Thesis Micropearls: a newly discovered biomineralization process in eukaryotes MARTIGNIER, Agathe Abstract The present work uncovers a potentially ancient but yet undescribed biomineralization process, in which unicellular planktonic eukaryotes produce multiple intracellular inclusions of near-amorphous calcium carbonate (ACC), called micropearls. This process is not linked to a specific habitat and occurs in multiple areas around the world in marine, brackish and freshwater environments. This unsuspected biomineralization capacity was discovered to be common in the Tetraselmis genus (Chlorodendrophyceae, Chlorophyta). In these organisms, the micropearls may be slightly enriched in strontium and their arrangement forms a pattern (alignements linked to the chloroplast shape), which is characteristic for a given species. Another organism forming micropearls highly enriched in barium was detected in Lake Geneva (Switzerland). It is still unidentified, but might belong to the zooplankton. All the micropearls seem to result of a biologically controlled process. They represent a new physiological feature for eukaryotes. Reference MARTIGNIER, Agathe. Micropearls: a newly discovered biomineralization process in eukaryotes. Thèse de doctorat : Univ. Genève, 2019, no. Sc. 5407 DOI : 10.13097/archive-ouverte/unige:129105 URN : urn:nbn:ch:unige-1291057 Available at: http://archive-ouverte.unige.ch/unige:129105 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département des Sciences de la Terre Directeur Prof. Daniel Ariztegui Co-directeur Dr Jean-Michel Jaquet Micropearls: a newly discovered biomineralization process in eukaryotes THÈSE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention sciences de la Terre par Agathe Martignier de Genève (Suisse) Thèse N° 5407 GENÈVE Atelier de reprographie ReproMail 2019 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES DOCTORAT ÈS SCIENCES, MENTION SCIENCES DE LA TERRE Thèse de Madame Agathe MARTIGNIER intitulée «Micropearls: a New Biomineralization Process in Eukaryotes» La Faculté des sciences, sur le préavis de Monsieur D. ARIZTEGUI, professeur associé et directeur de thèse (Département des sciences de la Terre), Monsieur J.-M. JAQUET, docteur et codirecteur de thèse (Département des sciences de la Terre), Madame M. FILELLA, docteure (Département F.-A. Farel des sciences de l'environnement et de l'eau), Monsieur F. BARJA, docteur (Département de botanique et biologie végétale), Monsieur A. IMMENHAUSER, professeur (Department of geology, mineralogy and geophysics, Ruhr-Universitat Bochum, Germany), autorise l'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées. Genève, le 1er novembre 2019 Thèse - 5407 - Le Doyen Publications déjà issues de ce travail: Intracellular amorphous carbonates uncover a new biomineralization process in eukaryotes. Martignier A., Pacton M., Filella M., Jaquet J.-M., Barja F., Pollok K., Langenhorst F., Lavigne S., Guagliardo P., Kilburn M.R., Thomas C., Martini R. and Ariztegui D., Geobiology 15, 240-253 (2017). Marine and freshwater micropearls: Biomineralization producing strontium-rich amorphous calcium carbonate inclusions is widespread in the genus Tetraselmis (Chlorophyta). Martignier A., Filella M., Pollok K., Melkonian M., Bensimon M., Barja F., Langenhorst F., Jaquet J.M. and Ariztegui D., Biogeosciences, 15, 6591-6605 (2018). Thesis director : Prof. Daniel Ariztegui Department of Earth sciences, University of Geneva, Swizterland Thesis co-director : Dr. Jean-Michel Jaquet Department of Earth sciences, University of Geneva, Swizterland Jury members : Dr. Montserrat Filella Department F.A. Forel for Environmental and Aquatic sciences, University of Geneva, Swizterland Dr. François Barja Microbiology unit, University of Geneva, Swizterland Prof. Adrian Immenhauser Institute for Geology, Mineralogy and Geophysics, Ruhr-Universität Bochum, Germany Abstract _____________________________ Calcium carbonates are the most abundant biogenic minerals, but intracellular inclusions of this same composition in unicellular organisms were generally thought to be rare. The present work uncovers a potentially ancient but yet undiscribed biomineralization process, in which unicellular planktonic eukaryotes produce multiple intracellular inclusions of near-amorphous calcium carbonate (ACC), called micropearls. The process is not related to a specific habitat and occurs in marine, brackish and freshwater environments. Micropearls have been identified in the water column of seven different lakes in Switzerland, France, Greece and Bolivia and literature mentions occurrences of the marine species producing them in multiple areas around the world. This phenomenon is therefore widespread geographically. The micropearls can be highly enriched in alkaline-earth elements such as strontium and barium, compared to the surrounding water: Sr/Ca ratio can be up to 87x higher in the micropearls and Ba/Ca ratio up to 800 000 times higher. In that case, these microspheres typically present nanometer-scale internal concentric zonation, expressing oscillatory variations of their chemical composition (Sr/Ca or Ba/Ca ratio). The process seems biologically controlled and the composition of the micropearls is characteristic for a given species: in the same culture medium, some will metabolize only Ca, some Ca and Sr, and others Ca, Sr and Ba. This unsuspected biomineralization capacity was discovered to be common in the Tetraselmis genus (Chlorodendrophyceae, Chlorophyta) and potentially in the whole class Chlorodendrophyceae, as micropearls were also observed in a species of the genus Scherffelia (S. dubia). The micropearls of Chlorodendrophyceae are mostly composed of pure calcium carbonate or enriched in strontium. The micropearls are not randomly scattered throughout the cell and their arrangement often forms a pattern (e.g. longitudinal alignments), which is characteristic for a given species. This arrangement pattern is linked to the chloroplast shape, as the mineral inclusions align themselves along the incisions between chloroplast lobes. Moreover, ITS and rbcL phylogenies suggest that this pattern may constitute a valid criterion 1 to distinguish different phylogenetic groups in the genus Tetraselmis: the species belonging to a given phylogenetic clade all seem to present similar micropearl arrangement patterns. In Lake Geneva (Switzerland), a different organism was discovered to produce near- amorphous micropearls with composition ranging from calcium carbonate highly enriched in barium to barium carbonate (up to > 90 Ba/Ca mol%). Although it is still unidentified, our results show that this organism is not a close relative of the Chlorodendrophyceae and even suggest that it might belong to the zooplankton or the mixotroph eukaryotes. Micropearls represent a new physiological feature for eukaryotes. It may seem puzzling that this had gone unnoticed, especially as they are widespread in the class Chlorodendrophyceae, which includes many well-studied organisms. In fact, early microscopists had spotted alignments of “refractive granules” in some Tetraselmis species, but without identifying their mineral nature. Later, micropearls have not been detected despite multiple electronic imagery observations of the organisms producing them, because they are easily dissolved by most sample preparation techniques used to fix the organic matter. The discovery and study of micropearls, in the present research, was due to an uncommon sample preparation technique originally meant for the observation of diatom frustules in the natural environment. It seems possible that calcium carbonate inclusions might have been dissolved by traditional sample preparation for electronic imagery in other samples too: other well-studied organisms may therefore also have unsuspected biomineralization capacities to produce micropearls or similar ACC inclusions. 2 Résumé _____________________________ Les carbonates de calcium sont les minéraux biogéniques les plus abondants. Cependant, jusqu’à récemment, les inclusions intracellulaires de cette composition étaient considérées comme rares dans les organismes unicellulaires. Le travail présenté dans ce manuscrit met au jour un processus de biominéralisation, potentiellement très ancien, qui n’avait pas encore été décrit: des organismes unicellulaires appartenant aux eucaryotes forment de multiples inclusions minérales intracellulaires, appelées microperles, qui sont constituées de carbonate de calcium amorphe (ACC) ou quasi amorphe. Ce processus ne semble pas être lié à un habitat spécifique et peut avoir lieu dans des environnements aussi bien marin que saumâtre ou encore d’eau douce. Jusqu’à présent, les microperles ont été détectées dans sept lacs différents en Suisse, France, Grèce et Bolivie. De plus, la littérature décrit que les organismes marins qui produisent des microperles ont été observés en de nombreux points du globe. Ce phénomène est donc largement répandu du point de vue géographique. Les microperles peuvent être fortement enrichies en éléments alcalino-terreux tels que le strontium et le baryum : le rapport Sr/Ca peut être 87 fois plus élevé dans les microperles que dans l’eau environnante et le rapport Ba/Ca jusqu’à 800 000 fois plus. Dans ce cas, les microsphères présentent typiquement des zonations internes concentriques de l’échelle du nanomètre qui expriment des variations de composition