comparison to chemically untreated cells [START_REF] Musat | The effect of FISH and CARD-FISH on the isotopic composition of 13 C-and 15 N-labeled Pseudomonas putida cells measured by nanoSIMS[END_REF]. After paraformaldehyde (PFA) fixation, a reduction of the 13 C fraction from 94% to 90% was observed. PFA dissolves some lipids in cellular membranes, which slightly damages the cell membrane integrity [START_REF] Cheng | Influence of fixation and permeabilization on the mass density of single cells: a surface plasmon resonance imaging study[END_REF], which may lead to leaking of labeled carbon that had not been fixed in high molecular weight molecules. Further decrease of the 13 C fraction to 80% was measured after FISH, while CARD-FISH decreased the fraction to 57% [START_REF] Musat | The effect of FISH and CARD-FISH on the isotopic composition of 13 C-and 15 N-labeled Pseudomonas putida cells measured by nanoSIMS[END_REF]. This was attributed by Musat et al. to the introduction of unlabeled carbon into the cells, diluting the 13 C proportion. The authors thus suggested to develop mathematical models to correct the reduction of labeled carbon observed after CARD-FISH labeling and quantitatively assess the importance of specific populations to biochemical processes [START_REF] Musat | The effect of FISH and CARD-FISH on the isotopic composition of 13 C-and 15 N-labeled Pseudomonas putida cells measured by nanoSIMS[END_REF].

To overcome the issues associated with chemical manipulation of the cells during CARD-FISH labelling, we could also imagine sequencing the three different size-groups sorted by flow cytometry. This would not enable to directly relate the function and identity of single cells like the first method considered, but it would give an idea of the key players within each size-group sorted.

However, this was not possible during MOBYDICK as samples used for NanoSIMS were fixed with PFA.

In situ CO2-fixation rates for model calibration Primary production rates attributed to different phytoplankton classes are key parameters required to validate or parameterize new biogeochemical models that explicitly incorporate distinct phytoplankton groups [START_REF] Aumont | Globalizing results from ocean in situ iron fertilization studies[END_REF]Uitz et al., 2009). In view of the relatively high homogeneity observed within small phytoplankton CO2-fixation rates, we suggested that mean fixation rates measured in this study for this size-class on and off-plateau could help to refine productivity models on and off naturally iron-fertilized areas of the SO. It would however be interesting to control the influence of specific abiotic factors on CO2-fixation rates, which were suggested to influence small phytoplankton taxonomic composition during MOBYDICK. We tried to model the influence of specific environmental parameters on growth rates, to explain the higher growth rates observed on-plateau in comparison to off-plateau. Nutrient concentrations (silicic acid, nitrate, phosphate) were lower on-plateau than off-plateau, but growth rates higher, which prevented to find any relation between nutrient concentration and growth rates. We thus assumed that the higher growth rates observed on-plateau were caused by continuous iron input. In Paper 1, we suggested iron, ammonium and silicic acid concentrations, could explain the spatial differences in the distribution of small phytoplankton taxa. The role of silicic acid depletion in limiting diatom's growth on-plateau was confirmed by the lower division rates of small diatoms in comparison to nonsilicified cells (Paper 2; Fig. S4). The relative importance of iron, silicic acid and ammonium in controlling small phytoplankton growth and species composition could further be explored experimentally.

Our results do not allow to refine the parametrization of growth rates for large diatoms, considering the high heterogeneity observed from one cell to the other. More measures are needed to assess the influence of active outliers on mean division rates. It could also be interesting to investigate the variability at the single-cell level in CO2-fixation rates within specific species or groups depending on silicic acid and iron concentration. Such in situ experiments may for example allow to check if pennate are indeed advantaged over centric diatoms in iron-limited environments. Incubation experiments with Si isotopic tracers could also confirm the role of Si-limitation on-plateau in initiating the quasi-dominance of diatom communities by Corethron inerme on-plateau. More importantly, understanding the drivers of the high variability observed in CO2-fixation rates within large diatoms would help calibrating models for this important group in SO productivity.

D. Summary and discussion

Small phytoplankton dominates phytoplankton biomass and photosynthetic activity in vast areas of the global ocean where nutrient availability is limiting the growth of larger cells [START_REF] Marañón | Phytoplankton size structure[END_REF]. This is the case in the SO, where phytoplankton communities are largely dominated by small cells (<20 µm) as a result of iron limitation in HNLC waters (e.g. 76% of phytoplankton biomass and 61% of CO2-fixation in the western Indian sector of the SO; Weber and El-Sayed, 1987). More recent studies found similar contribution of small cells to Chl a and underlined the dominance of haptophytes pigments in the small size fraction (Wright et al., 2010;[START_REF] Iida | Seasonal variability of phytoplankton biomass and composition in the major water masses of the Indian Ocean sector of the Southern Ocean[END_REF][START_REF] Nunes | Phytoplankton community structure in contrasting ecosystems of the Southern Ocean: South Georgia, South Orkneys and western Antarctic Peninsula[END_REF]. Consequently, model of primary production in the SO estimates that small phytoplankton contributes more to annual primary production (2.5 Gt C yr -1 ) than microphytoplankton (0.9 Gt C yr -1 ) (Uitz et al., 2010).

A shift in dominance towards larger size classes, mainly diatoms, is occasionally observed at spring and summer in productive areas naturally fertilized with iron such as the Kerguelen Plateau (Korb and Whitehouse, 2004;Blain et al., 2007). The magnitude and duration of these blooms dominated by large or chain-forming diatoms are dependent on seasonal factors (silicic acid and iron availability particularly), whereas small phytoplankton may constitute a more or less stable background community [START_REF] Smetacek | Seasonal and regional variation in the pelagial and its relationship to the life history cycle of krill[END_REF]) (Fig. D1).

Fig. D1: Conceptual scheme demonstrating the relationship between seasonality of the microbial network (regenerating system, thick line) with superimposed "blooms" (thin lines). The latter exhibit more variability as they are induced by weather conditions operating on a scale of weeks. They are followed by correspondingly variable sedimentation pulses (arrows). The microbial network presents less variability in terms of biomass, productivity and export. Modified from [START_REF] Smetacek | Seasonal and regional variation in the pelagial and its relationship to the life history cycle of krill[END_REF] Abundances of small phytoplankton rarely fall below concentrations of about 2-4 × 10 6 cells L -1 , which likely gives small phytoplankton a fundamental role within Antarctic food webs during winter and pre-bloom periods, when large and chain-forming diatoms are not abundant [START_REF] Detmer | Distribution patterns of autotrophic pico-and nanoplankton and their relative contribution to algal biomass during spring in the Atlantic sector of the Southern Ocean[END_REF]. Thus, microbial food webs, where primary production mainly originates from small phytoplankton and is characterized by high grazing pressure of microzooplankton and salps, -Inter-annual variability (biomass, occurrence) -Pulses of export may represent an important alternative energy flow to the classical food chain (diatoms-krillwhales) within marine ecosystems of the SO (Weber and El-Sayed, 1987;[START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]. Last but not least, higher carbon export efficiency was repeatedly measured in low productive ecosystems of the SO dominated by small phytoplankton in comparison with more productive ones dominated by large cells [START_REF] Savoye | 234 Th-based export fluxes during a natural iron fertilization experiment in the Southern Ocean (KEOPS)[END_REF][START_REF] Cavan | Attenuation of particulate organic carbon flux in the Scotia Sea, Southern Ocean, is controlled by zooplankton fecal pellets[END_REF]Planchon et al., 2015;Le Moigne et al., 2016). Efficient carbon export via fecal pellets of grazers, low bacterial remineralization in the mesopelagic, or direct export of heavy-silicified diatoms in these low productive regions may explain the high export efficiency observed in low productive ecosystems of the SO [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]Laurenceau-Cornec et al., 2015b;Le Moigne et al., 2016). Although the importance of small-sized protists in food webs, CO2-fixation and C-export in the SO is indisputable, only a few studies specifically explored their diversity [START_REF] Díez | Distribution of eukaryotic picoplankton assemblages across hydrographic fronts in the Southern Ocean, studied by denaturing gradient gel electrophoresis[END_REF][START_REF] Wright | Composition and significance of picophytoplankton in Antarctic waters[END_REF][START_REF] Thiele | Stable composition of the nano-and picoplankton community during the ocean iron fertilization experiment LOHAFEX[END_REF], and no study explored how diversity of small phytoplankton could impact the carbon cycling.

Deciphering which microorganisms are performing selected functions in the environment and what controls this function are among the most challenging tasks in microbial ecology [START_REF] Madsen | Identifying microorganisms responsible for ecologically significant biogeochemical processes[END_REF][START_REF] Gutierrez-Zamora | An appraisal of methods for linking environmental processes to specific microbial taxa[END_REF]. The two main objectives of this thesis -to explore the diversity of small phytoplankton communities and their contribution to CO2-fixation in contrasting environments -fit into this conceptual framework. Three questions were addressed in this project to answer these two objectives:

1. Does small phytoplankton community composition differ on and off the naturally ironfertilized plateau of Kerguelen after the diatom bloom?

2. What are the drivers of the balance between small cells and large diatoms throughout the season?

3. What is the contribution of different size and taxonomic groups of small cells to CO2-fixation on-and off-plateau in post-bloom conditions and what does this imply for the functioning of the biological carbon pump in the area?

In this chapter, I will briefly summarize the main conclusions of my different studies, widen the scope of the discussion in the context of global changes and draw a general scheme of the seasonal succession of phytoplankton communities and their influence on C-export around Kerguelen. Finally, general perspectives on the study of small phytoplankton in the C-cycling will be suggested.

First insights into small phytoplankton diversity around Kerguelen

The exploration of phytoplankton composition through the combination of pigment composition (CHEMTAX) and marker gene analyses (sequencing) clearly highlighted the important contribution of small phytoplankton -Phaeocystis antarctica-in particular, in phytoplankton communities on and off-plateau after the diatom bloom (Paper 1; Fig. 3, Fig. 6). Phaeocystis has been repeatedly reported as a key player of phytoplankton communities in HNLC, as well as in naturally ironfertilized waters of the SO (DiTullio et al., 2000;[START_REF] Wright | Composition and significance of picophytoplankton in Antarctic waters[END_REF]. The success of P. antarctica may be linked to its polymorphic life stages as single cells or large colonies, that represent different adaptation strategies to contrasting environmental controls. In iron-fertilized waters, the capacity to build large colonies, allows P. antarctica to partly escape microzooplankton grazing and form large blooms [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF]. Colony formation is triggered by iron availability [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF], but colony size can increase by 30% in the presence of grazers, which tends to confirm the ecological significance of colony formation as defense mechanism against small grazers [START_REF] Tang | Colony size of Phaeocystis antarctica (Prymnesiophyceae) as influenced by zooplankton grazers[END_REF]. As single cell, this species possesses different acclimation strategies to iron limitation by reducing its size, growth rate, chlorophyll a concentration and particulate organic carbon (POC) production [START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF]Koch et al., 2019). The importance of Phaeocystis under future oceanic conditions in the SO is likely to keep increasing as this species growth rates don't seem to be impacted by ocean acidification, CO2 partial pressure and temperature increases (Zhu et al., 2016;Koch et al., 2019).

Metabarcoding of small protist communities was essential to reveal variations in the distribution of other small phytoplankton taxa on-and off-plateau. Although not quantitative, metabarcoding was useful in revealing the presence of distinct taxonomic groups that shared a common cortege of accessory pigments and couldn't be differentiated with CHEMTAX analysis. For instance, pelagophytes, usually characterized by the presence of 19'-butanoyloxyfucoxanthin, could not be included in the CHEMTAX analysis. This accessory pigment is shared with Phaeocystis [START_REF] Wright | Fucoxanthin pigment markers of marine phytoplankton analysed by HPLC and HPTLC[END_REF], which prevented CHEMTAX software to find an optimum solution when including pelagophytes. CHEMTAX relies on prior knowledge of phytoplankton communities in the region. It may thus overestimate the contribution of groups already described in the area, like Phaeocystis, and overlook the contribution to Chl a of taxa not described in the area before, such as pelagophytes, which accounted for 5 to 25% of phytoplankton reads in the small size fraction at off-plateau stations during MOBYDICK (Paper 2; Fig. 3). Moreover, CHEMTAX was not able to discriminate between different species within a broad taxonomic group. For example, diatoms accounted for 36% and 26% of Chl a during MOBYDICK on and off-plateau, respectively. However, sequencing data highlighted that diatoms assemblages were significantly different on-and offplateau in relation with silicic acid availability (Paper 1; Fig. 4 &5). On-plateau, silicic acid concentrations were depleted after the bloom, and the lightly silicified Corethron inerme dominated diatom community, while in HNLC waters, diatoms mainly consisted of a diverse assemblage of Fragilariopsis, Thalassiosira, Chaetoceros and unidentified pennates spanning a broad sizespectrum (Paper 1; Fig. 3 &4). The importance of small-sized diatoms off-plateau was confirmed by NanoSIMS analysis that revealed abundant small diatoms (3.8 ± 1.5 µm equivalent spherical diameter) in nano-sized groups (Paper 2, Fig. 4b).

The distribution of other non-silicified small phytoplankton taxa seemed influenced by contrasting ammonium and iron concentrations on-and off-plateau (Paper 1; Fig. 5). High ammonium concentrations on-plateau favored the presence of an active and growing population of pico-sized Micromonas (Paper 2; Fig. 3 &4b), while iron limitation may explain the enriched contribution of pelagophytes off-plateau. These results are in line with pigment sampling at bloom end during CROZeX (Crozet Plateau, November to January 2004/05), that showed increasing contribution of pelagophytes to the pigment pool as iron was exhausted (Seeyave et al., 2007), and with CARD-FISH counts and sequencing during the artificial iron fertilization experiment LOHAFEX [START_REF] Thiele | Stable composition of the nano-and picoplankton community during the ocean iron fertilization experiment LOHAFEX[END_REF]. Thus, composition of small phytoplankton communities of the SO may change in relation to iron, ammonium and silicic acid concentrations.

Since no study explored in detail the taxonomic structure of small phytoplankton communities throughout the season before, we cannot exclude that different small phytoplankton may prevail later in the season. For example, PHYSAT model detected Phaeocystis in the SO mainly from November to March, whereas winter months were dominated by unspecified "nano" groups [START_REF] Alvain | Seasonal distribution and succession of dominant phytoplankton groups in the global ocean: a satellite view[END_REF]. Thus, eventhough P. antarctica didn't seem to be impacted by contrasting environmental conditions over our study area (Paper 1; Fig. 5), other small phytoplankton groups may be as or more competitive during autumn and winter. Mixed layer depth deepens considerably in the winter months and can be as deep as 185 m in August [START_REF] Park | Seasonal and interannual variability of the mixed layer properties and steric height at station KERFIX, southwest of Kerguelen[END_REF]. In the Arctic, a seasonal succession within pico-sized prasinophytes is observed from winter to spring.

Bathycoccus is more successful in winter than Micromonas, which is less adapted to dark winter conditions and more susceptible to viral suppression [START_REF] Joli | Seasonal patterns in Arctic prasinophytes and inferred ecology of Bathycoccus unveiled in an Arctic winter metagenome[END_REF]. In our study, prasinophytes accounted for 20% of Chl a in October off-plateau (R-2), when mixed layer (ML) depth was still over 100 m. Sequencing of protist communities during KEOPS2 cruise (summer) indicated that these corresponded to a mixed assemblage of Prasinoderma and Micromonas [START_REF] Georges | Protist community composition during early phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF]. Thus, specific groups of small phytoplankton, prasinophytes for example, may present special adaptations to low irradiance, enabling them to be as or even more competitive as Phaeocystis during winter. This study was the first assessment of the distribution of small phytoplankton taxa around Kerguelen and focused on the exploration of differences between the iron-fertilized plateau and HNLC waters. However, the localization of the Polar front (the natural boundary where Antarctic waters predominantly sink beneath the warmer sub-Antarctic waters) also likely drives small phytoplankton community composition at a smaller spatial scale in the area. Intriguingly, we observed different pelagophytes north and south of the Polar Front, with Pelagomonas being predominantly detected in Antarctic waters at M4 and M1 and Pelagococcus dominating in sub-Antarctic waters at M3 (Paper 2; Fig. 3). Prasinophytes species composition also differed between Antarctic and sub-Antarctic stations. Micromonas (Mamilellaceae family) was present at M2 in Antarctic waters south of the Polar Front, while Prasinoderma (Prasinococcales family) was observed north of the Polar Front at M3-1 (34% of the reads of small phytoplankton taxa)(Paper 2; Fig. 3). These two prasinophytes belong to different families (Mamiellales and Prasinococcales) and could occupy different ecological niches. Alternatively, global oceanic circulation may favor their dispersion from their main habitat north or south of the Polar Front.

Prasinoderma identified in our study presents a 100% similarity with sequences from Prasinoderma singularis. This species was described for the first time and sequenced since in the South-East Pacific Ocean [START_REF] Jouenne | Prasinoderma singularis sp. nov. (Prasinophyceae, Chlorophyta), a solitary coccoid prasinophyte from the South-East Pacific Ocean[END_REF]. In contrast, it was suggested that Micromonas in the SO was closely related to Arctic Micromonas and dispersed via deep currents and upwelling of deep waters on the Antarctic shelf in Antarctic waters [START_REF] Simmons | Intron invasions trace algal speciation and reveal nearly identical Arctic and Antarctic Micromonas populations[END_REF]. Further exploration of the biogeography and ecological drivers of small phytoplankton distribution in the SO is needed to explain these differences in species composition north and south of the Polar Front. As specific species of small phytoplankton, like Micromonas, could serve as "sentinels" of a warming ocean [START_REF] Demory | Picoeukaryotes of the Micromonas genus: sentinels of a warming ocean[END_REF], it may be interesting to further explore the detailed taxonomic composition of small phytoplankton taxa across frontal areas of the SO.

Seasonality and interannual variability of phytoplankton communities around Kerguelen

2.1.Seasonal succession from diatoms to small non-silicified cells

The comparison of MOBYDICK with KEOPS cruises enabled to describe the seasonal community succession from diatom to small non-silicified cells on-plateau (Paper 1, Fig. 6).

The bloom onset is triggered by a shallowing of the mixed layer, that reliefs phytoplankton from light limitation caused by deep mixing during winter [START_REF] Venables | Phytoplankton and light limitation in the Southern Ocean: learning from high-nutrient, high-chlorophyll areas[END_REF]. In a turbulent upper mixed layer, irradiance can fluctuate from full sunlight to complete darkness over just a few hours, limiting photosynthesis [START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF]. Consequently, the bloom onset is correlated with the decrease of the mixed layer depth starting in October [START_REF] Robinson | A tale of three islands: downstream natural iron fertilization in the Southern Ocean[END_REF]. At bloom onset, the combination of a relief in light limitation and maximum concentration of major inorganic nutrients and iron after winter mixing (maximum of 0.6 nM, reduced to 0.05 nM in summer) is expected to benefit both diatoms and small phytoplankton over the plateau (de Baar et al., 2005;[START_REF] Moore | Iron-light interactions during the CROZet natural iron bloom and EXport experiment (CROZEX) I: Phytoplankton growth and photophysiology[END_REF][START_REF] Mongin | Seasonality and scale of the Kerguelen plateau phytoplankton bloom: a remote sensing and modeling analysis of the influence of natural iron fertilization in the Southern Ocean[END_REF]. However, small phytoplankton are efficiently grazed by rapidly growing microzooplankton, preventing accumulation of small phytoplankton biomass during the bloom onset. In our study, a strong decrease of the pigments associated with prymnesiophytes and prasinophytes was observed on-plateau during KEOPS2 from October to November (Paper 1; Fig. 6). At the same time, ciliates biomass increased 7-fold [START_REF] Christaki | Microzooplankton community associated with phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean)[END_REF], exerting a strong control on small phytoplankton. The control of small phytoplankton biomass by microzooplankton was consistently observed over the different seasons. During the bloom decline, microzooplankton consumed daily up to 45% of the standing stock of small phytoplankton (KEOPS1), which represented more than small phytoplankton daily production [START_REF] Brussaard | Virioplankton dynamics and virally induced phytoplankton lysis versus microzooplankton grazing southeast of the Kerguelen (Southern Ocean)[END_REF]. During MOBYDICK, grazing experiments also showed that phytoplankton net growth rate was lower than microzooplankton grazing rate (Paper 3; Christaki et al. submitted). Microzooplankton grazers were however strongly controlled by copepod predation (Fig. D2) that, in return, likely controlled their impact on phytoplankton mortality. This can explain why abundances of small phytoplankton did not decline below 10 6 cell L -1 despite the high potential of microzooplankton grazing (Paper 1; Fig. 6).

Fig. D2: Positive (green) and negative (red) feedbacks in small phytoplankton, microzooplankton and copepods interactions. By controlling microzooplankton populations, copepod predation exerts an indirect positive feedback on small phytoplankton abundances (dashed green arrow).

As the bloom progresses, the sinking of biogenic silica produced by diatoms to build up their frustules diminishes over time the standing stock of silicic acid at surface to concentrations <2 µmol L -1 (Closset et al., 2014). The decrease of silicic acid concentrations during KEOPS1 and MOBYDICK was associated with a change in diatom community structure, shifting from an initial bloom of Chaetoceros Hyalochaete spp. to a quite monospecific bloom of Eucampia antarctica in February and Corethron inerme in March (Lafond et al., submitted;[START_REF] Armand | Late summer diatom biomass and community structure on and around the naturally iron-fertilised Kerguelen Plateau in the Southern Ocean[END_REF][START_REF] Lasbleiz | Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean[END_REF]. The shallow mixed layer depth during MOBYDICK further limited the amount of nutrients -silicate and iron particularly-being brought to the surface from deep waters.

In addition to nutrient limitation, biotic interactions such as grazing and parasitism may speed up the bloom decline. During MOBYDICK, infection of microphytoplankton by parasitic Syndiniales was likely low after the bloom, although an important parasitic infection may have occurred before sampling (Paper 4; [START_REF] Sassenhagen | Protist interactions and community structure during early autumn in the Kerguelen Region (Southern Ocean)[END_REF]. Indeed, a high proportion of parasitic Syndiniales (55-70% of the reads) was sequenced in the small size fraction during the first two visits on-plateau.

These sequences may be attributed to Syndiniales spores released after the host death, potentially reflecting the end of a parasitic infection. Co-variance network analyses also suggested that Rhizaria may be important grazers of a wide range of diatom taxa. Although biotic interactions certainly played a role in controlling diatoms after the bloom, Chl a and associated diatom pigments doubled at the last visit on-plateau after a storm (Paper 1; Fig. 6), highlighting the main bottom-up control of phytoplankton communities in late summer and the importance of transient mixing events to occasionally help diatoms in overcoming Si-and iron limitation [START_REF] Venables | Phytoplankton and light limitation in the Southern Ocean: learning from high-nutrient, high-chlorophyll areas[END_REF].

Microzooplankton Copepods Small phytoplankton

The diatom bloom is characterized by a large interannual variability, shown by the high standard deviation in surface Chl a observed over the bloom period (Paper 1, Fig. 6a). To note that diatom blooms were more intense than usual during KEOPS1 and KEOPS2, as indicated by the Chl a concentrations well above the seasonal average during these cruises. The atypical importance of diatom blooms during KEOPS cruises may have contributed to overlook the role of small phytoplankton in CO2-fixation and ecosystem functioning of the region. The reasons for the high interannual variability of the bloom magnitude remain unclear, but may result from changes in the importance of iron and/or silicate supply in this area characterized by dynamic fronts [START_REF] Mongin | Seasonality and scale of the Kerguelen plateau phytoplankton bloom: a remote sensing and modeling analysis of the influence of natural iron fertilization in the Southern Ocean[END_REF][START_REF] Robinson | A tale of three islands: downstream natural iron fertilization in the Southern Ocean[END_REF]. In this context, the position of the Polar Front North or South of the Kerguelen Plateau might be crucial, as sub-Antarctic waters north of the Polar Front are depleted in silicate. Indeed, the position of the Polar Front on the Kerguelen Plateau varies seasonally as warmer sub-Antarctic waters progress southward during summer (Pauthenet et al., 2018). This southward shift is even more pronounced when warm sea surface temperature anomalies occur [START_REF] Moore | Location and dynamics of the Antarctic Polar Front from satellite sea surface temperature data[END_REF]. It will intensify in the future as waters will warm up and become more stratified due to climate change, shifting the position of fronts southward in the SO [START_REF] Moore | Location and dynamics of the Antarctic Polar Front from satellite sea surface temperature data[END_REF][START_REF] Deppeler | Southern Ocean phytoplankton in a changing climate[END_REF][START_REF] Freeman | The variable and changing Southern Ocean silicate front: insights from the CESM Large Ensemble[END_REF].

In contrast, Chl a interannual variability is low before and after the bloom, (Paper 1, Fig. 6a).

Weather conditions in the SO constraint sampling in autumn and winter, but data from other cruises seem to confirm the dominance of small cells outside of bloom periods. For example, the deployment of BGC-Argo floats showed, based on optical properties of plankton communities, that nanophytoplankton dominated before the bloom on the plateau of Kerguelen [START_REF] Rembauville | Plankton assemblage estimated with BGC-Argo Floats in the Southern Ocean: implications for seasonal successions and particle export[END_REF]. Pico-and nanophytoplankton, mostly haptophytes and prasinophytes, also represented over 70% of the phytoplankton biomass before the diatom bloom in the Antarctic Peninsula [START_REF] Rozema | Summer microbial community composition governed by upper-ocean stratification and nutrient availability in northern Marguerite Bay, Antarctica[END_REF] and in the marginal ice zone (Wright et al., 2010). Thus, it is likely that before and after the bloom, productive ecosystems of the Southern Ocean are consistently dominated by overlooked small phytoplankton cells.

2.3.Ecological consequences of a shift towards smaller cells

Intensified stratification projected with southward shift in the position of the Polar Front will reduce the delivery of nutrients to surface water. This may shift the size-spectrum of phytoplankton towards smaller cells, as observed during MOBYDICK.

Changes in the frequency and magnitude of diatom blooms over the plateau is likely to impact the entire food chain. Similar shifts from diatoms to small non-silicified cells (cryptophytes) has caused a decline of krill populations and increase of gelatinous salps along the Antarctic Peninsula (Moline et al., 2004). This trend has been confirmed by long term observations in the SO with negative feedbacks on the ecology of krill consumers, like penguins and seals [START_REF] Atkinson | Long-term decline in krill stock and increase in salps within the Southern Ocean[END_REF].

Our study showed that Phaeocystis is likely to be the winner in case of more intensified limitation of diatoms around Kerguelen. The impact of a shift towards smaller phytoplankton cells on higher trophic levels has not been explicitly investigated around Kerguelen yet. However, two elements indicate that such a shift may have similar ecological implications as the ones described in the above paragraph. First, salps populations during MOBYDICK were at least as abundant (up to 119 ind. 1000 m -3 on-plateau) as more frequently sampled areas of the Southern Ocean, particularly at the most productive stations M2 and M1 (Henschke et al., submitted). Similar abundances were described on-plateau during February and April 1987 and were associated with an influx of warmer sub-Antarctic waters. In the following summer (February 1988), the Polar Front passed to the north of the Kerguelen Islands, and salps abundances to the south-east of Kerguelen Islands were lower than the previous year [START_REF] Hunt | Comparative analysis of the 1980s and 2004 microzooplankton composition and distribution in the vicinity of Kerguelen and Heard Islands: seasonal cycles and oceanographic forcing of long-term change[END_REF]. Secondly, past southward anomalies of the location of the Polar Front in link with warm sea surface temperatures were associated to decreasing breeding success of seabirds usually foraging in Antarctic waters, while opposite trends were observed for birds foraging in sub-Antarctic waters [START_REF] Inchausti | Inter-annual variability in the breeding performance of seabirds in relation to oceanographic anomalies that affect the Crozet and the Kerguelen sectors of the Southern Ocean[END_REF]. This suggests that increasing importance of small cells on the Kerguelen Plateau could deeply impact the food webs and affect the survival of emblematic predators of the area.

3. Impact of surface phytoplankton community structure and productivity regime on the biological carbon pump 3.1.Importance of small phytoplankton in CO2-fixation rates during MOBYDICK and implications for C-export

During MOBYDICK, CO2-fixation rates showed that small cells were actively growing, whereas large diatoms presented low and heterogeneous growth rates (Paper 2; Fig. 4). These contrasted patterns of small phytoplankton and large diatoms photosynthetic activity provide a bottom-up explanation for the seasonal succession from large diatoms to smaller cells on-plateau. Increasing relative importance of small phytoplankton in CO2-fixation is mainly caused by diatoms limitation in post-bloom conditions, and not by increasing activity of small cells over the season. Indeed, mean growth rate of small cells were steady over the repeated visits at M2 (0.37 ± 0.13 division d -1 ) and compared well with growth rates of a 3 µm-sized phytoplankton population measured at bloom decline (KEOPS1) with the dilution technique (from 0.42 to 0.24 d -1 ) [START_REF] Brussaard | Virioplankton dynamics and virally induced phytoplankton lysis versus microzooplankton grazing southeast of the Kerguelen (Southern Ocean)[END_REF]. The ability of small cells to maintain steady growth rates -as opposed to large diatoms-explained the strong increase in the contribution of small cells to CO2-fixation from 10-20% at bloom decline (KEOPS1) (Uitz et al., 2009) to 40-60% after the bloom during MOBYDICK (Paper 2; Fig. 6a). Two elements indicate that Si-limitation, rather than iron limitation, is the principal nutrient limiting diatom's growth on-plateau. First, higher growth rates of non-silicified cells were observed onplateau in comparison to off-plateau stations (Paper 2; Fig. 4b). We can thus hypothesize that the plateau still benefited from continuous iron input or regeneration that sustained primary production.

Secondly, small diatoms were characterized by significantly lower division rates than small nonsilicified cells on-plateau (Paper 2; Fig S4 ), confirming that Si-limitation was limiting diatom's growth on-plateau.

In theory, small cells dominating phytoplankton in the SO are assumed to be characterized by little export, because of rapid recycling of the bulk of organic matter in the euphotic zone [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF]. As such, a larger contribution of small phytoplankton was expected to lower the C-export in comparison to previous cruises KEOPS1 and 2. Reduced export at 200m was observed during MOBYDICK in comparison to export at bloom decline (KEOPS1), but export after the bloom was higher than at bloom onset (KEOPS2) (7,[START_REF] Foster | Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses[END_REF] genus could be incorporated in the marine snow as aggregates or may be ingested by grazers in the mesopelagic. The study of pigment vertical distribution further suggested that small nonsilicified taxa were rapidly ingested by grazers, especially on-plateau and at M1, where dense salps populations occured (Paper 2; Fig. 2). To date, no technique can quantitatively assess the contribution of small phytoplankton to C-export. In contrast to large diatoms, for which the silica frustules can easily be observed in sediment traps or on polyacrylamide gels, small non-silicified cells lack characteristic features to identify them in the marine snow. Thus, most studies that discuss the contribution of small phytoplankton to C-export rely on indirect clues, based on modelling or qualitative observations. "Vigorous" export (>50 mmol C m -2 d -1 ) is thought to occur in summer in the sub-Antarctic zone south of Australia, a region dominated by small phytoplankton with few diatoms [START_REF] Cassar | The influence of iron and light on net community production in the Subantarctic and Polar Frontal Zones[END_REF](Cassar et al., , 2015)). In this area, grazing was positively correlated with export fluxes (Cassar et al., 2015).

The method used in these studies was however indirect, as it assumed that C-export equaled net community production (NCP). NCP corresponds to gross primary productivity (GPP) minus community respiration (i.e. the sum of auto-and heterotrophic respiration) within the surface ocean.

When the organic carbon pool is at steady state, little particulate organic carbon is stored in the euphotic zone and it can be considered that NCP approximates organic C-export [START_REF] Falkowski | Phytoplankton and their role in primary, new, and export production[END_REF]. During MOBYDICK, NCP was appr. 4 to 14 times higher than export at 200 m on-and offplateau (Paper 5; Christaki et al. 2020). Steady state conditions required for this method were likely not met around the Kerguelen Plateau, especially on-plateau, where rapid changes in autotrophic biomass were observed (Paper 1; Fig. 6a). Inverse food web models are another indirect way to evaluate the contribution of small phytoplankton to C-export (Richardson and Jackson, 2007). To our knowledge, this method hasn't been applied to evaluate the impact of phytoplankton sizestructure on C-export in the SO yet. In the Arctic, inverse modelling highlighted that a shift from diatoms to Phaeocystis/nanoflagellates dominated phytoplankton communities, would not necessarily lead to a reduction in C-export [START_REF] Vernet | Models of plankton community changes during a warm water anomaly in Arctic waters show altered trophic pathways with minimal changes in carbon export[END_REF]. However, the pathway of C-export through the food web should be longer when Phaeocystis dominated, with increasing importance of microzooplankton and detritus in carbon transfer to mesozooplankton [START_REF] Vernet | Models of plankton community changes during a warm water anomaly in Arctic waters show altered trophic pathways with minimal changes in carbon export[END_REF].

Observational techniques, such as identification of specific pigments or enumeration by flow cytometry of small cells in deep samples, provide direct evidence of the presence of small phytoplankton in sinking material (Roca-Martí et al., 2017). However, these approaches are not quantitative as they underestimate the contribution of small phytoplankton to export via grazing or detritus. Only one study effectively showed, thanks to a combination of sediment trap gels, videography, and confocal microscopy, that a fraction of picoplankton was incorporated as abundant intact cells (1.15 10 11 prokaryotic picoplankton cells m -2 yr -1 ) into sinking organic aggregates via inefficient grazing from salps and copepods in sub-Antarctic HNLC waters [START_REF] Waite | Mass sedimentation of picoplankton embedded in organic aggregates[END_REF]. This method presented the advantage unequivocally prove the contribution of small cells to export via both grazing and aggregation mechanisms, but can't be considered as quantitative, since it can only quantify cells incompletely digested. To conclude, a set of observations suggests that small phytoplankton is of importance in C-export where it dominates phytoplankton biomass and CO2-fixation, but for the moment, only models can propose estimates of small phytoplankton C-export. Output and accuracy of these models will however depend on detailed knowledge of production, growth, and grazing rates of all biological compartments included in the model, as well as experimental examination of the origin, composition, and fate of detrital material [START_REF] Stukel | Contribution of picophytoplankton to carbon export in the equatorial Pacific: a reassessment of food web flux inferences from inverse models[END_REF].

3.2.Lessons from other Si-limited, iron-fertilized areas of the SO

Predicting the consequences of a more intense Si-limitation of larger diatoms on-plateau on CO2fixation and C-export is challenging. Looking at past fertilization experiments in Si-limited waters can bring valuable insights. During LOHAFEX -an artificial iron fertilization in Si-limited waters (<2 μmol L -1 ) in the Atlantic region of the SO-Chl a and primary productivity doubled after artificial fertilization, mainly due to the increasing importance of small non-silicified cells, while diatoms accounted for <10% of phytoplankton biomass [START_REF] Martin | Iron fertilization enhanced net community production but not downward particle flux during the Southern Ocean iron fertilization experiment LOHAFEX[END_REF]. The unusually high biomass attained and maintained by small flagellates was attributed to the initial absence of salps and to constraints on microzooplankton grazers by heavy predation exerted by the large copepod stock [START_REF] Schulz | Remarkable structural resistance of a nanoflagellate-dominated plankton community to iron fertilization during the Southern Ocean experiment LOHAFEX[END_REF]. The increase of primary production at surface did not enhance downward particle flux, that remained low throughout the experiment. Nevertheless, many small flagellates and coccoid cells, belonging to the pico-and nanoplankton, were found in sediment traps, and these small, soft-bodied cells probably contributed to the majority of downward POC flux via mesozooplankton grazing and fecal pellet export [START_REF] Ebersbach | Particle flux characterisation and sedimentation patterns of protistan plankton during the iron fertilisation experiment LOHAFEX in the Southern Ocean[END_REF]. During MOBYDICK, dense salp populations were observed on-plateau and at M1 (Henschke et al., submitted), potentially adding a one-step export pathway of small phytoplankton export as compared to LOHAFEX in addition to the two-step microzooplankton-copepod grazing and subsequent fecal pellet production (Paper 2).

Investigations of phytoplankton community structure, productivity and export in naturally ironfertilized Si-limited waters around Crozet during CROZeX showed a more complex situation. In naturally iron-fertilized waters dominated by Phaeocystis, productivity was five times higher from early November to mid-December [START_REF] Poulton | Phytoplankton community composition around the Crozet Plateau, with emphasis on diatoms and Phaeocystis[END_REF]Seeyave et al., 2007) and C-export three times greater than in adjacent HNLC area [START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF]. However, in both iron-enriched and iron-limited waters, most of the POC export over the season was the consequence of pulsed export of diatoms [START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF]. It was suggested that the developing stage of the bloom, that occurred before the cruise, was dominated by large diatom Eucampia antarctica (Seeyave et al., 2007). This species may have produced resting spores early in the season as a result of Silimitation, that accounted for over 90% of the POC export [START_REF] Salter | Diatom resting spore ecology drives enhanced carbon export from a naturally iron-fertilized bloom in the Southern Ocean[END_REF]. Interestingly, in HNLC waters off Crozet, large, grazing resistant, heavy silicified diatoms also greatly contributed to C-export [START_REF] Pollard | Southern Ocean deep-water carbon export enhanced by natural iron fertilization[END_REF]. "Giant" heavy silicified diatoms, developing slowly in HNLC waters like Thalassiotrix and Fragilariosis kerguelensis were referred to as "silica sinkers" by [START_REF] Smetacek | The role of grazing in structuring Southern Ocean pelagic ecosystems and biogeochemical cycles[END_REF], as they allegedly contributed mostly to silicon export as empty cells and not to C-export.

However, [START_REF] Quéguiner | Iron fertilization and the structure of planktonic communities in high nutrient regions of the Southern Ocean[END_REF] suggested they could rapidly sink as intact full cells in late summer and contribute significantly to C-export in HNLC waters. These observations underline the great contribution to export of heavy silicified and spore-producing diatoms in Si-limited environments.

During MOBYDICK, Odontella, Eucampia and Chaetoceros were well represented in sequencing data below 125m, suggesting that these spore-producing genera were efficiently exported onplateau. Off-plateau, large heavy silicified diatoms like Fragilariopsis and Thalassiosira were also common in sequencing results at 300m (Paper 1; Fig. 7). Thus, even when small phytoplankton greatly contributes to phytoplankton biomass and productivity, large diatoms may still contribute disproportionally to C-export.

3.3.Can phytoplankton assemblages predict C-export over the Kerguelen Plateau?

We showed that the contribution of small phytoplankton to CO2-fixation increased after the bloom due to silicic acid and iron limitation of large diatoms, and that both small phytoplankton and diatoms could contribute to C-export via direct sinking and indirect pathways through grazing. The fate of the carbon produced at surface by phytoplankton depends mainly on other biological processes involving a large diversity of trophic levels (e.g. prokaryotes, microzooplankton, mesozooplankton and higher trophic levels). In an attempt to understand the significance of this shift in phytoplankton community composition on the annual C-export over the Kerguelen Plateau, I summarized the available data collected during diverse cruises that occurred at different seasons over the last decades.

Prokaryotic communities on-plateau changed from bloom onset to bloom decline as a function of phytoplankton assemblages and community succession [START_REF] Liu | Seasonal dynamics of prokaryotes and their associations with diatoms in the Southern Ocean as revealed by an autonomous sampler[END_REF]. In addition to changes in bacterial composition, the efficiency of bacterially-mediated carbon transfer to higher trophic levels also shifted throughout seasons (Paper 5;Christaki et al. 2020). Marked losses of bacterial carbon were attributed to respiration and viral lysis during spring and summer, while grazing from heterotrophic nanoflagellates sustained a more efficient microbial food web after the bloom (Paper 5; Christaki et al. 2020). Microzooplankton community composition and biomass was also characterized by a high seasonality. A shift from large dinoflagellates (e.g. Gyrodinium, Amphidinium and Protoperidinium), able to feed on large diatoms, to a dominance of small mixotrophic dinoflagellates like Gymnodinium was observed from bloom onset until bloom end (Paper 3;Christaki et al. submitted, 2015). This change in community composition affected microzooplankton biomass that strongly increased (10-fold) at bloom onset, but was back to prebloom levels during MOBYDICK (116 to 15.4 mmol C m 2 )(Paper 3; Christaki et al. submitted, 2015). Mesozooplankton biomass -mainly associated to copepods-increased continuously from mid-October to early February (×9 on-plateau and ×2.5 off-plateau), and may have constrained diatom and microzooplankton biomasses during the decline of the bloom [START_REF] Carlotti | Mesozooplankton structure and functioning during the onset of the Kerguelen phytoplankton bloom during the KEOPS2 survey[END_REF]. At bloom onset, salps occurred in low numbers on-plateau and were almost absent off-plateau [START_REF] Carlotti | Mesozooplankton structure and functioning during the onset of the Kerguelen phytoplankton bloom during the KEOPS2 survey[END_REF]. They reached important densities during MOBYDICK (Henschke et al. submitted), although their importance on-plateau seems to vary from one year to the other depending on the position of the Polar Front [START_REF] Hunt | Comparative analysis of the 1980s and 2004 microzooplankton composition and distribution in the vicinity of Kerguelen and Heard Islands: seasonal cycles and oceanographic forcing of long-term change[END_REF].

The quality of the exported organic matter during the different seasons reflected the succession of phytoplankton and zooplankton assemblages. Phytoplankton aggregates were dominant at bloom onset (Laurenceau-Cornec et al., 2015b), when grazers were still in a developing phase.

Productivity regime was high (GCP 344 mmol m -2 d -1 ) when export efficiency was very low (1%), suggesting a "retention" system characterized by high bacterial remineralization and biomass accumulation at surface (Laurenceau-Cornec et al., 2015b;Christaki et al., 2020). In contrast, large fecal pellets (>100 µm) dominated the export flux during bloom decline (KEOPS1), after important mesozooplankton communities had developed [START_REF] Ebersbach | Sinking particle properties from polyacrylamide gels during the KErguelen Ocean and Plateau compared Study (KEOPS): zooplankton control of carbon export in an area of persistent natural iron inputs in the Southern Ocean[END_REF]. At this time, export efficiency over the plateau was maximum (26%). After the bloom (MOBYDICK), particles < 50 µm represented almost the whole POC biomass down to 300 m at all stations (Planchon, personal communication). This feature suggests a greater role in downward flux of fecal pellets of smaller grazers (microzooplankton and small copepods), as well as of easily fragmentable salp fecal pellets (Iversen et al., 2017). Export efficiency dropped down to 5% in post-bloom conditions (Christaki et al., 2020), which tends to confirm that export efficiency is lower when small phytoplankton and grazers are important, than when large diatoms and copepods dominate during bloom decline. This typical succession was confirmed by deployment of sediment traps over the season (Rembauville, Salter, et al., 2015). This technique showed that fecal pellets contribution to annual carbon flux reached its seasonal maximum in autumn and winter (>80%). Composition of fecal pellets types evolved throughout the season from small copepods in spring to large euphausiids and copepods in summer, to salps in autumn and winter (Rembauville, Salter, et al., 2015).

Considering this seasonality, it could be tempting to associate characteristic phytoplankton and zooplankton assemblages to specific export regimes and export efficiencies and extrapolate these measures to an annual cycle. However, annual sediment traps also highlighted that Chaetoceros Hyalochaete spp. and Thalassiosira antarctica resting spores were responsible for more than 60% of the annual POC flux (Rembauville, Blain, et al., 2015). Export pulses from resting spores of the same two species accounted for 42% of annual POC flux in the productive waters downstream of South Georgia, whereas diatom vegetative stages only contributed to 5% of POC fluxes [START_REF] Rembauville | Strong contribution of diatom resting spores to deep-sea carbon transfer in naturally iron-fertilized waters downstream of South Georgia[END_REF] and the importance of Eucampia spores in iron-fertilized waters off

Crozet was already mentioned previously [START_REF] Salter | Diatom resting spore ecology drives enhanced carbon export from a naturally iron-fertilized bloom in the Southern Ocean[END_REF]. Integrative cruises like MOBYDICK are of great interest to understand pelagic ecology in contrasted productivity regimes and try to assess the impact of climate change on the different compartment of the ecosystem. However, temporal variability of C-export flux in the SO with importance of short flux events of a few diatom spore-resting species precludes extrapolation of discrete measurements to estimate seasonal or annual C-export (Rembauville et al. 2015a). As these fluxes can occur in Si-limited waters [START_REF] Salter | Diatom resting spore ecology drives enhanced carbon export from a naturally iron-fertilized bloom in the Southern Ocean[END_REF], where diatoms growth is limited, it makes it even more speculative to predict the Fully appreciating the links between phytoplankton community composition and biogeochemistry requires to (i) characterize phytoplankton diversity, that encompasses many taxonomic and functional groups, (ii) measure the activity of different functional groups, and (iii) understand the importance of these groups for other biological compartments and the feedbacks exerted by these other compartments on phytoplankton. Specific methods are associated with each of these issues, and accessing at the same time diversity and measurable functions (in our case CO2-fixation) remains challenging in microbial ecology.

The advent of new sequencing technologies represented a huge technical advance to describe the diversity of small eukaryotes. Issues associated with PCR biases in metabarcoding studies will likely be circumvented in a near future by a more generalized use of metagenomic. For example, metagenomic studies can limit the common overestimation of dinoflagellates and underrepresentation of diatoms observed in metabarcoding studies [START_REF] Genitsaris | Marine microbial community structure assessed from combined metagenomic analysis and ribosomal amplicon deep-sequencing[END_REF].

Identification of 18S rDNA sequences within metagenomes is a PCR-free alternative to metabarcoding to study microbial diversity, with the potential of also assessing functional and metabolic diversity. However, retrieving precise taxonomical classification from short Illumina metagenomic reads (100-250 bp) remains challenging, especially when targeting the 18S rDNA gene that contains a mosaic of conserved and highly variable regions [START_REF] Breitwieser | A review of methods and databases for metagenomic classification and assembly[END_REF].

Nano-pore based sequencing has the potential to overcome limitations due to short reads by allowing for sequencing of long fragments (>10 000 bp) that potentially can contribute to higher taxonomic resolution and functional characterization [START_REF] Reddington | Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function[END_REF]. Methodological developments are still desirable to use this sequencing technology in complex environments, by deepening the sequence coverage, reducing the sequencing error rate and ensuring results replicability [START_REF] Brown | MinION TM nanopore sequencing of environmental metagenomes: a synthetic approach[END_REF]. Another main limitation of metagenomic methods is the lack of reference sequences in databases, although the reconstruction of metagenome-assembled genomes from metagenomic data could partly overcome this issue.

Metagenomic or transcriptomic data can inform on metabolism of uncultured taxonomic groups and, therefore, shed light on their effects on biogeochemistry [START_REF] Caron | Probing the evolution, ecology and physiology of marine protists using transcriptomics[END_REF][START_REF] Grossart | Linking metagenomics to aquatic microbial ecology and biogeochemical cycles[END_REF]. However, they do not inform on fluxes and need to be combined with in situ measurements to confirm hypothetic roles of taxonomic groups in the environment. In our study, we established that CO2-fixation was more homogeneously distributed within small cells belonging to various taxa, than within larger cells affiliated to the same taxonomic group (large diatoms). This suggests the validity of considering small phytoplankton as a unique compartment in primary production models.

In contrast, taxonomy (at the species level) may be determinant for larger diatoms, for which species-specific responses to iron and silicic acid availability may significantly impact CO2-fixation rates. Once in situ measurements establish which descriptors are the best suited to accurately represent CO2-fixation by phytoplankton in the system, these can be used to calibrate models. We used single-cell data and scaled them up to understand the contribution of small phytoplankton to CO2-fixation around Kerguelen. The next step would be to incorporate such data into large-scale biogeochemical models [START_REF] Mayali | NanoSIMS: microscale quantification of biogeochemical activity with largescale impacts[END_REF]. In situ measurements of photosynthetic activity are relatively direct. They constitute the first step in understanding the functioning of the BCP but need to be concomitantly completed with in situ measures on micro-and mesozooplankton grazing to understand the processing of photosynthetically-fixed carbon by heterotrophs. Future research should also focus on feedbacks of heterotrophic activity on bottom-up control of phytoplankton growth rates. Indeed, in addition to being a major cause of phytoplankton mortality, grazing was shown to largely control phytoplankton growth rates by recycling nutrients in the euphotic layer (Laws, 2013). For example, grazing by small copepods is considered in the SO as a major driver of iron recycling [START_REF] Laglera | Iron partitioning during LOHAFEX: copepod grazing as a major driver for iron recycling in the Southern Ocean[END_REF], whereas salps fast sinking fecal pellets could actively contribute to iron depletion in surface waters [START_REF] Cabanes | First evaluation of the role of salp fecal pellets on iron biogeochemistry[END_REF]. More integrative studies are needed to assess the transfer of elements throughout the food web and highlight possible competition or positive interactions between autotrophs and heterotrophs. This can for example be achieved by following with NanoSIMS and large-geometry SIMS the fixation and transfer of isotopic tracers between autotrophs and heterotrophs forming the basis of the food web [START_REF] Adam | N2-fixation, ammonium release and N-transfer to the microbial and classical food web within a plankton community[END_REF].

Methodological considerations still limit our ability to assess directly the actual contributions of large and small phytoplankton to export flux. Direct measures of phytoplankton export as single cells or aggregate in sediment traps may help in assessing the relative contribution of small and large phytoplankton via sedimentation. Unsurprisingly, large cells are most commonly observed this way.

Consequently, large cells are assumed in most models to contribute disproportionately to export in comparison with smaller ones. However, fecal pellets can contribute from <1 to 100% of export fluxes [START_REF] Turner | Zooplankton fecal pellets, marine snow, phytodetritus and the ocean's biological pump[END_REF] and can constitute an important export pathway of small phytoplankton carbon. Inverse food-web models may be used to reconcile in situ measurements of primary production, grazing and export and provide a schematic overview of the processing of photosynthetically-fixed carbon via the BCP. These models highlighted the importance of small cells to export when they contributed importantly to net primary production (Richardson and Jackson, 2007;[START_REF] Stukel | Contribution of picophytoplankton to carbon export in the equatorial Pacific: a reassessment of food web flux inferences from inverse models[END_REF][START_REF] Vernet | Models of plankton community changes during a warm water anomaly in Arctic waters show altered trophic pathways with minimal changes in carbon export[END_REF]. However, sensitivity of model results to input parameters must be evaluated with care to potentially highlight important knowledge gaps, in particular regarding the origin, composition, and fate of detrital material of varying size [START_REF] Stukel | Contribution of picophytoplankton to carbon export in the equatorial Pacific: a reassessment of food web flux inferences from inverse models[END_REF]. More collaboration between scientists of different fields are needed to explore these promising research avenues that will contribute to a more mechanistic and predictive understanding of the links between phytoplankton diversity, structure and biogeochemical cycles.

Related papers as co-author

In the frame of the MOBYDICK project, I was co-author on three papers, presented in this section as Paper 3, 4 and 5.

Paper 3 deals with microzooplankton diversity, grazing potential on phytoplankton and seasonal evolution. During MOBYDICK, microzooplankton community composition was similar on-and offplateau and dominated by small cells (<20µm). Microzooplankton grazing rates were higher than phytoplankton growth rates at all stations, but microzooplankton biomass low. Consequently, it was hypothesized that microzooplankton biomass was kept in check by copepod predation. This paper highlights the important role of microzooplankton for channeling the carbon produced by small phytoplankton to higher trophic levels.

Paper 4 explores the spatial structure and temporal dynamics of large-sized protist communities during MOBYDICK and identify potential abiotic and biotic drivers of these assemblages. Main findings of this study were that an important parasitic infection by Syndiniales may have occurred before MOBYDICK. Co-variance network analysis also suggested that Rhizaria may be important grazers of a wide range of diatom taxa and that Syndiniales may be associated with diatoms.

Paper 5 reviewed the seasonal dynamics of microbial loop fluxes in the Kerguelen area. During spring and summer, respiration and viral lysis represented important loss terms of bacteriallymediated carbon. After the bloom (MOBYDICK), grazing by heterotrophic nanoflagellates was the main loss process of heterotrophic bacterial production. This resulted in a more efficient transfer of primary production to bacteria, heterotrophic nanoflagellates and higher trophic levels.

Contributions:

Paper 3: sequencing and pigment data analysis Paper 5: Seasonal microbial food web dynamics in contrasting Southern Ocean productivity regimes (Christaki et al., 2020)

Fig. A2 :

 A2 Fig. A2: Schematic phylogenetic tree representing the distribution of phytoplanktonic taxa across eukaryote lineages (in color). Illustrations of (a) Chlorophyceae, (b) Pseudoscourfieldia sp., (c) Porphyridium cruentum, (d) Gymnochlora dimorpha, (e) Dinoflagellates, (f) Odontella sp. (g) Bolidomonas pacifica, (h) Dictyocha sp., (i) Aureococcus anophagefferens, (j) Heterosigma akashiwa, (k) Pinguiochrysis pyriformis, (l) Ochromonas sp., (m) Nannochloropsis salina, (n) Calcidiscus sp., (o) Cryptomonas sp., (p) Euglenids. (Source :Not et al., 2012)

Fig. A4 :

 A4 Fig. A4: Estimated contribution of phytoplankton size-groups to Chl a for the period of winter 2010-2011. (Source:[START_REF] Roy | The global distribution of phytoplankton size spectrum and size classes from their light-absorption spectra derived from satellite data[END_REF]

Fig. A5 :

 A5 Fig. A5: Surface dominance (fraction of total Chl a > 55%) of phytoplankton groups for July 2003 and January 2004 according to three different models (PHYSAT;[START_REF] Alvain | Seasonal distribution and succession of dominant phytoplankton groups in the global ocean: a satellite view[END_REF], the Darwin-MITgcm version of[START_REF] Dutkiewicz | Capturing optically important constituents and properties in a marine biogeochemical and ecosystem model[END_REF] and[START_REF] Losa | On modeling the Southern Ocean phytoplankton functional types[END_REF] . "SCL" represents Synechococcus like prokaryotic phytoplankton. Simulated haptophytes include coccolithophores and Phaeocystis. The dominance between diatoms and small phytoplankton (haptophytes, Phaeo and small eukaryotes) varies greatly between models. However, in all models, the dominance of small phytoplankton over diatoms is observed during winter months (July).

Fig. A6 :

 A6 Fig. A6: Schematic representation of water masses, frontal systems and transport of nutrients in the SO. PF: Polar Front, STF: Sub-Tropical Front.Through the upwelling of the Circumpolar Deep Water (CPDW) at the Antarctic Divergence, the surface waters are enriched in nutrients (nitrate, phosphate and silicic acid in particular). This favors the growth of diatoms and intense biogenic silica production, which triggers abundant export of biogenic silica to deep waters, ultimately depositing opal on abyssal sediments (mostly south of the PF). Unused nutrients are exported to the rest of the world ocean through the Sub-Antarctic Mode Water (SAMW), the Antarctic Intermediate Water (AAIW), and the Antarctic Bottom Water (AABW). (Source[START_REF] Tréguer | The Southern Ocean silica cycle[END_REF]

Fig. A7 :

 A7 Fig. A7: Phylogenetic tree of haptophyte algae showing major groups. (Source:[START_REF] Tsuji | Chapter seven -Biology of haptophytes: complicated cellular processes driving the global carbon cycle[END_REF]

Fig. 7 :

 7 Fig. 7 : Micrographs of (a) a single Phaeocystis in cell culture and (b) Phaeocystis colonies in a Ross Sea bloom.

Fig. A8 :

 A8 Fig. A8: Pictures of (a) a single Phaeocystis in cell culture and (b)Phaeocystis colonies in a Ross Sea bloom. (Source:[START_REF] Bender | Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry[END_REF]

  photosynthetic electron transport chains (e.g. PSI, PSII, the Cytochrome b6-f-FeS complex; Behrenfeld and Milligan, 2013). As a consequence of the lack of iron, phytoplankton cannot completely utilize the available macronutrients (particularly nitrate) for photosynthesis, and the primary production is relatively low in vast areas of the SO, despite high availability of macronutrients. The implications of the "iron hypothesis" in a context of climate change are huge. After John Martin stated during a lecture, "give me a half tanker of iron, and I will give you an ice age", iron-addition

Fig. A10 :

 A10 Fig. A10: The anti-correlated data that inspired the iron hypothesis. (a) Measurements of air bubbles trapped in cores drilled from the Antarctic ice sheet show that atmospheric levels of carbon dioxide were significantly lower during the coldest periods (shaded regions) than during modern times (CO2 concentrations are shown in parts per million by volume; p.p.m.v.). (b) The ice-core records also reveal that more iron was transported to the Southern Ocean in wind-blown dust during the coldest periods than during warmer times (iron flux is measured in micrograms per square metre per year). (Source:Stoll 2020)

  compared the effects of iron fertilization in low Si sub-Antarctic (Si[OH]4 < 3 µmol L -1 ) waters north of the Polar Frontal and in high silicate waters south of the Southern Antarctic Circumpolar Current Front (Si[OH] 4 >60 µmol L -1 ). A mixed community of dinoflagellates, Phaeocystis and large diatoms

4. 1

 1 Oceanic circulation in the study area The Kerguelen Plateau (southeast of the Kerguelen Island) is a major bathymetry feature within the Indian Ocean sector of the Southern Ocean, extending from 46°S to 64°S at the 3000 m isobath. It forms a major barrier to the eastward flowing Antarctic Circumpolar Current (ACC), with most of the flow being deflected by the topography to the north of the plateau, and the substantial remainder to the south (Fig. A11)(Park et al., 2008). The Polar Front is a major circulation feature within the region, flowing close to the southeast Kerguelen Island. The position of the Polar Front presents large seasonal variations, shifting southward from spring to summer, being pushed south by warmer and fresher

Fig. A11 :

 A11 Fig. A11: Schematic of the geostrophic circulation over and around the Kerguelen Plateau based on the synthesis of our major findings. Thin arrows with Arabic numerals stand for directly measured time-mean current vectors averaged over the upper 500 m layer. (Source:[START_REF] Park | Large-scale circulation over and around the Northern Kerguelen Plateau[END_REF]

Fig. B1 :

 B1 Fig. B1: Overview of the different steps involved in metabarcoding of planktonic communities. Adapted from http://www.naturemetrics.co.uk

Fig. B2 :

 B2 Fig. B2: Epifluorescence microscopy of small phytoplankton sampled during MOBYDICK. Hybridization with a combination of EUK 1209R, NCHLO01 and CHLO01 aimed at targeting all eukaryotes. Scale bar = 10 μm.

  13 C) date back to the 1950s, when Steemann[START_REF] Steemann Nielsen | The use of radio-active carbon (C 14 ) for measuring organic production in the sea[END_REF] established the principles of 14 C radiotracer labelling. The method developed then is still the same and consists in incubating a phytoplankton community for several hours in the presence of a known quantity of 13 C or 14 C-DIC. At the end of the experiment, the metabolic activity of phytoplankton is stopped, and the incorporated labeled carbon ( 13 C or 14 C-POC) is measured into particulate organic matter. The initial DIC concentration of the sample being known as well as the added amount of 14 C-DIC, the measurement of labeled POC makes it possible to determine the amount of DIC fixed during the experiment. This method assumes that the fixation of 13 C or14 C during an incubation is proportional to the fixation of dissolved inorganic carbon (DIC) naturally present in the water column. The fixation rate of DIC into particulate organic carbon (POC) is then considered as a proxy of primary production.

Fig. B3 :

 B3 Fig. B3: Picture of the on-deck incubators in the vicinity of the Kerguelen islands. In situ temperature was reproduced in the incubator by a constant flow of sub-surface seawater. In situ light intensity of the sampling depth (10 m) was mimicked using blue light screens attenuating direct sunlight by approximately 50%.

Fig. B4 :

 B4 Fig. B4: Principle of SIMS image acquisition. This simplified scheme of SIMS acquisition used during MOBYDICK depicts the sputtering of nano-sized phytoplankton cells by a Cs + primary ion beam and the resulting secondary electron (SE) and secondary ion images of various masses (12C -, 13 C -, 28 Si). In this example, plankton communities were incubated with H 13 CO3 -from dawn to dusk. The resulting ratio image of 13 C/12 C was used to quantify CO2fixation of individual cells sorted by flow cytometry.28 Si was used to identify the silicified frustule of diatoms. Modified from[START_REF] Musat | Detecting metabolic activities in single cells, with emphasis on nanoSIMS[END_REF].

  Fig. S1. Evenness for the entire dataset according to different size fractions and locations on or off the plateau, and for each depth of the different size fractions. Pairwise significant differences (Tukey HSD, P<0.05) are indicated. Pielou's index is constrained between 0 and 1 (lowest to highest possible evenness).

Fig. S3 :

 S3 Fig. S3: Correlation between Chl a and small phytoplankton abundances measured by flow cytometry. Correlation was significant off plateau only.

Fig. S4 .

 S4 Fig. S4. Example of typical Phaeocystis antarctica flagellated cell as seen by SEM in MOBYDICK samples. Scale bar = 1 µm. Copyright: Lucie Courcot (LOG)

  13 C after incubation (A sample POC ) of the T0 non-enriched samples (A control POC ) of the enriched DIC source pool (A enriched DIC ) and of the natural DIC pool (A natural DIC ).

Fig. 1 :

 1 Fig.1: Map of the study area. Surface chlorophyll a concentrations correspond to AQUA/ MODIS average values for March 2018. The yellow dashed line indicates the position of the polar front after Pauthenet et al. (2018)[START_REF] Pauthenet | Seasonal meandering of the Polar Front upstream of the Kerguelen Plateau[END_REF].

Fig. 2 :Fig. 3 :

 23 Fig. 2: Chemotaxonomic vertical profiles of the contribution of major phytoplankton groups to Chl a concentration. The black line indicates Phaeopigment:Chl a ratio. Phaeopigments correspond to degraded and Chl a to fresh pigment material. The dashed black line corresponds to a ratio of 1. In (a), stations have Phaeo/Chl a ratio above 1 at 200m depth, whereas in (b) stations the ratio is <1.

Fig. 4 :

 4 Fig. 4: Boxplot of the daily CO2-based cell-specific division rates. Each dot corresponds to the division rate of a single cell measured with NanoSIMS for cells <20 µm (a,b) or with SIMS for diatoms >20 µm (c). Diamonds indicate mean division rates and inactive cells are colored in black. Significant differences (pairwise Mann-Whitney test with p<0.05) in division rates between stations (a) or between size-groups on-and off-plateau (b) are indicated by letters above the boxplots (ranked by alphabetical order from highest to lowest division rates). The interquartile range is indicated as a measure of dispersion of the division rates. Outliers correspond to the larger points.

Fig. 5 :

 5 Fig. 5: Single-cell daily uptake rate of carbon (pg C d -1 ) vs. single-cell volume for diatoms and non-silicifed cells measured with NanoSIMS on-(a) and off-plateau (b). Empty circles correspond to inactive cells. Scaling exponents have been obtained by linear least-squares fitting of log-transformed data. Consequently, the amount of CO2 fixed at the single cell level (C-fix) scaled with cell volume (V) according to the power law C-fix= a.V α where a is a constant that differed on-and off-plateau and α is the scaling exponent.

Fig. 6 :

 6 Fig. 6: Estimated relative (a) and absolute (b) contribution of the different groups of small phytoplankton to bulk CO2-fixation (measured by EA-IRMS). Contribution of each group was obtained by multiplying mean CO2-fixation rates (NanoSIMS) by the abundance of the groups (flow cytometry enumeration).

Fig. S2 :

 S2 Fig. S2: Step by step protocol for the gating and sorting of small phytoplankton groups. Pigmented cells were first gated based on their chlorophyll (PC5.5-H) and forward scatter (FSC-H) (a). Only pigmented cells were plotted and Synechococcus cells gated based on their higher phycoerythrin signal (PE-H) (b). Pico group was created to group Synechococcus cells with pico-eukaryotes in the same size range (c). The rest of the autotrophs were separated in two groups (Nano1 and Nano2) based on their FSC-H (d). All groups were plotted based on their Chl a and forward scatter signal (Syn and pico-eukaryotes in pink and light green; Nano1 in turquoise and Nano2 in dark blue) (e).

Fig. S3 :Fig

 S3 Fig. S3: Surface abundance of different groups of small non-silicified autotrophs assessed by CARD-FISH labeling with probes PRAS04 (prasinophytes, Mamiellaceae family), PRYM02 (haptophytes) and PELA01 (pelagophytes). Abundance of Synechococcus obtained by flow cytometry is also indicated (Syn). Total abundances of small autotrophs enumerated by flow cytometry are indicated at the top of each column with a grey dot. Counts with the CARD-FISH method confirmed the importance of haptophytes (2-5µm in size) on-and off-plateau (735 -4950 cells mL-1 depending on the station).Prasinophytes (<2µm in size) from the Mamiellaceae family were more abundant on-plateau (750-2300 and 70-115 cells mL-1 respectively), while pelagophytes (appr. 3 µm in size) were mostly observed off-plateau. These three groups accounted for 58-87% and 22-61% of small phytoplankton abundances enumerated by flow cytometry on-and off-plateau, respectively.

Fig. S5 :

 S5 Fig. S5: Histogram of measured division rates of small cells on (a) and off-plateau (b) and large diatoms (c).The best distribution was selected from a predefined family of distributions(normal, logistic, exponential, log-normal, uniform, gamma) based on the lowest AIC. Division rates of small cells were best fitted by a logistic distribution on and off-plateau with mean division rates µ=0.37 and µ=0.23 on-and off-plateau respectively. Division rates of large diatoms fitted a lognormal distribution characterized by skewed distributions to the left (low division rates) with mean division rates µ=0.09.

  Fig. S6: Single-cell volume-specific C-fixation rates (i.e. the daily carbon fixation rate expressed in fg C divided by the cell volume). Significant differences are indicated by letters above the boxplots ranked by alphabetical order from highest to lowest mean volume-specific C-fixation rates (pairwise Mann-Whitney test with P<0.05)

  and 4 mmol m -2 d -1 , respectively) (Paper 5; Christaki et al. 2020). Thus, small phytoplankton may contribute to C-export, via grazing or aggregation mechanisms, as suggested by several findings in this thesis. The high proportion of Phaeocystis reads observed off-plateau at 300 m in the large size fraction (Paper 1; Fig. 3) indicated that this

  outcome on annual C-export of a shift in size of phytoplankton community composition from diatoms towards small non-silicified cells. This variability also highlights the idiosyncratic dimension of the links between pelagic ecology and ocean biogeochemistry and the difficulty for time-limited cruises to use natural or artificial fertilization experiments to understand the functioning of the biological carbon pump and serve as analogues for past and future climate-induced changes in Cexport magnitude. Considering the remoteness and central role of the SO in the global C-cycle, this emphasizes the need to develop more remote automated sampling devices to explore the seasonality, recurrence and environmental links between phytoplankton surface communities and associated export.

4 .

 4 Outlook for future studies on the role of small phytoplankton in the marine Ccycle

Paper 4 :

 4 help with developing the bioinformatic pipeline, analyzing and interpreting sequencing data. Paper 5: flow cytometry enumeration of FLBs and viruses, analysis of environmental data. Paper 3: Microzooplankton diversity and role in carbon cycle in contrasting Southern Ocean productivity regimes (Christaki et al., submitted to Journal of Marine Systems) Paper 4: Protist interactions and community structure during early autumn in the Kerguelen Region (Southern Ocean)[START_REF] Sassenhagen | Protist interactions and community structure during early autumn in the Kerguelen Region (Southern Ocean)[END_REF]

Table B1 :

 B1 Summary of the objectives and methods used to describe the diversity, abundance and biomass of phytoplankton communities, focusing on small phytoplankton groups Molecular exploration of small phytoplankton diversity with 18S rDNA metabarcoding Environmental rDNA metabarcoding has opened a new way of assessing the diversity of the smallest organisms in natural environments. The method provides diversity data on many organisms that are difficult to identify by microscopy observation, hard to grow in culture, fragile, or

	1.1 rare. Consequently, metabarcoding studies have revealed previously hidden taxonomic richness,
	including rare species and parasites (Pedrós-Alió, 2007; Logares et al., 2014), and provided much
	higher biodiversity estimates than traditional microscopy-based methods
	Objectives			Method		Sampling
	1. Molecular diversity of	-Metabarcoding (18S rRNA	-2 size fractions (0.2-20 µm and
	small (<20 µm) and large	gene)			20-100 µm)
	(>20 µm) phytoplankton					-4 depths (10, 60, 125, 300 m)
						-After the bloom (MOBYDICK)
	2. Abundance of specific	-CARD-FISH labelling of	-Surface only
	taxonomic groups of small	prymnesiophytes, prasinophytes	-After the bloom (MOBYDICK)
	phytoplankton		and pelagophytes
	3. Biomass of small cells and	-HPLC	and	CHEMTAX	-Surface down to 50 m
	importance	in	analysis			-3 cruises:
	phytoplankton					Bloom onset (KEOPS 2)
	communities throughout				Bloom decline (KEOPS 1)
	the season					After the bloom (MOBYDICK)

Table B3 :

 B3 FISH probes used in this study

	Probe name	Target group	Source

PRAS04

Mamiellophyceae with a few exception (e.g. Dolichomatix)

(Not et al., 2004

) PELA01 Pelagophyceae (Simon et al., 2000) PRYM02 Haptophyta (Simon et al., 2000) Euk1209+NChlo01+Chlo02 Eukarya

(Not et al., 2002)

Table S1 .

 S1 Initial pigment:Chl a ratios used in CHEMTAX analysis of pigment data.

Table 1 :

 1 Main surface biogeochemical parameters of the stations sampled. Nutrients and Chl a concentration indicated in the table were sampled at 10 m for plateau stations and 25 m for off-plateau stations. The mean mixed layer depth (difference in sigma of 0.02 to the surface value) and euphotic layer depth (1% light depth) of all CTD casts performed during the occupation of the stations is given.

		Station	Sampling date	Temperature (°C)	NH4 + (nmol L -1 )	NO3 -(µmol L -1 )	PO4 3-(µmol L -1 )	Si(OH)4 (µmol L -1 )	Mixed layer depth (m)	Euphotic layer (m)	Chl a (mg m -3 )
	Off	M1	10/03/2018 5.08	421	24.76	1.63	6.49	27	80	0.31
	plateau
		M3_1	05/03/2018 5.60	501	23.39	1.62	2.31	65	93	0.19
		M4_1	01/03/2018 4.49	354	25.50	1.72	4.13	49	96	0.18
		M4_2	10/03/2018 4.47	481	24.79	1.71	4.80	87	100	0.22
	Plateau M2_1	26/02/2018 5.21	704	21.62	1.45	1.17	62	64	0.28
		M2_2	07/03/2018 5.24	1090	21.25	1.47	1.29	61	61	0.32
		M2_3	17/03/2018 5.18	899	21.75	1.51	2.60	68	58	0.58

Table 2 :

 2 Contribution of small phytoplankton to CO2-fixation on and off iron-fertilized areas of the Southern Ocean. During artificial fertilization studies, the initial contribution of small phytoplankton corresponds to the HNLC value. This contribution decreased throughout the fertilization experiments as the contribution of larger phytoplankton increased.

	Study area (experiment	Type of Fe-	Small	HNLC	Fe-fertilized	Month	Method	Source
	name)			fertilization	phytoplankton
					size
	Crozet (CROZEX)	Natural Fe	<20µm	66%	53%	November -January	Size-fractionation	[82]
	Kerguelen (KEOPS1)	Natural Fe	<10µm	68%	10 -20	February	Size-fractionation	[12]
	Amundsen Sea		Natural Fe	<5µm	50.8%	14.9%	January	Size-fractionation	[83]
	South Georgia		Natural Fe	<12µm	>60%	<20%	January	Size-fractionation	[84]
	South (SOIREE)	of	Australia	Artificial	<20µm	>60%	decrease during the experiment to <40%	February	Size-fractionation	[85]
	Atlantic sector of the so (EISENEX)	Artificial	<20µm	70-90%	decrease during the experiment to <50%	November Size-fractionation	[86]
	Kerguelen (MOBYDICK) Natural Fe	<20µm	43-70%	41-61%	March	NanoSIMS	This study

Table S4 :

 S4 Compiled literature on division rates of open-ocean Sothern Ocean phytoplankton depending on iron availability. When available, standard deviation is indicated in brackets.

			Fragilariopsis kerguelensis	0.18	0.08	cell counts		3°C	cultures	[7]
			Fragilariopsis kerguelensis	0.38	0.14	cell counts		4°C	cultures	[15]
	Pico Nano1 Nano-sized flagellates M2-1 M2-2 M2-3 M1 M3-1 M4-1 M4-2 M2-1 M2-2 M2-3 M1 Haptophytes	-1 ) Natural assemblage of small Pseudo-nitzschia 0.28 1.12 0.23 1.09 0.20 0.86 Species Pseudo-nitzschia subcurvata Thalassiosira 0.17 1.03 0.08 0.54 0.15 1.02 0.11 0.88 0.55 2.55 0.57 2.71 0.62 2.58 0.39 2.85 flagellates dominated by P. antarctica Thalassiosira antarctica Eucampia antarctica P. antarctica (strain CCMP #1871) Eucampia antarctica P. antarctica Eucampia antarctica Phaeocystis antarctica (clone AA1) Proboscia inermis Proboscia inermis	Pg Division rate [d -1 ] C-fixed 0.67 0.27 biomass 0.25 0.21 Fe-replete Fe-limited / 0.37-0.57 0.19-0.26 0.28 0.08 0.23 0.17 0.15 0.14 0.12 0.22 0.21 0.24 0.14 0.37 (0.12) 0.23 (0.09) 0.15 0.05 0.34 0.19 0.38 (0.025) 0.19 (0.006) 0.31 0.18 0.33 0.1 0.26 0.14 0.52 0.28 0.47 0.29 0.56 0.34	fluorometry Method cell counts cell counts cell counts 13 C incubations and NanoSIMS cell counts Chl a cell counts concentration cell counts fluorimetry cell counts cell counts cell counts	1°C Temperature 0-4°C 4°C 3°C 4-5°C 3°C 2°C 3°C 3°C 3°C 3°C 3°C	cultures cultures Comments cultures cultures incubation of nat. communities Fe-replete corresponds to plateau cultures samples, Fe-limited to HNLC stations culture cultures incubation of nat. communities, Antarctic Circumpolar Current cultures cultures Cultures from isolates (Polar Frontal Zone, December 2001) cultures	[13] Reference [8] [15] [7] This study [7] [9] [5] [10] [6] [7] [7] [9]
	Nano2	M3-1 M4-1 M4-2 M2-1 M2-2 M2-3	0.49 0.54 0.32 0.74 4.37 P. antarctica Proboscia inermis Phaeocystis antarctica (clone 3.16 3.10 2.59 7.41 28.10 AA1) Chaetoceros Phaeocystis antarctica (clone AA1) Chaetoceros brevis (4-6µm) 3.08 17.82 Phaeocystis antarctica (clone SX9) Chaetoceros dichaeta (60-80µm) 0.55 0.15 0.17 0.12 0.10 0.37-0.40 0.44 0.50 0.45-0.54 0.33 0.39 (0.09) 0.16 0.17 0.41	0.14-0.17 0.30 0.20 0.15-0.21 0.15 0.39 (0.09) 0.14 0.12	cell counts fluorimetry cell counts cell counts fluorometry cell counts cell counts cell counts		0-4°C 3°C 3°C 0-4°C 3°C 0-3°C 3°C 0-3°C	cultures cultures cultures cultures cultures cultures cultures cultures	[8] [10] [9] [8] [10] [16] [9] [16]
		M1 M3-1	1.92 2.01 P. antarctica Chaetoceros dichaeta 18.71 11.63	0.10 0.17 0.36 0.43	0.24 0.18	cell counts fluorometry		2°C 1°C	cultures cultures	[11] [13]
		M4-1	2.58 P. antarctica Chaetoceros simplex	14.04	0.18 0.42 0.5	0.24 0.23	cell counts cell counts		2°C 2°C	cultures cultures	[12] [14]
		M4-2	1.39 P. antarctica Chaetoceros debilis	15.27	0.09 0.58 0.37	0.18 0.19	fluorometry cell counts		1°C 2°C	cultures cultures	[13] [12]
	Cryptophytes		Geminigera cryophila Actinocyclus		0.26 0.3	0.05 0.05	cell counts cell counts		2°C 4°C	cultures cultures	[14] [15]
	Diatoms		Corethron pennatum		0.32	0.09	cell counts		4°C	cultures	[15]
			Corethron criophilum Natural assemblage of small diatoms (<20µm) Odontella	0.37 0.24 (0.14) 0.23	0.1 0.22 (0.1) 0.03	fluorometry 13 C incubations and NanoSIMS fluorometry	1°C 4-5°C 1°C	incubation of nat. communities cultures Fe-replete corresponds to plateau samples, Fe-limited to HNLC stations cultures	[13] This study [13]
	Size fractionation		Natural assemblage of large diatoms (>20µm) pennates <5µm pennates 5-20µm >20µm	0.11 (0.14) 0.2 0.5 0.27 0.23 0.55	0.09 (0.11) 0.16 0.2 0.2 0.05 0.06	13 C incubations and SIMS cell counts cell counts 14 C incubations	4-5°C -2.8°C	incubation of nat. communities Fe-replete corresponds to plateau samples, Fe-limited to HNLC stations incubation of nat. communities, Ross sea (oceanic) In situ incubations of an artificial Fe-fertilization experiment incubation of nat. communities, Antarctic Circumpolar Current	This study [6] [17] [6]
			Fragilariopsis cylindrus	0.33-0.64	0.2-0.43	cell counts		0-4°C	cultures	[8]
			Fragilariopsis cylindrus	0.16 (0.046)	0.05 (0.001)	Chl concentration	a	2°C	cultures	[5]

Remerciements

Acknowledgement

We thank B. Quéguiner, the PI of the MOBYDICK project, for providing us the opportunity to participate in this cruise; the captain and crew of the R/V Marion Dufresne for their enthusiasm and support aboard during the MOBYDICK-THEMISTO cruise (https://doi.org/10.17600/18000403) and the chief scientist I. Obernosterer. We thank Hélène Timpano and Maria Ciobanu working at UNICELL platform, Orsay, for their help with flow cytometry sorting before NanoSIMS analysis. We also thank the NanoSIMS team of the French National Ion Microprobe Facility hosted by the Muséum National d'histoire Naturelle (Paris) and the SIMS team from the Centre de Recherches Pétrographiques et Géochimiques (CRPG) in Nancy for precious advice and assistance during the analysis. This work was supported by the French oceanographic fleet ("Flotte océanographique française"), the French ANR ("Agence Nationale de la Recherche", AAPG 2017 program, MOBYDICK Project number: ANR-17-CE01-0013), and the French Research program of INSU-CNRS LEFE/CYBER ("Les enveloppes fluides et l'environnement" -"Cycles biogéochimiques, environnement et ressources"). We also thank the French Ministry of Higher Education and the Region des Hauts de France for funding the PhD grant to S. Irion. The French Research program of INSU-CNRS LEFE/CYBER funded the project 'ACTIVEUK' that supported preliminary experiments that led to use of stable isotope tracers and the subsequent set up of protocols to concentrate cells for NanoSIMS analyses.

Appendix

Appendix C1: Example of the comparative analysis of diatom communities obtained with microscopy and sequencing. At station M2-2 (a) Corethron, Proboscia and Guinardia dominated both biomass estimates (microscopy) and sequencing of the >20 µm size fraction. As small diatoms were rare at M2, sequencing of small diatom communities was not congruent with microscopy counts. At station M4-1 (b), dominant taxa in microscopy counts were Fragilariopsis, Thalassiosira, Proboscia, Rhizosoloenia, Chaetoceros and unidentified raphid pennates. These genera were also dominant in sequencing results of the two size-fraction. Cylindrotheca was abundant in microscopy counts at all stations but rarely sequenced.To note also the important proportion of unidentified "Bacillariophyta" especially in the large sizefraction. Microscopy counts of diatom communities were conducted at the MIO by Justine Legras and Augustin Lafond.

Appendix C2: Maximum Likelihood phylogeny of 18S rRNA gene of diatoms built with RAxML. Sequences marked with ASV in red originate from MOBYDICK community sequencing data. All other sequences represent references from PR2 and NCBI databases. ASVs in red were assigned to Hemiaulus, which is not reported in the SO. Building a tree illustrated the close relatedness of Hemiaulus and Dactyliosolen and supported the adaption of the genus name Dactyliosolen, which was frequently observed by microscopy, for the ASVs in question.

Appendix C3: Seasonal evolution of the relative abundance of the major protist class to total protist community composition determined by Illumina sequencing of 18S rDNA V4 region of RAS samples. The use of HgCl2 lead to unexpected and probably erroneous contribution of specific groups, like Centroheliozoa. Samples preserved with glutaraldehyde presented however some interesting patterns. For example, the two peaks in relative abundance of diatoms (Bacillariophyta) corresponded to the two bloom periods, whereas Prymnesiophyceae relative abundance increased before and after the bloom period. Interestingly, the relative abundance of phytoplankton grazers (Dinophyceae and Spirotrichea) increased shortly after the increase of diatoms. The considerable increase of Syndiniales reads after the bloom is also interesting as this group also contributed to a high proportion of reads on-plateau in the small size fraction during MOBYDICK. It was suggested that it could reflect the release of Syndiniales spores after a parasitic infection, which may have played a role in bloom termination (Paper 4;[START_REF] Sassenhagen | Protist interactions and community structure during early autumn in the Kerguelen Region (Southern Ocean)[END_REF].

Competing Interests

The authors declare no conflict of interest.

Supplementary material

Catalyzed reporter deposition of Fluorescent In Situ Hybridization (CARD-FISH)

Fluorescent in situ hybridization with horseradish peroxidase-labelled probes was performed as detailed in Not et al. [1] to quantify the abundances of the major phytoplankton phylogenetic groups. Diverse probes (EUK1209r, NCHLO01 and CHLO02) were combined to target all eukaryotes and a set of probes (PRAS04, PRYM02 and PELA01) were used separately to target most of the prasinophytes, prymnesiophytes, and pelagophytes [1][2][3]. In brief, filter pieces were incubated in the hybridization buffer

(2 µl of oligonucleotide probe at 50 ng µl -1 stock, 40% formamide, 0.9 M NaCl, 20 mM pH 7.5 Tris-HCl, 0.01% (w/v) sodium dodecyl sulphate (SDS) -Sigma-Aldrich) and incubated at 35°C for 3 h.