Je Remercie

Yamama Naciri

Xavier Vekemans

Jerome Duminil

Emilie Lecompte

Christophe Thebaud D'avoir

Merci À Pierre-Olivier Cheptou

Rafael Rubio

D E Casas

Sylvain Billiard

Pierre Saumitou-Laprade

Philippe Vernet

Herve Philippe

Pascal-Antoine Christin

Je

Céline Van De Paer
email: vandepaer.celine@gmail.com

Cynthia Hong-Wa

Céline Jeziorski

Guillaume Besnard
email: guillaume.besnard@univ-tlse3.fr

Mohamed Mensous
email: mmensous@ymail.com

Sophie Manzi

& Olivier Bouchez

& Djamel Baâli-Cherif

V Torɵlis Raddiana

V Seyal

V Flava

V Niloɵca Tomentosa

comités de thèse pour leurs remarques et leurs suggestions constructives

Keywords: De novo assembly Herbarium specimen Hesperelaea palmeri Lamiales mitogenome, mitochondrial genome, NGS, Next Generation Sequencing, plastome, plastid or chloroplast genome, RFLPs, Restriction Fragment Length Polymorphisms, rRNA, ribosomal RNA, SD, standard deviation, tRNA, transfer RNA Acacia . Chloroplast genome . Hoggar . Microsatellite . Nuclear ribosomal DNA . Phylogeny . Phylogeography . Population genetics . Positive selection

Merci pour ta bonne humeur, ta bienveillance et ton optimisme. Je remercie également PHILIPPE VERNET : travailler avec vous deux, Pierre et Philippe, a été particulièrement enrichissant pour moi. Je tiens aussi à remercier le laboratoire de Lille pour m'avoir accueillie en stages (obligatoires et volontaires) pendant plusieurs années.

Travailler sur les Oleaceae m'a également permis de me réconcilier avec les équations. Je tiens en effet à remercier SYLVAIN BILLIARD pour m'avoir ouvert au monde de la modélisation. Je souhaite à tout étudiant (souhaitant faire de la modélisation ou non) d'être encadré par une personne comme toi. Merci pour ton sens de la pédagogie, merci pour ta patience et pour le temps que tu as toujours pris pour expliquer, voire ré-expliquer, certains principes, même les plus simples. Merci pour ton écoute ; merci d'être ouvert à toutes les questions, même celles qui nous paraissent les plus bêtes et naïves. Merci pour ton savant dosage entre la part d'encadrement et la part de liberté et d'autonomie accordées à tes étudiants. Travailler à tes côtés a été vraiment stimulant scientifiquement et intellectuellement. Merci pour nos discussions diverses ; c'est toujours un plaisir de discuter avec toi de sujets scientifiques, ou simplement de parler de la vie en général. Merci Sylvain.

Ce sont donc

Remerciements

Mes premiers remerciements s'adressent à mon directeur de thèse GUILLAUME BESNARD. Tout d'abord, merci Guillaume de m'avoir proposé ce sujet de thèse sur la famille des Oleaceae. Merci pour ton enthousiasme et ta passion pour la recherche qui m'ont véritablement portée pendant ces trois années. C'était un vrai plaisir de travailler à tes côtés. Moi qui étais focalisée sur les systèmes de reproduction chez les plantes, tu m'as ouvert à un nouveau domaine de la recherche tout aussi intéressant qui est l'évolution moléculaire (même si je suis loin de maîtriser tous les concepts...). Tu as toujours été bienveillant et à l'écoute, je t'en remercie. Je te remercie d'avoir été aussi présent et de m'avoir épaulée lorsque j'en avais besoin. J'ai aussi beaucoup apprécié ta rigueur scientifique. Je te remercie pour ton investissement en termes de temps et d'énergie et pour tes retours constructifs lors de l'écriture des papiers ou du manuscrit. Merci de m'avoir fait confiance. Je te remercie de m'avoir donné la possibilité de faire des expérimentions à Montpellier lors de ma première année de thèse et de m'avoir permis de travailler sur d'autres projets tels que celui des acacias et des aulnes. Merci également de m'avoir fait partager les petits moments de bonheur/malheur et les aléas de la vie de chercheur, quasi quotidiennement (sûrement parfois au grand désespoir de mes chers collègues de bureau). Et puis, une thèse, c'est aussi la rencontre entre un doctorant et un directeur de thèse, c'est la rencontre de deux personnes et c'est la rencontre de deux caractères. Les nôtres sont assez différents, mais c'est aussi la complémentarité de nos caractères qui a fait que ces trois ans se sont aussi bien passés. Alors une dernière fois merci Guillaume, ces trois ans ont été pour moi une des expériences les plus enrichissantes scientifiquement, mais aussi humainement. Et puis j'espère que tu continueras à me raconter tes petites histoires d'Oleaceae, de graminées, de tortues, de pigeons, ... bref, tes petites histoires de science! *** Si je me suis lancée dans le projet fou de faire une thèse, ce n'est pas tout à fait par hasard, surtout une thèse sur les Oleaceae! Parfois, tout se joue à un détail … Tout a commencé en 2011. Petite stagiaire de L2 au laboratoire de Lille (Evo-Eco-Paléo, qui s'appelait alors GEPV), je travaillais sur l'auto-fécondation chez la Silene. Une partie de mon travail consistait à peser des feuilles de Silene et à préparer des plaques d'extraction. Alors que je pesais mes petites feuilles de Silene, un certain Pierre est arrivé dans la salle de manip et m'a gentiment fait remarquer que ma balance était mal calibrée et qu'il fallait que je travaille un peu plus proprement. Un détail. Un détail, certes, mais un détail qui témoignait d'une rigueur scientifique et qui m'a donné envie de travailler avec ce Pierre, que je ne connaissais absolument pas. L'année suivante, c'est donc tout naturellement que j'ai décidé d'effectuer mon stage de L3 avec Pierre Saumitou-Laprade. J'ai d'abord travaillé sur la répartition racinaire d'un sol calaminaire (en extrayant à la pince quelques milliers de racines de blocs de terre), puis j'ai rapidement bifurqué sur les questions liées aux systèmes de reproduction chez les Oleaceae.

C'est ainsi que je tiens à remercier PIERRE SAUMITOU-LAPRADE. Merci Pierre de m'avoir fait partager ta passion pour la recherche et de m'avoir fait découvrir l'histoire étonnante et fascinante des systèmes de reproduction chez les Oleaceae. Merci pour ton écoute, ta rigueur scientifique et ta patience. Merci de m'avoir permis de faire des stages passionnants à tes côtés pendant plusieurs étés. Merci pour ces sorties terrains dans des lieux magnifiques : que ce soit dans la baie de Canche, dans le Platier d'Oye ou dans les Cévennes. Merci à JAN. J'aurais tellement de remerciements à t'adresser... Si je suis arrivée au bout de cette thèse, c'est aussi grâce à ton écoute, tes encouragements, ta patience et ta bienveillance. Tu m'as épaulée au quotidien. Merci de m'avoir supportée, encouragée et soutenue lors de mes (nombreux) moments de doutes. Tu m'as apporté l'équilibre dont j'avais besoin. J'ai été heureuse de partager l'aventure de cette thèse, ou plutôt de nos thèses, avec toi et j'espère encore partager de nombreuses aventures à tes côtés.

Enfin, je tiens tout particulièrement à remercier MES PARENTS. Bien que vous ne les connaissez pas personnellement, les noms de « Pierre », «Philippe », « Sylvain » ou « Guillaume » vous sont familiers depuis plus ou moins longtemps. Le monde de la recherche vous était inconnu, mais vous avez toujours fait l'effort de découvrir et de connaître mon univers et d'essayer de comprendre ce sur quoi je travaillais (en m'écoutant patiemment parler de « systèmes d'incompatibilités », de « génomes » ou de « publications »). Je vous en suis reconnaissante. Vous avez toujours été présents pour partager mes joies, pour me redonner confiance et m'encourager quand j'avais des doutes ou pour me réconforter dans les moments les plus difficiles. Pour tout cela, je vous remercie. Merci de m'avoir fait confiance et de m'avoir toujours soutenue, quels que soient mes choix. Merci d'avoir toujours été là pour moi.

INTRODUCTION GENERALE

Les organismes vivants présentent des génomes très diversifiés en termes de taille, de structure, d'évolution et de fonction. Déterminer et comprendre les facteurs génétiques, écologiques et évolutifs qui gouvernent cette diversité génomique est un enjeu majeur en évolution moléculaire. Le génome des animaux, des plantes et des champignons est partitionné dans différents compartiments au sein de la cellule. Le noyau contient une large partie du génome et le reste du génome est contenu dans les mitochondries chez les eucaryotes mais aussi dans les chloroplastes chez les plantes. Les chloroplastes et les mitochondries sont les seuls organites (i.e. compartiments intracellulaires différenciés et spécialisés) à posséder leur propre génome. Les génomes nucléaire, mitochondrial et chloroplastique co-évoluent et interagissent afin de coordonner l'expression génique et assurer les fonctions métaboliques nécessaires au bon fonctionnement de la cellule. Chez les plantes, la dynamique structurelle et l'histoire évolutive concertée des génomes mitochondriaux et chloroplastiques sont encore mal connues. L'objectif de ce travail de thèse est d'étudier la diversité structurelle et l'évolution des génomes mitochondriaux et chloroplastiques chez une famille de plantes à fleurs, les Oleaceae, en combinant une approche génomique et phylogénétique. Bien qu'il existe d'autres organites que les chloroplastes et les mitochondries, le terme "organite" sera utilisé dans ce manuscrit pour désigner les chloroplastes et les mitochondries uniquement.

I/ Origines et évolution des organites chez les plantes à fleurs 1. Origines des organites

1.1. Apparition de la photosynthèse et de la respiration La photosynthèse est un processus permettant la synthèse de molécules organiques complexes en utilisant de l'énergie lumineuse. La photosynthèse oxygénique entraîne la fixation du dioxyde de carbone (CO 2 ) et la libération d'oxygène (O 2 ). L'apparition de la photosynthèse est estimée entre 2,4 et 2,1 milliards d'années [START_REF] Lyons | The rise of oxygen in Earth's early ocean and atmosphere[END_REF]Soo et al. 2017). Jusqu'alors, l'oxygène était présent en quantité infime dans l'atmosphère terrestre (~0,0001%) et le métabolisme des organismes vivants était anaérobie (i.e. qui ne nécessite pas d'oxygène). Les premiers organismes photosynthétiques sont les cyanobactéries, appartenant au domaine des bactéries [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF]Blankenship 2017 ;Soo et al. 2017). L'activité photosynthétique des cyanobactéries entraîna une augmentation significative de la concentration de l'oxygène dans l'atmosphère [START_REF] Lyons | The rise of oxygen in Earth's early ocean and atmosphere[END_REF]). Certaines bactéries s'adaptèrent à ce changement de composition atmosphérique en utilisant l'oxygène dans leur métabolisme : c'est l'apparition de la respiration aérobie [i.e. qui nécessite de l'oxygène; [START_REF] Lyons | The rise of oxygen in Earth's early ocean and atmosphere[END_REF] ; Soo et al. (2017)].

Origines des chloroplastes et des mitochondries par endosymbiose

Une endosymbiose est une relation durable et bénéfique entre deux organismes vivants, où l'un des deux organismes (appelé endosymbiote) est présent à l'intérieur de l'autre organisme (appelé hôte). Les chloroplastes et les mitochondries sont issus d'endosymbioses indépendantes (Fig. 1). Cependant, ces événements d'endosymbioses chloroplastique et mitochondriale sont fondamentalement similaires (Fig. 1), impliquant un hôte eucaryote (i.e. dont le noyau contenant l'ADN est séparé du reste de la cellule par une membrane) ou procaryote et un endosymbiote procaryote [i.e. une cellule dépourvue de noyau et de structures internes ; [START_REF] Margulis | Origin of eukaryotic cells: evidence 5404 (1.25[END_REF]]. La théorie endosymbiotique de l'origine des organites stipule que l'endosymbiose mitochondriale a précédé l'endosymbiose chloroplastique (Soo et al. 2017). L'origine endosymbiotique des mitochondries [START_REF] Martin | The origin of mitochondria[END_REF]Gray & Archibald 2012). On distingue deux principales théories sur l'origine des mitochondries par endosymbiose, qui s'opposent sur la nature de l'hôte. La théorie dite traditionnelle suppose une endosymbiose entre un hôte eucaryote anaérobie (un protiste amitochondrial) et un endosymbiote aérobie de type bactérien (du groupe α-Proteobacteria ; Fig. 1). La mitochondrie (i.e. l'endosymbiote) permet alors de détoxifier l'oxygène de l'hôte en l'utilisant pour produire de l'énergie ; en contrepartie, la mitochondrie reçoit au sein de l'hôte la protection et les nutriments nécessaires à son fonctionnement. Cette théorie est remise en cause du fait qu'aucune lignée d'eucaryotes n'a pu être identifiée comme ayant divergé avant l'acquisition symbiotique de la mitochondrie. Une hypothèse inhérente à cette théorie est que l'ensemble des descendants issus de la lignée hôte s'est éteint, à l'exception de ceux porteurs de mitochondries. Une théorie alternative postule que l'hôte impliqué dans l'endosymbiose de la mitochondrie était un procaryote de type archéobactérien aérobie facultatif (i.e. capable de vivre avec ou sans oxygène). Il est supposé que cet hôte archéobactérien était H 2 -dépendant et aurait pu bénéficier de la production d'H 2 par la mitochondrie pour produire de l'énergie. Cette théorie suppose que les eucaryotes ont évolué à partir d'une endosymbiose mitochondriale dans un hôte procaryote, ce qui expliquerait l'ubiquité des mitochondries au sein des eucaryotes.

L'origine endosymbiotique des chloroplastes (Keeling 2010 ;Jensen & Leister 2014). La théorie de l'endosymbiose des chloroplastes (appelée aussi endosymbiose primaire) implique un hôte eucaryote et une cyanobactérie photosynthétique (Figs 1 et 2). Cette théorie, largement acceptée, repose notamment sur les larges similitudes entre les génomes des chloroplastes et des cyanobactéries. La plupart des reconstructions phylogénétiques regroupent les gènes chloroplastiques avec les gènes homologues (i.e. descendant d'un même gène ancestral) de cyanobactéries. Trois lignées d'eucaryotes ont dérivé de l'acquisition primaire d'une cyanobactérie : les glaucophytes, les algues rouges et les algues vertes (à partir desquelles les plantes terrestres ont dérivé ; Fig. 2). Les reconstructions phylogénétiques suggèrent que ces lignées dérivent d'un seul événement d'endosymbiose primaire, bien que différents événements d'endosymbioses rapprochés sont quasiment impossibles à distinguer d'un unique événement. La plupart des autres lignées d'algues sont issues d'endosymbioses secondaires impliquant un hôte eucaryote et une algue eucaryote photosynthétique (i.e. une algue rouge ou une algue verte ; Fig. 2). Ces événements d'endosymbioses secondaires ont eu lieu à de multiples occasions (entre deux et sept fois) au cours de l'évolution des algues (Delwiche 1999;Cavalier-Smith 2000).

Structure et évolution des génomes cytoplasmiques chez les plantes à fleurs

Compte tenu de leurs histoires évolutives similaires, les chloroplastes et les mitochondries partagent de nombreuses caractéristiques génomiques en commun. Les chloroplastes et les mitochondries possèdent en partie leur propre génome, relique des endosymbioses bactériennes à partir desquelles ils ont évolué. Suite aux événements d'endosymbioses, les chloroplastes et les mitochondries ont subi une réduction drastique de leurs génomes et la plupart de leurs gènes ont été transférés vers le génome nucléaire. Par ailleurs, les chloroplastes et les mitochondries ont suivi indépendamment des chemins évolutifs semblables, comme c'est le cas pour leurs stratégies d'expression génique et de réplication, leur mode de transmission ou encore certaines de leurs caractéristiques structurelles. Le génome nucléaire codant pour des protéines cytoplasmiques pourrait lier les génomes chloroplastiques et mitochondriaux et favoriser l'évolution convergente des deux organites. Néanmoins, on observe de nombreux traits divergents entre les chloroplastes et les mitochondries, tant au niveau de leur structure que de leur évolution.

Structure et organisation des génomes cytoplasmiques

Structure des génomes chloroplastiques [START_REF] Wicke | The evolution of the plastid chromosome in land plants: gene content, gene order, gene function[END_REF]Jansen & Ruhlman 2012 ;Tonti-Filippini et al. 2017). Les génomes chloroplastiques (appelés aussi plastomes) chez les plantes à fleurs sont hautement conservés en termes de structure, de taille et de contenu en gènes. Les plastomes sont généralement des génomes circulaires de ~140-160 kilobases (kb) et quadripartites incluant deux régions répétées et inversées (les "Inverted Repeats", notées IR) séparées par une petite et une grande régions en simple copie ["Small Single Copy" (SSC) et "Large Single Copy" (LSC) ; Fig. 3]. On peut néanmoins retrouver des espèces dont la taille du plastome est extrêmement réduite, notamment chez les plantes parasites nonphotosynthétiques (appelées holoparasites). Le plus petit plastome connu, d'environ 11 kb, est celui de l'holoparasite africain Pilostyles aethiopica (Bellot & Renner 2015). A l'inverse, certaines espèces telles que Pelargonium×hortorum (Geraniaceae) possèdent des plastomes de tailles exceptionnellement larges [parfois supérieures à 200 kb; Chumley et al. (2006)]. Une extension des "Inverted Repeats" et des duplications partielles ou complètes de gènes peuvent entraîner une telle augmentation de la taille du plastome. En termes de contenu génique, le plastome chez les plantes à fleurs contient entre 100 et 120 gènes, codant principalement pour des protéines et des ARN [de transfert (ARNt) ou ribosomiques (ARNr)] impliqués dans la photosynthèse, la transcription et la translation. Structure des génomes mitochondriaux (Burger et al. 2003 ;Mower et al. 2012 ;Gualberto et al. 2014 ;Smith & Keeling 2015). Contrairement aux plastomes, les génomes mitochondriaux (appelés aussi mitogénomes) sont extrêmement variables en termes de structure, de taille et d'organisation au sein des plantes à fleurs. Le mitogénome des plantes présente une structure complexe, en sous-cercles d'ADN (Fig. 4). De nombreuses régions répétées (allant jusqu'à plusieurs kb) sont généralement présentes dans un mitogénome de plante. Des événements de recombinaison active entre ces régions répétées entraînent une structure multipartite du mitogénome, i.e. la formation de configurations multiples et interchangeables du mitogénome. La représentation physique du mitogénome en molécule circulaire, appelée "master-cercle", habituellement utilisée est donc factice et n'est qu'une séquence consensus résumant le contenu génomique de la mitochondrie. La taille du mitogénome des plantes à fleurs est extrêmement variable entre espèces, voire à l'échelle intra-spécifique. Le mitogénome des plantes présente une taille comprise entre ~200 kb chez Brassica rapa (Brassicaceae) et ~11 megabases (Mb) chez Silene conica (Caryophyllaceae). Ces grandes variations de taille des mitogénomes sont essentiellement dues aux nombreuses séquences répétées et aux régions non-codantes. Bien qu'extrêmement larges, les mitogénomes chez les plantes à fleurs ne contiennent qu'entre 50 et 60 gènes. Les gènes mitochondriaux codent pour des protéines, des ARNt et des ARNr impliqués dans la respiration et/ou la phosphorylation oxydative (qui permet la phosphorylation de l'ADP en ATP), la maturation des ARN, l'importation de protéines, la translation et occasionnellement la transcription. Les événements de recombinaison au sein du mitogénome sont à l'origine de nombreux réarrangements dans l'ordre des gènes.

Évolution des génomes cytoplasmiques

Vitesse d'évolution des régions codantes (Wolfe et al. 1987 ;[START_REF] Palmer | Mitochondrial DNA in plant systematics: Applications and limitations[END_REF][START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]. Bien que le mitogénome des plantes à fleurs varie rapidement en termes de structure, la séquence nucléotidique mitochondriale évolue globalement très lentement. Le taux de substitutions synonymes (d S ) dans les gènes mitochondriaux est trois à quatre fois plus faible que dans les gènes chloroplastiques et près de 12 fois plus faible que dans le génome nucléaire. En revanche, le taux de substitutions non-synonymes (d N ) est similaire dans les gènes chloroplastiques et mitochondriaux. Les taux de substitutions nucléotidiques peuvent varier entre gènes selon leur position au sein du génome. Les gènes chloroplastiques localisés dans les régions en simple copie présentent un taux de substitutions synonymes ~3,7 fois plus élevé que les gènes situés dans les "Inverted Repeats". Néanmoins, il a été montré que trois lignées de plantes à fleurs (Pelargonium, Plantago et Silene) présentent un taux de substitutions synonymes légèrement plus élevé (de 1,4 à 2,1 fois) dans les gènes des "Inverted Repeats" que ceux situés dans les régions en simple copie [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]. Les taux de substitutions nucléotidiques peuvent être également très variables parmi les gènes et entre espèces (Jansen & Ruhlman 2012). [START_REF] Maréchal | Recombination and the maintenance of plant organelle genome stability[END_REF]. La recombinaison homologue correspond à l'échange de matériel génétique entre des séquences nucléotidiques identiques ou similaires. La recombinaison homologue est impliquée dans la réparation des dommages subis par l'ADN et assure la stabilité des génomes. Un système actif de recombinaison homologue a été mis en évidence dans les plastomes et les mitogénomes chez les plantes à fleurs. Ces événements de recombinaison jouent un rôle important dans la dynamique de la structure du génome des organites et dans leur évolution.

Séquences dupliquées et recombinaison homologue

-Dans le plastome des plantes, la recombinaison entre les "Inverted Repeats" génère deux types d'isoformes différant selon l'orientation des régions en simple copie. Mis à part les "Inverted Repeats", le plastome des plantes présente très peu de séquences répétées. Il a été montré que des répétitions suffisamment larges pour recombiner ont un effet déstabilisant délétère pour le plastome et seraient contre-sélectionnées.

-Le mitogénome des plantes présente un système de recombinaison particulièrement actif, du fait des nombreuses séquences répétées réparties dans le génome. Les larges répétitions (>1 kb) recombinent fréquemment au sein du mitogénome, entraînant la structure multipartite du génome. Les réarrangements du mitogénome sont souvent à l'origine de nouveaux cadres de lecture ouverts (dits ORFs : Open Reading Frames) codant pour des gènes chimériques. Ces gènes chimériques peuvent générer des incompatibilités cytoplasmiques telles que la stérilité mâle cytoplasmique (dite CMS : Cytoplasmic Male Sterility). Des recombinaisons homologues moins fréquentes ont lieu entre les petites répétitions du mitogénome et génèrent de petites molécules d'ADN appelées sublimons. Chez le millet perlé et le maïs, il a été montré que la recombinaison entre des petites répétitions mitochondriales peut entraîner la restauration de la fertilité chez des plantes initialement mâle-stériles (Schardl et al. 1985;Feng et al. 2009).

Transferts de séquences d'ADN entre organites [START_REF] Wang | Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA[END_REF]Xia et al. 2013 ;Sloan & Wu 2014). Les régions non-codantes du mitogénome des plantes à fleurs sont en partie composées d'ADN d'origine chloroplastique. Le mitogénome des plantes contient entre 1% (~4 kb) chez Arabidopsis et 11,5% (~113 kb) chez Curcubita de régions dérivées du plastome [START_REF] Unseld | The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides[END_REF][START_REF] Alverson | Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)[END_REF]. L'ADN d'origine chloroplastique retrouvé dans le mitogénome peut provenir de différentes régions du plastome. Ces régions dérivées du plastome sont généralement des pseudogènes ou des séquences non-codantes, bien que des gènes codant pour des ARNt restent fonctionnels. Dans de rares cas, des séquences d'origine chloroplastique sont intégrées dans la région codante de gènes mitochondriaux fonctionnels. Le contenu en séquences d'origine chloroplastique peut fortement varier entre espèces, voire au niveau intra-spécifique. Les mitogénomes acquièrent et perdent fréquemment ces régions dérivées du plastome. Les séquences d'origine chloroplastique n'étant pas forcément orthologues (i.e. issues d'un ancêtre commun) au sein d'une même lignée (e.g. au sein d'une espèce, d'un genre ou d'une famille), il est souvent difficile d'éclaircir l'histoire évolutive de ces régions. Contrairement au mitogénome, le transfert de séquences mitochondriales vers le plastome se produit rarement. Des événements d'intégration de régions mitochondriales dans le plastome ont été reportés chez la carotte (Iorizzo et al. 2012), l'herbe aux perruches [Asclepias syriaca ;[START_REF] Straub | Horizontal transfer of DNA from the mitochondrial to the plastid genome and its subsequent evolution in milkweeds (Apocynaceae)[END_REF]] et quelques graminées (Ma et al. 2015 ;[START_REF] Burke | Evolutionary relationships in panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae)[END_REF].

Transferts réciproques de séquences entre les génomes cytoplasmiques et le génome nucléaire. Alors que le transfert de gènes mitochondriaux vers le génome nucléaire a été décrit dans de nombreux cas chez les plantes à fleurs, l'intégration de gènes chloroplastiques dans le génome nucléaire n'a été que rarement reportée (Arthofer et al. 2010). Les rares cas de transfert de gènes chloroplastiques vers le génome nucléaire reportés concernent les gènes rpl22 chez le pois [START_REF] Gantt | Transfer of rpl22 to the nucleus greatly preceded its loss from the chloroplast and involved the gain of an intron[END_REF], infA chez Arabidopsis et le soja [START_REF] Millen | Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus[END_REF], rpl32 chez le peuplier [START_REF] Cusack | When gene marriages don't work out: divorce by subfunctionalization[END_REF][START_REF] Ueda | Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus[END_REF]) et accD chez le trèfle (Magee et al. 2010) et les Campanulaceae [START_REF] Rousseau-Gueutin | Potential functional replacement of the plastidic acetyl-CoA carboxylase subunit (accD) gene by recent transfers to the nucleus in some angiosperm lineages[END_REF]. Dans le mitogénome, le contenu génique est relativement stable parmi les lignées de plantes à fleurs. Seize gènes mitochondriaux semblent néanmoins être fréquemment perdus parmi les lignées d'angiospermes [START_REF] R E F E R E N C E S Adams | Evolution of mitochondrial gene content: Gene loss and transfer to the nucleus[END_REF]) : l'ensemble des 14 gènes ribosomiques et deux gènes codant pour la succinate déshydrogénase (sdh3 et sdh4). Alors que la plupart des lignées basales des angiospermes possèdent les 40 gènes présents chez l'ancêtre commun des plantes à fleurs, certaines lignées [telles que Allium (l'oignon), Lachnocaulon et Erodium] ont perdu tous, ou presque tous, les 14 gènes ribosomiques ainsi que sdh3 et sdh4. Ces lignées convergent vers une même réduction de leur contenu génique. On distingue trois raisons majeures de la perte de gènes au sein du mitogénome : (i) la fonction du gène n'est plus nécessaire et le gène est complètement perdu de la cellule (cet événement n'arrive que rarement) ; (ii) la "substitution de gènes", ou "remplacement de gènes", i.e. un gène préexistant est recruté pour assurer la fonction du gène perdu ; (iii) le gène perdu a été transféré vers le génome nucléaire en conservant sa fonction. Différentes études ont mis en évidence le transfert indépendant de 10 gènes ribosomiques et des gènes cox2, sdh3 et sdh4 du mitogénome vers le génome nucléaire au sein de différentes lignées d'angiospermes [START_REF] R E F E R E N C E S Adams | Evolution of mitochondrial gene content: Gene loss and transfer to the nucleus[END_REF]. Réciproquement, il a été montré que les régions intergéniques du mitogénome des plantes à fleurs peuvent également être en partie composées de séquences dérivées du génome nucléaire. Le contenu en séquences dérivées du génome nucléaire est très variable entre espèces [START_REF] Kubo | Organization and variation of angiosperm mitochondrial genome[END_REF]. Chez les graminées, les séquences dérivées du génome nucléaire représentent 13,4% du mitogénome du riz [START_REF] Notsu | The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: Frequent DNA sequence acquisition and loss during the evolution of flowering plants[END_REF] et moins de 0,2% des mitogénomes du maïs et du blé [START_REF] Notsu | The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: Frequent DNA sequence acquisition and loss during the evolution of flowering plants[END_REF]Ogihara et al. 2005). Nécessitant le séquençage complet du génome nucléaire et du mitogénome d'une même espèce, peu de données sont actuellement disponibles quant au contenu en séquences nucléaires au sein des mitogénomes.

Transferts horizontaux vers les génomes cytoplasmiques (Bock 2009). Le transfert horizontal d'ADN correspond à l'acquisition de matériel génétique d'un autre organisme (pouvant appartenir à une autre espèce), sans en être son descendant. Le transfert horizontal de séquences mitochondriales entre plantes a été reporté chez plusieurs plantes à fleurs, alors qu'aucun cas d'échange d'ADN chloroplastique n'est actuellement connu. L'importante activité de recombinaison homologue des mitochondries chez les plantes et leur faculté de pouvoir fusionner sont deux caractéristiques qui peuvent faciliter l'échange horizontal d'ADN. La plupart des transferts horizontaux d'ADN mitochondrial concernent des plantes parasites ou épiphytes (i.e. poussant sur une autre plante en s'en servant comme support) et leur plante hôte. De larges régions d'ADN, voire le mitogénome complet, peuvent être transférées et intégrées dans le mitogénome de la plante hôte par fusion et recombinaison des deux mitogénomes. Bien que les transferts horizontaux d'ADN mitochondrial se passent généralement intra-genre, des cas de transferts horizontaux d'une angiosperme vers une fougère ou d'une mousse vers une angiosperme ont été reportés. L'angiosperme basal Amborella trichopoda (Amborellaceae) présente le plus grand nombre de transferts horizontaux détectés dans un mitogénome de plante [START_REF] Bergthorsson | Massive horizontal transfer of mitochondrial genes from diverse land plant donors to the basal angiosperm Amborella[END_REF][START_REF] Rice | Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella[END_REF]. Cette espèce est un arbuste tropical généralement couvert de plantes épiphytes, dont diverses espèces de mousses (Fig. 5). Il a été montré que le mitogénome d'A. trichopoda de ~3,9 Mb a acquis de larges séquences mitochondriales d'algues, de mousses et de plusieurs angiospermes. Le mitogénome d'A. trichopoda contient notamment le mitogénome complet d'une mousse et de trois algues vertes. Par ailleurs, il est possible que les régions intergéniques des mitogénomes de plantes à fleurs contiennent un grand nombre de régions intégrées horizontalement, mais leur identification reste encore difficile dans la mesure où peu de génomes sont encore complètement séquencés.

Transmission des organites chez les plantes à fleurs

L'ensemble des informations composant cette partie reposent sur la revue de Greiner et al. (2014).

Prédominance de la transmission uniparentale maternelle des organites

Chez la plupart des plantes à fleurs, la transmission des organites est uniparentale et maternelle. La transmission uniparentale des organites implique une ségrégation non-Mendélienne, avec l'absence de recombinaison. A la différence du génome nucléaire transmis par le pollen et par les graines, la valeur sélective du génome des organites transmis maternellement dépend directement de la qualité et de la quantité de graines produites. Par ailleurs, les génomes nucléaires et cytoplasmiques présentent généralement des structures génétiques de population différentes, reflétant des modes de dispersion contrastés (via le pollen et les graines versus les graines uniquement). Bien que la transmission uniparentale maternelle est dominante chez les angiospermes, le passage occasionnel ou régulier d'organites dans le pollen a été décrit chez de nombreuses espèces.

Forces évolutives entraînant l'apparition et le maintien d'une transmission uniparentale des organites

Une hypothèse proposée pour la dominance d'une transmission uniparentale des organites est l'évitement de conflits entre le génome des organites paternels et maternels, qui seraient délétères pour le génome nucléaire. L'une des incompatibilités cyto-nucléaires les plus connues est la stérilité mâle cytoplasmique, qui interfère avec le développement des anthères et du pollen pour rendre des individus hermaphrodites fonctionnellement femelles. Par ailleurs, le génome des organites provenant des deux parents ne subissant pas de recombinaison, ces derniers pourraient entrer en compétition directe. Un des deux génomes pourrait par exemple acquérir un avantage compétitif lui permettant de se répliquer plus rapidement que l'autre génome. La sélection naturelle favoriserait la transmission du génome le plus compétitif à la génération suivante. Une telle compétition entre organites serait désavantageuse pour le génome nucléaire. Un organite se répliquant plus rapidement que le génome nucléaire serait mal adapté à la cellule et entraînerait une réduction de la valeur sélective. Une autre possibilité de conflits cyto-nucléaires serait le cas où les organites paternels et maternels porteraient des allèles différents au même locus et que leur combinaison hétéroplasmique serait mal adaptée.

Forces évolutives entraînant la prédominance de la transmission maternelle des organites

La prédominance de la transmission maternelle des organites chez les plantes à fleurs pourrait être liée à un fardeau mutationnel plus élevé dans la cellule spermatique du pollen que dans l'ovule. Les organites du pollen mobile seraient davantage sujets à des dommages oxydatifs que les organites de l'ovule sessile. Par ailleurs, les cellules de la lignée germinale maternelle sont protégées du stress oxydatif par des tissus spécialisés. Enfin, le nombre de copies du génome des organites est généralement plus faible dans la cellule spermatique du pollen que dans l'ovule. En conséquence, les génomes cytoplasmiques paternels seraient davantage soumis à la dérive génétique. Chez des espèces dont la transmission des organites est paternelle ou biparentale telles que Pelargonium, le melon ou la luzerne, il a été montré que le nombre de copies des génomes cytoplasmiques dans le pollen est plus élevé que chez les espèces à transmission maternelle, ce qui pourrait limiter la dérive génétique (Mogensen 1996;[START_REF] Paterson | Insights from the comparison of plant genome sequences[END_REF]. Par ailleurs, on peut supposer que la dispersion à faible distance des organites transmis par les graines peut favoriser l'adaptation locale, ce qui pourrait être bénéfique dans un contexte environnemental stable.

La transmission uniparentale maternelle des organites : un trait évolutivement instable

La transmission uniparentale des organites exclut toute recombinaison entre les organites provenant des deux parents. L'absence de recombinaison est susceptible d'entraîner une accumulation de mutations délétères au fil des générations [processus appelé cliquet de Muller, Muller (1964)]. Les mécanismes d'évitement d'un fardeau de mutations délétères chez les organites sont encore mal connus. Le passage occasionnel d'organites par la voie paternelle via le pollen pourrait favoriser la recombinaison entre les copies maternelles et paternelles des organites, et ainsi contrebalancer le cliquet de Muller. Du "paternal leakage" ou de la transmission biparentale ont été détectés chez de nombreuses espèces. Il apparaît que la transmission biparentale ait évolué indépendamment et de manière répétée au cours de l'évolution des plantes à fleurs. Néanmoins, les conséquences génomiques d'un "paternal leakage" étant peu connues, ces hypothèses nécessitent davantage d'études et de tests expérimentaux.

3.5. Une hypothèse consensus de l'évolution de la transmission uniparentale maternelle proposée par Greiner et al. (2014) Dans leur revue, Greiner et al. (2014) proposent un modèle d'évolution de la transmission uniparentale maternelle des organites, en tenant en compte des différentes hypothèses théoriques et données expérimentales (Fig. 6). La transmission uniparentale des organites pourrait évoluer afin d'éviter la propagation d'éléments dits "égoïstes" (e.g. des organites se répliquant plus rapidement), qui entraîneraient de mauvaises adaptations ou des incompatibilités avec le génome nucléaire. Néanmoins, la transmission uniparentale est évolutivement instable du fait de l'accumulation de mutations délétères liée à l'absence de recombinaison. Afin de contrebalancer le cliquet de Muller, la recombinaison sexuelle pourrait avoir lieu via des événements épisodiques ou réguliers de transmission biparentale. A nouveau, la propagation d'éléments égoïstes favorisée par la transmission biparentale entraînerait un retour vers la transmission uniparentale des organites. La transmission uniparentale à prédominance maternelle pourrait être due à un fardeau mutationnel plus élevé chez les organites paternels, causé par des dommages oxydatifs et/ou par la dérive génétique. Ce fardeau de mutations délétères est d'autant plus important que le nombre de copies des génomes cytoplasmiques est faible dans la cellule spermatique du pollen.

Bien que partageant un certain nombre de caractéristiques communes, les données actuelles montrent que les génomes chloroplastiques et mitochondriaux présentent d'importantes différences structurelles et évolutives. Les mitogénomes des plantes à fleurs, en particulier, sont caractérisés par une large diversité de taille, de structure, de contenu génique et de substitutions nucléotidiques. Néanmoins, il est nécessaire d'accumuler davantage de données génomiques couvrant un plus large échantillonnage taxonomique. Par ailleurs, les conséquences génomiques d'un passage occasionnel ou régulier d'organites par la voie paternelle sont encore mal connues. Des approches phylogénétiques basées sur des données génomiques toujours plus abondantes pourraient apporter de réelles avancées dans la détermination des forces sélectives responsables de la diversité structurelle et de l'évolution des génomes cytoplasmiques.

II/ De la phylogénie à la phylogénomique 1. La notion de phylogénie

La notion de phylogénie repose sur la théorie de l'évolution développée par Charles Darwin (1859) dans L'origine des espèces qui stipule que l'ensemble des organismes vivants dérivent d'un ancêtre commun et sont génétiquement apparentés. Un arbre phylogénétique est une représentation graphique (sous forme d'arbre) et simplifiée des liens de parenté entre des espèces, des organismes ou des gènes et de leur évolution à partir d'un ancêtre commun. Une reconstruction phylogénétique repose sur l'identification de caractères homologues (i.e. descendant d'un ancêtre commun) partagés par différents organismes, d'une part, et sur l'application de méthodes d'inférence phylogénétique, d'autre part. Jusqu'à la moitié du XX ème siècle, les reconstructions phylogénétiques se basaient sur des caractères homologues morphoanatomiques. La découverte de la structure de l'ADN par Watson & Crick (1953) et le développement des techniques moléculaires dans les années 1970 ont révolutionné les méthodes de reconstructions phylogénétiques. Les reconstructions phylogénétiques se basent désormais sur des séquences d'ADN ou de protéines. Ceci permet notamment de limiter la subjectivité liée à l'identification de caractères morpho-anatomiques et d'augmenter considérablement le nombre de caractères homologues.

Séquençage d'ADN de nouvelle génération (NGS)

Le séquençage Sanger était la méthode de séquençage d'ADN la plus utilisée en biologie (Sanger et al. 1977), jusqu'à l'arrivée du séquençage de nouvelle génération (dit NGS : Next-Generation Sequencing). Le séquençage Sanger présente certaines limites. D'une part, il ne permet pas de séquencer des fragments d'ADN dont la longueur est supérieure à un kb. L'utilisation d'une telle technique est inenvisageable pour séquencer des génomes entiers de plusieurs milliers de paires de bases (pb), qui nécessiteraient de multiples séquençages. Une autre limite du séquençage Sanger est son coût. A environ 1 $ par kb, le séquençage d'un génome d'un gigabase (Gb) avec une couverture de séquençage de 10× couterait 10,000,000 $. Cela signifie que la plupart des laboratoires de recherche seraient contraints de séquencer des génomes à l'échelle de la mégabase.

Le séquençage de nouvelle génération (NGS), dit aussi séquençage à haut débit, a révolutionné la recherche en génomique, en médecine, en biologie moléculaire et évolutive ainsi que dans bien d'autres domaines. Les technologies NGS peuvent générer en parallèle des millions voire des milliards de petits fragments d'ADN d'une centaine de paires de bases en un seul cycle pour un coût relativement faible. Alors que le séquençage du génome humain (~3 Gb) avec la méthode Sanger a coûté 3 milliards de dollars et s'est effectuée entre 1990 et 2003 avec la collaboration de plusieurs laboratoires, il est désormais possible de séquencer le génome humain avec les techniques NGS en une journée pour un coût de 1,000 $. Les premières technologies NGS sont apparues en 2005 avec le séquençage Roche 454. Depuis, de nombreuses technologies NGS gagnant en efficacité et en rapidité pour un moindre coût ont vu le jour et se font concurrence sur le marché du séquençage. On distingue deux méthodes majeures de séquençage : la méthode globale si on veut séquencer l'ensemble du génome et la méthode ciblée si on veut séquencer uniquement certaines régions du génome, par exemple uniquement les régions codantes. La technologie Illumina, qui utilise une méthode de séquençage globale du génome, est actuellement la technologie de séquençage la plus couramment utilisée sur le marché du séquençage à haut débit.

Principe du séquençage aléatoire avec la technologie Illumina (Metzker 2009). La technologie Illumina utilise un séquençage aléatoire (ou séquençage "shotgun") qui permet de séquencer des fragments d'ADN au hasard, qui sont ensuite assemblés pour reconstruire de longs fragments appelés contigs (dont la longueur est supérieure à 1 kb), voire des génomes entiers. Cette méthode de séquençage est dite aléatoire car elle ne permet pas de cibler des régions spécifiques à séquencer. Le séquençage s'effectue par fragmentation au hasard du génome en de multiples petites séquences d'ADN. La fragmentation est réalisée par sonication (i.e. des ultra-sons envoyés à une certaine fréquence découpent l'ADN en fragments de taille donnée) ou par des enzymes de restriction qui découpent l'ADN au niveau de certains motifs [e.g. RAD-Seq (Davey & Blaxter 2010) ou GBS (Elshire et al. 2011)]. Des adaptateurs sont ensuite fixés à chacune des extrémités des fragments, ce qui permet l'immobilisation des fragments sur un support solide par hybridation d'une amorce avec l'un des deux adaptateurs (Fig. 7A). La deuxième extrémité de chacun des fragments s'hybride ensuite avec l'amorce complémentaire pour former un pont. Le brin complémentaire au fragment est alors synthétisé. Cette opération sera répétée un grand nombre de fois afin d'obtenir plusieurs centaines de millions de fragments. Au cours du séquençage, l'ADN polymérase va incorporer des nucléotides associés à des fluorochromes spécifiques à chacun des quatre nucléotides (Fig. 7B). Les signaux lumineux émis par les fluorochromes sont détectés par le séquenceur et traduits en nucléotides. L'incorporation d'un nucléotide fluorescent dans la séquence est réversible. Ceci permet à la réaction de séquençage de continuer, jusqu'à l'incorporation d'un nouveau nucléotide fluorescent dans la séquence en cours de synthèse. Les fragments obtenus (appelés "reads") sont de petites tailles (quelques dizaines ou centaines de bases). L'étape suivante est l'assemblage qui consiste à reconstituer des contigs, voire le génome entier, à partir de ces petits fragments.

Assemblage. Le séquençage d'ADN génère une importante redondance de séquences, un nucléotide pouvant être séquencé plusieurs fois. La partie du génome ciblée, voire le génome entier, peut être séquencée aléatoirement x fois en de courts fragments, c'est ce qu'on appelle la profondeur de séquençage. De plus, les fragments obtenus peuvent se chevaucher du fait de la nature aléatoire du séquençage. C'est le chevauchement de deux fragments contigus qui permet de constituer un plus grand fragment (i.e. un contig). De chevauchement en chevauchement, les fragments s'assemblent en un contig de plus en plus long, jusqu'à obtenir la partie du génome ciblée, voire le génome complet. L'assemblage est d'autant plus facile que la profondeur de séquençage est élevée. On distingue deux approches d'assemblage : (i) l'assemblage avec référence, lorsque l'assemblage est initié par la projection des petits fragments contre une séquence (ou un génome) similaire de référence (e.g. d'une espèce proche), et (ii) l'assemblage de novo, utilisé quand aucune séquence similaire n'est disponible dans les bases de données. La combinaison de ces deux approches peut également être utilisée pour assembler une séquence. Limites. L'une des limites majeures du séquençage à haut débit concerne l'étape d'assemblage du génome. La présence de régions répétées dans le génome est souvent à l'origine de cassures dans l'assemblage. Les régions répétées entraînent l'alignement de mêmes "reads" au niveau de différentes zones du génome. Ces "reads" ne s'alignent pas forcément à la bonne place dans le génome, ce qui peut entraîner une reconstruction erronée des zones adjacentes à ces répétitions. La difficulté à assembler ces zones répétées dépend essentiellement de la taille des "reads". L'assemblage de ces zones est plus aisé lorsque les "reads" comprennent la région répétée et une partie des séquences adjacentes à cette région. Néanmoins, d'autres stratégies d'assemblage restent à être développées afin d'assembler de larges régions répétées (e.g. transposons, duplications, gènes en tandem, répétitions >1 kb présents dans le mitogénome de certaines plantes).

Une autre limite concerne la profondeur de séquençage qui peut être insuffisante pour assembler certaines zones du génome. Une profondeur de séquençage minimale est requise afin de couvrir l'ensemble du génome et de distinguer une erreur de séquençage d'une substitution nucléotidique. Le coût du séquençage étant d'autant plus élevé que la profondeur de séquençage est grande, il convient d'estimer une profondeur de séquençage minimale et optimale. La détermination de la profondeur de séquençage minimale dépend de différents facteurs tels que le taux d'erreurs de la méthode de séquençage, les algorithmes d'assemblage utilisés, la longueur des "reads" ou encore le contenu en régions répétées du génome. Par ailleurs, la profondeur de séquençage dépend de la qualité et de la quantité d'ADN utilisé pour le séquençage. Le séquençage d'ADN de spécimens provenant d'herbiers ou de collections de musées peut être difficile du fait de la dégradation de l'ADN ou de la contamination avec d'autres organismes tels que des bactéries ou des moisissures.

Enfin, le séquençage haut débit a permis d'enrichir considérablement nos connaissances quant à la structure et l'évolution des génomes d'organites. Depuis le développement des technologies NGS en 2005, le nombre de plastomes complets disponibles dans les bases de données publiques (e.g. GenBank) ne cesse d'augmenter. En août 2017, on compte près de 1455 plastomes complets de plantes terrestres dans GenBank. Les assembleurs sont de plus en plus performants et spécialisés et permettent de reconstruire relativement facilement des molécules circulaires contenant des "Inverted Repeats" telles que les plastomes. Néanmoins, le mitogénome des plantes reste très compliqué à assembler, du fait de sa structure complexe. Il n'existe aujourd'hui aucune méthode d'assemblage des mitochondries de plantes standardisée et automatisée. Seuls 210 mitogénomes de plantes terrestres sont actuellement disponibles dans GenBank. La présence de nombreuses séquences répétées et de multiples régions dérivées du plastome requiert une intervention manuelle pour allonger et relier les contigs. L'assemblage manuel du mitogénome est cependant une étape laborieuse. Avec les nouvelles technologies de séquençage produisant des "reads" toujours plus longs, on peut imaginer que l'assemblage du mitogénome des plantes sera plus aisé dans les années futures.

Inférences phylogénétiques

Identification de sites homologues

La première étape des inférences phylogénétiques est l'identification de caractères homologues à partir des séquences d'ADN. En supposant une corrélation entre homologie et similarité, cette étape implique une comparaison des similarités entre les séquences des différents taxons. La comparaison entre séquences s'effectue en alignant les séquences entre elles. L'objectif de l'alignement de séquences est de déterminer la meilleure hypothèse d'homologie de chacun des sites nucléotidiques des séquences. Depuis la divergence avec leur ancêtre commun, les séquences ont subi des substitutions nucléotidiques, des délétions ou des insertions de séquences, des duplications voire des réarrangements de séquences. Le principe de l'alignement est d'ajouter des espaces dans les séquences d'ADN afin d'aligner les sites homologues des séquences (Fig. 8). On obtient une matrice dont les lignes correspondent aux séquences de chacun des taxons et les colonnes correspondent aux sites nucléotidiques homologues entre les différentes séquences. Chaque colonne contient les différents états de caractères du site homologue. L'alignement de sites homologues est d'autant plus difficile que les séquences d'ADN ont divergé.

Méthodes d'inférence phylogénétique

La seconde étape d'une reconstruction phylogénétique consiste à appliquer une méthode d'inférence phylogénétique à partir de l'alignement de séquences. Le taux d'évolution des séquences n'étant pas forcément constant au cours du temps, le simple calcul des différences entre séquences ne permet pas toujours de déterminer leur degré de proximité phylogénétique. Différentes méthodes statistiques sont utilisées pour reconstruire l'arbre phylogénétique qui décrit au mieux l'évolution et les relations phylogénétiques de la matrice de séquences. On distingue quatre principales méthodes d'inférence phylogénétique : les méthodes basées sur les distances, la méthode de maximum de parcimonie, les méthodes de maximum de vraisemblance et les méthodes bayésiennes [START_REF] Holder | Phylogeny estimation: traditional and Bayesian approaches[END_REF][START_REF] Delsuc | Phylogenomics and the reconstruction of the tree of life[END_REF].

Méthodes basées sur les distances. Les méthodes basées sur les distances convertissent la matrice de séquences en une matrice de distances, représentant les distances évolutives entre chaque paire de séquences. Ces méthodes ne tiennent pas en compte des possibles cas d'homoplasie, i.e. l'apparition indépendante de caractères similaires suite à des substitutions multiples. Ce type de méthode s'applique uniquement à des séquences ayant peu divergé.

Méthode de maximum de parcimonie. La méthode de maximum de parcimonie sélectionne l'arbre phylogénétique qui présente le moins de changements de caractères pour expliquer les séquences observées. Un des désavantages de cette méthode est qu'elle ne prend pas en compte les possibles variations du taux d'évolution le long des branches de l'arbre.

Méthodes de maximum de vraisemblance. Les méthodes de maximum de vraisemblance sélectionnent l'arbre qui a la probabilité la plus élevée de produire les séquences observées. Ces méthodes permettent d'incorporer des modèles d'évolution de séquences. Ce type de méthodes nécessite de lourds calculs informatiques, notamment pour évaluer la robustesse de l'arbre phylogénétique, ce qui peut être un facteur limitant pour de grands jeux de données (>100 séquences).

Ces trois types de méthodes permettent de générer un arbre phylogénétique optimal mais la robustesse des relations phylogénétiques entre taxons n'est pas évaluée. La méthode la plus courante pour évaluer statistiquement la robustesse des branches de l'arbre est le "bootstrap". Le "bootstrap" est une méthode de ré-échantillonnage qui permet de créer des pseudo-matrices de séquences de même taille que la matrice initiale. Il s'agit d'un tirage aléatoire avec remise des différentes positions de l'alignement, répété x fois. Pour chaque pseudo-matrice créée, un arbre phylogénétique est reconstruit. Le soutien d'une branche interne de l'arbre initial est estimé à partir du nombre de fois où cette branche interne est retrouvée dans les x arbres phylogénétiques reconstruits à partir des pseudo-matrices. Si la branche interne est retrouvée dans 100% des arbres, la valeur de "bootstrap" est de 100. Le calcul des valeurs de "bootstrap" étant relativement lent pour les grands jeux de données, les méthodes bayésiennes peuvent constituer une alternative. Les méthodes bayésiennes calculent directement les valeurs de "bootstrap" en estimant des distributions de probabilité postérieure.

Méthodes bayésiennes. Les méthodes bayésiennes sélectionnent l'arbre phylogénétique qui maximise la probabilité postérieure de l'arbre. La probabilité postérieure d'un arbre phylogénétique correspond à la probabilité que cet arbre soit vrai sachant les données (i.e. les séquences d'ADN). Alors que les méthodes de maximum de vraisemblance cherchent la probabilité d'observer des données D sachant un arbre phylogénétique T [Pr(D|T)], les méthodes bayésiennes cherchent la probabilité d'un arbre phylogénétique T sachant les données D [Pr(T|D)]. La probabilité postérieure Pr(T|D), calculée selon le théorème de Bayes, est une fonction de la vraisemblance des données D sachant l'arbre T [Pr(D|T)] et de la probabilité a priori de l'arbre phylogénétique Pr(T). Une des difficultés des méthodes bayésiennes est le choix a priori de la distribution de probabilité des différents paramètres. La spécification de la distribution a priori nécessite de prendre en compte l'ensemble des connaissances sur les paramètres avant le recueil des données (e.g. études antérieures). Une distribution uniforme (i.e. toutes les valeurs du paramètre sont possibles) est souvent spécifiée pour les paramètres où aucune connaissance a priori n'est disponible.

Les reconstructions phylogénétiques permettent d'éclairer l'histoire évolutive de groupes d'organismes vivants et de tester des hypothèses spatio-temporelles quant à la divergence de ces groupes. Par ailleurs, les arbres phylogénétiques peuvent être utilisés dans l'étude des changements de pressions de sélection exercées sur les génomes au cours de leur évolution.

Apports de la phylogénomique dans l'étude des génomes cytoplasmiques

La phylogénomique

La phylogénomique correspond à l'étude des relations phylogénétiques entre taxons à partir de données génomiques. Avant l'arrivée des technologies de séquençage à haut débit, la plupart des études phylogénétiques reposaient sur un ou plusieurs gènes. Il était souvent difficile de résoudre les relations phylogénétiques des taxons ayant divergé récemment ou ayant subi une radiation récente. Mais depuis l'émergence des technologies NGS, la phylogénomique permet d'étudier les relations de parenté entre espèces à partir de génomes complets (ou de la concaténation de multiples séquences d'ADN codantes ou non-codantes). L'analyse de génomes complets augmente considérablement le nombre de caractères informatifs, ce qui permet de clarifier le signal phylogénétique et de résoudre certaines polytomies. Par ailleurs, l'étude de la structure du génome (contenu génique, ordre des gènes, perte/gain d'introns, taille du génome, composition nucléotidique) peut permettre de résoudre certains problèmes phylogénétiques [START_REF] Delsuc | Phylogenomics and the reconstruction of the tree of life[END_REF].

Les reconstructions phylogénomiques sont basées sur l'alignement multiple de séquences, i.e. alignement de plusieurs gènes ou régions orthologues. On distingue deux approches phylogénomiques d'alignement de séquences : l'approche super-matrice et l'approche super-arbre [START_REF] Holder | Phylogeny estimation: traditional and Bayesian approaches[END_REF][START_REF] Delsuc | Phylogenomics and the reconstruction of the tree of life[END_REF]. L'approche super-matrice (la plus utilisée) consiste à concaténer plusieurs gènes orthologues pour créer une seule matrice, sur laquelle sont appliquées les méthodes phylogénétiques standard. Les séquences de gènes absents chez certains taxons sont notées comme des données manquantes. Les données manquantes n'interfèrent pas avec la reconstruction phylogénétique si elles représentent moins de 20-25% du jeu de données. La tolérance d'un certain seuil de données manquantes permet d'intégrer un grand nombre de taxons dans la matrice. La deuxième approche est l'approche super-arbre qui consiste à reconstruire un arbre individuellement pour chaque gène puis à combiner l'ensemble de ces arbres individuels en un seul super-arbre.

La phylogénomique à partir de données génomiques cytoplasmiques

La reconstruction d'arbres phylogénétiques à partir de données génomiques cytoplasmiques a permis d'éclairer l'histoire évolutive de nombreux groupes de plantes. Dans le cas d'une transmission maternelle des organites, l'utilisation de génomes cytoplasmiques dans les études moléculaires, et particulièrement dans les analyses phylogéographiques, présente plusieurs intérêts : (i) les génomes cytoplasmiques reflètent la dispersion des graines; (ii) leur taille de population efficace étant divisée par deux par rapport aux génomes diploïdes, les génomes cytoplasmiques sont davantage soumis à la dérive génétique, et (iii) la dispersion des génomes cytoplasmiques par les graines est limitée, les graines étant généralement dispersées à des distances plus courtes que le pollen. Ces deux dernières caractéristiques conduisent généralement à des patrons de différentiation inter-population très marqués.

Les arbres phylogénétiques de groupes de plantes sont essentiellement basés sur le plastome et reflètent uniquement l'histoire évolutive de ce génome. Peu d'études se sont intéressées à l'évolution d'un groupe de plantes en utilisant des données mitogénomiques. La faible utilisation du mitogénome pour reconstruire des phylogénies moléculaires est due à son évolution lente et à sa complexité d'assemblage. Par ailleurs, les mitochondries et les chloroplastes sont transmis maternellement chez la plupart des plantes et sont supposés être en fort déséquilibre de liaison. Par conséquent, les mitochondries et chloroplastes pourraient partager la même histoire évolutive. Pourtant, l'intérêt potentiel des données mitogénomiques en phylogénie pourrait être sous-estimé. D'une part, le mitogénome pourrait apporter davantage de sites informatifs et permettre de résoudre certaines relations phylogénétiques non-résolues avec la seule utilisation du plastome. D'autre part, l'utilisation de données mitogénomiques pourrait être pertinente dans le cas d'une transmission contrastée des mitochondries et des chloroplastes. Aussi, des processus tels que des événements d'hybridation inter-spécifique peuvent créer une incongruence phylogénétique entre les génomes cytoplasmiques et le génome nucléaire, d'où l'intérêt de comparer les reconstructions phylogénétiques basées sur des marqueurs chloroplastiques, mitochondriaux et nucléaires.

La phylogénomique pour tester les changements de pressions de sélection au sein des génomes cytoplasmiques

La reconstruction de l'histoire évolutive de groupes de plantes à partir de données génomiques permet également d'étudier les changements de pressions de sélection qui s'opèrent au sein des génomes. L'approche la plus couramment utilisée pour caractériser les régimes de sélection impliqués dans l'évolution des gènes codant pour des protéines est l'estimation du ratio entre taux de substitutions non-synonymes et taux de substitutions synonymes (d N /d S , noté omega ω). Le ratio ω indique une sélection purifiante (ω < 1), neutre (ω = 1) ou positive (ω > 1). L'estimation du ratio ω a notamment été utilisée sur des gènes chloroplastiques dans l'étude des changements de pressions de sélection au cours de l'évolution de la photosynthèse C 4 chez les Poaceae. Une évolution convergente du gène rbcL codant pour la large sous-unité de la RubisCO a été mise en évidence au sein des lignées de graminées à photosynthèse C 4 et plusieurs changements parallèles d'acides aminés ont pu être identifiés comme des déterminants majeurs de l'affinité pour le CO 2 et l'activité catalytique de l'enzyme (Christin et al. 2008). Par ailleurs, Piot et al. (2018) ont récemment montré qu'un tiers des gènes chloroplastiques sont sous sélection positive dans un clade de graminées (PACMAD), mais les pressions environnementales induisant ces changements sont encore mal identifiés. Une sélection positive a aussi été détectée chez plusieurs gènes chloroplastiques présentant une accélération du taux d'évolution dans certaines lignées. En particulier, une divergence élevée associée à de la sélection positive a été détectée chez les gènes clpP, ycf1, ycf2 et chez plusieurs gènes ribosomiques au sein de certaines lignées de Silene (Sloan et al. 2012), acacias (Mensous et al. 2017), Campanulaceae (Barnard-Kubow et al. 2014), Geraniaceae (Guisinger et al. 2008) et chez OEnothera (Erixon & Oxelman 2008).

Les nouvelles technologies de séquençage associées à des algorithmes de traitement de données génomiques toujours plus performants ont permis de réelles avancées dans l'étude de la diversité structurelle et l'évolution des génomes cytoplasmiques. Néanmoins, l'évolution concertée des génomes cytoplasmiques et la diversité structurelle des mitogénomes en particulier restent encore mal connues. Ce travail de thèse s'est intéressé à une famille de plantes à fleurs, les Oleaceae, qui présentent différents intérêts dans l'étude des génomes cytoplasmiques.

III/ Modèle d'étude : la famille de l'olivier, les Oleaceae 1. Caractéristiques générales

La famille des Oleaceae est une famille de plantes qui comprend plus de 600 espèces d'arbres et d'arbustes répartis en 25 genres (Wallander & Albert 2000 ;Green 2004). On y retrouve des espèces présentant un intérêt économique, culturel ou ornemental telles que l'olivier (Olea europaea), le frêne (Fraxinus spp.), le jasmin (Jasminum spp.), le forsythia (Forsythia spp.), le troène (Ligustrum spp.) ou encore le lilas (Syringa spp.). Les espèces d'Oleaceae sont distribuées sur l'ensemble des continents, à l'exception de l'Antarctique, et sont retrouvées dans des régions aussi bien tropicales que tempérées et à des altitudes très variables (Wallander & Albert 2000). Beaucoup d'espèces ou de lignées d'Oleaceae sont relativement difficiles à échantillonner du fait de leur rareté. C'est notamment le cas pour les espèces présentes dans des régions tropicales ou subtropicales isolées (e.g. Madagascar, Papouasie, Andes). En conséquence, les études phylogénétiques de la famille restent encore non exhaustives.

L'une des synapomorphies de la famille des Oleaceae est la présence de feuilles opposées (à l'exception de quelques Noronhia et Jasminum), simples ou composées. Les Oleaceae, et particulièrement la tribu des Oleeae, sont caractérisés par une grande diversité florale (Fig. 9), associée à divers modes de pollinisation [i.e. pollinisation par le vent (anémophile) ou par les insectes (entomophile), voire les deux (ambophile)] et de dispersion des graines (Wallander & Albert 2000). On y retrouve divers types de graines tels que des capsules, des schizocarpes, des samares, des baies ou encore des drupes. Les Oleaceae sont également caractérisés par une large diversité de systèmes de reproduction, allant de l'hermaphrodisme à la dioécie en passant par des états intermédiaires tels que l'androdioécie, la trioécie ou la polygamie.

Systématique

La famille des Oleaceae appartient à l'ordre des Lamiales. A partir de données moléculaires, Wallander & Albert (2000) ont révisé la systématique des Oleaceae en divisant la famille en cinq tribus : les Myxopyreae, Fontanesieae, Forsythieae et Jasmineae qui constituent les "lignées diploïdes", et les Oleeae qui sont issus d'un ancêtre allopolyploïde [i.e. paléopolyploïde : ancien polyploïde dont le génome ségrége comme un diploïde; Taylor (1945)]. Cet événement de polyploïdie est associé à une duplication du nombre de chromosomes, passant de x = 11-14 chez les "lignées diploïdes" à x = 23 chez les Oleeae. La tribu des Oleeae comprend près de la moitié des espèces d'Oleaceae et se divise en quatre sous-tribus: Ligustrinae, Schreberinae, Fraxininae et Oleinae. Les travaux de Wallander & Albert (2000), basés sur deux marqueurs chloroplastiques non-codants (l'intron de rps16 et l'espace inter-génique trnL-trnF), ont permis de clarifier la systématique des Oleaceae à l'échelle des tribus et des sous-tribus (Fig. 10). Néanmoins, la plupart des relations phylogénétiques intra-génériques ne sont pas résolues. Plusieurs études utilisant des marqueurs nucléaires (ETS et ITS) et/ou chloroplastiques se sont attachées à résoudre les relations phylogénétiques au sein de différents genres tels que Olea (Besnard et al. 2009), Noronhia et Chionanthus (Hong-Wa & Besnard 2013, 2014), Fraxinus [START_REF] Jeandroz | Phylogeny and phylogeography of the circumpolar genus Fraxinus (Oleaceae) based on internal transcribed spacer sequences of nuclear ribosomal DNA[END_REF]Wallander 2008 ;Arca et al. 2012 ;[START_REF] Hinsinger | The phylogeny and biogeographic history of ashes (Fraxinus, Oleaceae) highlight the roles of migration and vicariance in the diversification of temperate trees[END_REF][START_REF] Hinsinger | The phylogeography of Eurasian Fraxinus species reveals ancient transcontinental reticulation[END_REF], Syringa et Ligustrum [START_REF] Kim | A chloroplast DNA phylogeny of lilacs (Syringa, Oleaceae): plastome groups show a strong correlation with crossing groups[END_REF][START_REF] Li | Phylogenetics and diversification of Syringa inferred from nuclear and plastid DNA sequences[END_REF][START_REF] Lendvay | Phylogeography of Syringa josikaea (Oleaceae): early Pleistocene divergence from East Asian relatives and survival in small populations in the Carpathians[END_REF] obtenue est fortement soutenue avec des valeurs de soutien supérieures à 90% pour la plupart des noeuds. Ces analyses ont permis de confirmer la monophylie des quatre sous-tribus composant la tribu des Oleeae (Ligustrinae, Schreberinae, Fraxininae et Oleinae). Par ailleurs, de nombreuses paraphylies et polyphylies ont été détectées à l'échelle du genre, comme c'est le cas pour les genres Chionanthus, Osmanthus, Nestegis, Olea ou encore Syringa.

Transmission des organites chez les Oleaceae

La transmission des chloroplastes et des mitochondries chez les Oleaceae est peu connue. Néanmoins, une transmission maternelle stricte des organites a été mise en évidence dans le genre Syringa et chez l'olivier, alors qu'une potentielle transmission biparentale est supposée chez deux lignées indépendantes (les Ligustrinae et les Jasmineae).

3.1. Transmission maternelle des organites chez le lilas commun et chez l'olivier Corriveau & Coleman (1988) ont détecté par microscopie à fluorescence une transmission maternelle des organites chez Syringa vulgaris. Aucun chloroplaste, ni aucune mitochondrie n'a été observé dans la cellule germinative des grains de pollen de S. vulgaris. Liu et al. (2004) ont confirmé ces observations et ont également détecté par microscopie à fluorescence une transmission maternelle des organites chez six autres accessions de Syringa appartenant aux séries Syringa et Pinnatifoliae (sous-genre Syringa). Par ailleurs, Besnard et al. (2000) ont montré à partir de croisements que les organites sont transmis maternellement dans le complexe de l'olivier (Olea europaea) et qu'il existe un fort déséquilibre de liaison entre les polymorphismes chloroplastiques et mitochondriaux chez cette espèce (Besnard et al. 2002). Une stérilité mâle cytoplasmique a également été mise en évidence chez une des lignées (lignée E3) d'O. e. subsp. europaea (Besnard et al. 2000).

3.2. Transmission potentiellement biparentale occasionnelle chez deux lignées indépendantes : les Ligustrinae et les Jasmineae Liu et al. (2004) ont observé par microscopie à fluorescence la présence de chloroplastes et de mitochondries dans la cellule germinative de grains de pollen de 15 espèces de la sous-tribu Ligustrinae (Ligustrum et Syringa appartenant au sous-genre Ligustrina et aux séries Villosae et Pubescentes du sous-genre Syringa). Par une technique d'immuno-marquage aux particules d'or, ils ont détecté une diminution du nombre de mitochondries et une augmentation du nombre de chloroplastes au cours de la maturation de la cellule germinative du pollen. Des mitochondries et des chloroplastes ont également été observés dans la cellule germinative mature de grains de pollen de deux espèces de jasmin phylogénétiquement éloignés ; Jasminum officinale et J. nudiflorum (tribu des Jasmineae ; Sodmergen et al. 1998). Ces observations laissent supposer le passage occasionnel de chloroplastes dans les grains de pollen de certaines espèces de Ligustrinae et de chloroplastes et de mitochondries dans les grains de pollen de Jasminum.

IV/ Plan de la thèse

*Licences des photographies utilisées

Introduction

Recent advances in high-throughput DNA sequencing, so-called Next Generation Sequencing (NGS), have revolutionized phylogenetics by generating massive genomic resources [START_REF] Harrison | Next-generation sequencing and systematics: what can a billion base pairs of DNA sequence data do for you?[END_REF]. In particular, the genome skimming approach (i.e. low coverage shotgun sequencing) allowed the assembly of the most represented genomic regions such as the nuclear ribosomal cluster and cytoplasmic genomes (e.g. [START_REF] Cronn | Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology[END_REF]Straub et al., 2012;Besnard et al., 2013;[START_REF] Malé | Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family[END_REF]. In plants, genome skimming has been mainly used to generate chloroplast genomes (hereafter plastomes) and nearly complete nuclear ribosomal sequences (Straub et al., 2012), but the method can also be used to assemble sequences from the mitochondrial genome (hereafter mitogenome) (e.g. [START_REF] Ma | Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses[END_REF][START_REF] Bock | Genome skimming reveals the origin of the Jerusalem Artichoke tuber crop species: neither from Jerusalem nor an artichoke[END_REF][START_REF] Malé | Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family[END_REF]. In addition, the genome skimming method was successfully applied to low-quality DNA extracted from herbarium specimens, which are generally not amenable to classical PCR (e.g. [START_REF] Staats | Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens[END_REF]Besnard et al., 2014;Bakker et al., 2016;Zedane et al., 2016). This approach thus offers the possibility to include in phylogenetic analyses rare and extinct species from natural history collections.

Both plastomes and mitogenomes are maternally inherited in numerous plant groups, in particular in angiosperms, but there are a few exceptions (e.g. Corriveau and Coleman, 1988;[START_REF] Birky | Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution[END_REF]. Polymorphism of the plastome has been extensively used in phylogeographic studies, in contrast with the mitogenome (e.g. [START_REF] Ruhlman | The plastid genomes of flowering plants[END_REF]. The general lack of interest in plant mitogenomes for phylogenetic analyses mainly stems from their supposed low sequence variation (Wolfe et al., 1987;Smith and Keeling, 2015). Consequently, the assembly of plant mitochondrial sequences from shotgun data was reported only in a few studies (e.g. [START_REF] Ma | Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses[END_REF][START_REF] Bock | Genome skimming reveals the origin of the Jerusalem Artichoke tuber crop species: neither from Jerusalem nor an artichoke[END_REF][START_REF] Malé | Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family[END_REF]. In addition, reconstructing full mitogenome sequences is challenging due to the presence of both long duplications and plastid-derived regions (Mower et al., 2012b;Smith and Keeling, 2015). These characteristics are lineage-specific and make the assembly of plant mitogenome from short sequence reads very complex, with no standard procedure (as proposed for reconstructing plastomes; Straub et al., 2012;Bakker et al., 2016). Yet, mitogenomic variation in plants may be valuable in investigating not only phylogenetic relationships among species, but also in studying patterns of substitution (e.g. Zhu et al., 2014) or the genetic determinism of maternally inherited traits such as the cytoplasmic male sterility (e.g. [START_REF] Liu | Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility (CMS) line and its maintainer line[END_REF]Mower et al., 2012a;[START_REF] Hiroshi | Complete mitochondrial genome sequence of black mustard (Brassica nigra; BB) and comparison with Brassica oleracea (CC) and Brassica carinata (BBCC)[END_REF][START_REF] Touzet | Cytoplasmic male sterility and mitochondrial metabolism in plants[END_REF].

Here, we describe the assembly of the first mitogenome of Oleaceae (Lamiales). We applied our approach to degraded DNA extracted from an old herbarium specimen collected 141 years ago on Guadalupe Island, Mexico. This specimen represents the only known remain of the genus Hesperelaea, which is now considered extinct [START_REF] Watson | Botanical contributions. I. On the flora of Guadalupe Island, Lower California[END_REF]Zedane et al., 2016). Our study demonstrates that shotgun sequencing data, even from poorly preserved DNA, can be used to generate mitochondrial DNA datasets. Molecular analysis of available mitogenomes of Lamiales reveals deep structural reorganizations and heterogeneous evolutionary rates between mitochondrial genes. Finally, we discuss the implication of our results for further studies on the Oleaceae functional mitogenomics and phylogenetics.

Material and methods

Study model and DNA sequencing

Hesperelaea palmeri A. Gray belongs to the tribe Oleeae (Wallander and Albert, 2000). A recent phylogenetic study indicates that it was a paleoendemic lineage of an American group that includes Forestiera and Priogymnanthus (Zedane et al., 2016). The complete plastome of this species has been sequenced previously using a genome skimming approach (Zedane et al., 2016). Here, we reused these shotgun data produced with the HiSeq technology (Illumina Inc., 2010), consisting of 10,694,511 paired-end reads of 100 bp (Zedane et al., 2016). The mean size of inserts (DNA fragments used for library construction) was estimated at ca. 120 bp, reflecting that the DNA extracted from a herbarium leaf is highly fragmented (for more details see Zedane et al., 2016). We first removed duplicates (identical reads) with FASTUNIQ (Xu et al., 2012). These duplicates were likely resulting from library amplification before sequencing (see Zedane et al., 2016). Second, overlapping paired-end reads were merged using BBMERGE [BBTOOLS implemented in GENEIOUS v. 9.0.5 (Biomatters Ltd., 2015)]. Read merging allowed us to align reads and their reverse complements to create a single consensus read. This step also allowed improving the sequence quality of overlapping parts. In cases of non-overlapping paired-end reads, reads were not merged and were used independently. We thus obtained 9,620,010 merged reads (with a mean length of 120 bp, SD = 27.6) and 2,011,824 unmerged reads (with a length of 100 bp).

Mitogenome assembly

We used an approach combining reference-guided and de novo assembly. To discriminate plastid and mitochondrial sequences, merged and unmerged reads were first mapped simultaneously to the plastome of H. palmeri [GenBank: NC_025787] and to the mitogenome of Boea hygrometrica (Lamiales: Gesneriaceae, [NC_016741]) using GENEIOUS. Due to large genomic rearrangements relative to the mitochondrial reference genome, we obtained 76 non-overlapping contigs that had N500 bp (up to 3459 bp). These contigs were then extended independently using GENEIOUS by mapping all merged and unmerged reads to each of them with series of 100 iterations. Coverage depth was relatively constant across the genome but we observed peaks of coverage corresponding to duplicated and plastid-derived regions: (i) in long duplicated regions, the depth-coverage was, as expected, twice as high as in single-copy regions (ca. 70 × vs. 35 ×; see below); (ii) in plastidderived regions, plastid reads mapped to their homologous mitochondrial regions, leading to an increase of the coverage depth. In the first case, as mentioned by Mower et al. (2012a), some reads contiguous to the duplicated regions allowed different possible paths of assembly, reflecting several alternative conformations. We thus arbitrarily resolved the path of assembly when we encountered long repeated sequences (e.g. [START_REF] Cahill | Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies[END_REF]. In the second case, as the sequencing depth of the plastome was higher than that of the mitogenome (ca. 300 × vs. 35×), we resolved these regions manually (using GENEIOUS) by identifying mitochondrial reads taking into account polymorphisms (i.e. single nucleotide polymorphisms and indels) with the lowest coverage.

During the process of assembly, we regularly checked whether the extended contigs could be joined, taking into account the sequence similarity between their terminal regions using a de novo analysis in GENEIOUS with a minimum overlap of 100 bp. In this manner, several circular DNA sequences can be obtained. Duplicated regions (with higher sequencing depth; see above) that are shared by such circular sequences allowed their integration in a "master circle". Even if the master circle could not be an accurate representation of the mitochondrial structure in vivo due to alternative genomic conformations (e.g. Mower et al., 2012b;[START_REF] Warren | Linear plasmids and the rate of sequence evolution in plant mitochondrial genomes[END_REF], it is a convenient mitochondrial consensus sequence representing the genomic content. A last verification of the assembly was performed by mapping all paired-end reads simultaneously to the plastome of H. palmeri and to the mitochondrial consensus sequence to check for abnormal variation of coverage. The average coverage depth of the mitogenome was estimated on non-plastid-derived regions.

Identification of genes, ORFs, duplicated regions and plastid-derived regions

Coding-protein genes and rRNA were annotated with GENEIOUS by transferring the annotations of available Lamiales mitogenomes of Boea hygrometrica, Mimulus guttatus, Ajuga reptans and Salvia miltiorrhiza (GenBank accession numbers given in Table 1). Start and stop codons were manually adjusted. Annotation of tRNA was performed with tRNASCANSE (http://lowelab.ucsc.edu/tRNAscanSE). Additional open reading frames (ORFs) with minimum length of 300 bp were detected with GENEIOUS.

Table 1

Comparison of genomic content (sequence length in base pairs and percent of the complete mitogenome sequence given in brackets; GC content in percent) among the mitogenomes of Lamiales and Nicotiana tabacum (Solanales). Annotations of the mitogenomes were extracted from GenBank. To estimate the plastid-derived content of the mitogenome of each species, we used their corresponding complete plastome available in GenBank as query. For A. reptans and M. guttatus whose complete plastomes were not available, we used the complete plastome of Lavandula angustifolia (Lamiaceae) and Lindenbergia philippensis (Orobanchaceae), respectively.

Hesperelaea palmeri (KX545367) a

Boea hygrometrica (NC_016741) Repeats with a 40-bp minimum length and with at least 95% identity were identified using VMATCH (Kurtz, 2016). Identification of plastidderived regions was then carried out using BLASTN with the H. palmeri plastome as a query sequence. We listed plastid-derived regions with a minimum length of 100 bp and with at least 80% of identity with the H. palmeri plastome.

Mimulus guttatus (NC_018041) Salvia miltiorrhiza (NC_023209) Ajuga reptans (NC_023103) Nicotiana tabacum (KR780036)

Phylogenetic analyses and estimation of substitution rates

A phylogeny of Lamiales including Hesperelaea was reconstructed based on mitochondrial gene sequences, in order to compare the evolutionary rates of these genes in this plant group. We used the four available Lamiales mitogenomes (see above) with the mitogenome of Nicotiana tabacum (Solanales) as outgroup. From each mitogenome, we extracted nucleotide sequences for all protein-coding genes and introns. We excluded genes that were either absent or present as pseudogenes in several mitogenomes. Each gene and intron was aligned individually using MUSCLE (Edgar, 2004) with default parameters. The alignments of protein-coding genes were manually adjusted taking into account their amino-acid translation. All aligned protein-coding genes and introns were then concatenated. Maximum-Likelihood (ML) analyses were performed using RAXML V.8 (Stamatakis, 2014) in Fig. 1. Master circle of the Hesperelaea palmeri mitogenome (658,522 bp). The outer circle represents the gene content, with protein-coding genes, rRNA and tRNA genes. The inner circle represents repeated sequences with a minimum length of 100 bp in the mitogenome. The map was performed with OGDRAW V1.2 (Lohse et al., 2007) and CIRCOS (Krzywinski et al., 2009).

the CIPRES platform. We applied the GTR + G model and the rapid bootstrap algorithm with 1000 iterations. We visualized the best likelihood phylogenetic tree using FIGTREE V1.4.2 (Rambaut, 2014). Nonsynonymous and synonymous substitution rates (d N and d S ) were calculated individually on each coding-protein gene using codeml in PAML V.4.8 (Yang, 2007). These gene divergence rates were estimated for the five Lamiales mitogenomes by comparing each of them to the mitogenome of N. tabacum as a reference (outgroup).

Results and discussion

Mitogenome assembly, content and annotation

Our approach allowed us to reconstruct a master sequence of the H. palmeri mitogenome (EMBL accession no: KX545367; mitogenome length: 658,522 bp; GC content: 44.50%). About 5% of this genome is composed of duplicated regions, with one large repeat of 7975 bp and 151 small repeats ranging from 40 to 496 bp (Tables 1 andS1). We identified 21 plastid-derived regions with a length N 100 bp and with a minimum identity of 80% with the H. palmeri plastome [covering in total 57,617 bp (8.75%) of the mitogenome; Tables 1 andS2]. Fifteen of these regions (54,996 bp) have an identity of N 96% with the plastome, and two of them are part (1722 bp) of the long duplicated region. We thus estimated that about 41% of the plastome (130,108 bp when excluding one inverted repeat) have a highly homologous region in the mitogenome. The existence of sequences with variable identity with the plastome (96-99% vs. 80-84%; Table S2) suggests at least two rounds of integration of plastid sequences into the mitogenome. The analysis of the evolution (e.g. multiple integrations, degeneration) of such genomic regions however requires an extended sample of Oleaceae mitogenomes (e.g. Hao and Palmer, 2009). Excluding these plastid-derived regions, the average coverage depth of the H. palmeri mitogenome was estimated at 34.6× (SD = 11).

The largest part of the H. palmeri mitogenome corresponds to nonannotated sequences (592,970 bp). Annotated gene regions represent 65,552 bp (9.95%) and comprise 46 protein-coding genes with introns, three rRNA genes and 19 tRNA genes (Fig. 1; Tables 1 andS3). Two copies of nad6 were detected in the H. palmeri mitogenome, but with different start codons (i.e. ATG or GTG). We also identified a partially duplicated copy of rps12; it is very close to its paralogous functional copy but appears to be truncated at the end of the sequence as similarly reported in other genes of plant mitogenomes (e.g. rps3 duplicates in Gossypium; [START_REF] Tang | Rapid evolutionary divergence of Gossypium barbadense and G. hirsutum mitochondrial genomes[END_REF]. Lastly, we identified 171 ORFs with lengths ranging from 300 to 1000 bp and 14 additional ORFs ranging from 1000 to 2757 bp, for a total of 185 ORFs detected (Table S4). The longest ORFs (N1000 bp) were not observed in other Lamiales mitogenomes and may not be functional.

The H. palmeri mitogenome is larger ~659 kb than that of other Lamiales (ranging from ~352 to 526 kb; Table 1) but within the range reported for mitogenomes of angiosperms [from ~200 kb in Brassica rapa (Brassicaceae) to ∼ 11 Mb in Silene conica (Caryophyllaceae); Smith and Keeling, 2015]. Gene content in Lamiales mitogenomes varies between species (from 54 in B. hygrometrica to 71 in S. miltiorrhiza; Table S3) due to the transfer of mitochondrial genes to the nuclear genome (e.g. ribosomal protein genes; Adams and [START_REF] R E F E R E N C E S Adams | Evolution of mitochondrial gene content: Gene loss and transfer to the nucleus[END_REF]. Variable gene content, however, does not explain the large mitogenomic size difference in Lamiales (Table 1). As in most angiosperms, the mitogenome of H. palmeri has a complex organization with a large portion of noncoding regions, plastid-derived regions and repeated sequences (see above), and the difference of genome size in Lamiales appears to be due to a large variation in these non-annotated regions (Table 1). In higher plant mitogenomes, numerous repeated sequences found in the non-coding regions could promote recombination events and lead to sequence duplication [START_REF] Satoh | The Owen mitochondrial genome in sugar beet (Beta vulgaris L.): possible mechanisms of extensive rearrangements and the origin of the mitotype-unique regions[END_REF]. Degenerated duplicated sequences could diverge though continuous reshuffling and contribute to the extension of the genome size. This could explain mitogenome size variation in related species. The amount of plastid-like sequences in the H. palmeri mitogenome also partly explains its larger size relative to that of the other Lamiales available so far (Table 1).

Phylogenetic position of H. palmeri in Lamiales

A phylogeny of Lamiales including a Solanales as outgroup was reconstructed for six species with a concatenated alignment of 28 mitochondrial protein-coding genes and 13 introns (with a total of 56,448 aligned bp; Fig. 2). We excluded 18 protein-coding genes (ccmC, petL, rpl2, rpl5, rpl10, rpl16, rpl23, rps7, rps10, rps14, sdh3, sdh4, orf101, orf111a, orf117a, orf121, orf122c andorf182a) from the analysis due to their absence or their presence as a pseudogene in at least two species. The best ML tree (scored under the GTR + G model) was well supported with all bootstrap values reaching 100%. Hesperelaea palmeri appears to have a basal position in the Lamiales (Fig. 2). Such a phylogenetic placement of Oleaceae (Hesperelaea) as sister to a clade including Gesneriaceae (Boea), Phrymaceae (Mimulus) and Lamiaceae (Ajuga, Salvia) is congruent with other phylogenies reconstructed on cytoplasmic genes [START_REF] Wortley | How much data are needed to resolve a difficult phylogeny? Case study in Lamiales[END_REF][START_REF] Refulio-Rodriguez | Phylogeny of Lamiidae[END_REF]. The branch leading to Hesperelaea is however shorter than that of other Lamiales [especially Ajuga (Zhu et al., 2014)], suggesting lower rate of evolution in the Oleaceae lineage compared to other families within this plant order (see below).

Substitution rates in Lamiales mitogenomes

Non-synonymous and synonymous substitution rates for proteincoding genes of Lamiales species were estimated against those of N. tabacum. Being absent in the mitogenome of N. tabacum, atp8 was excluded from the analysis that was thus performed on 27 genes (Figs. 3 andS1). As shown by Zhu et al. (2014), protein-coding genes of A. reptans evolve particularly quickly compared to the other Lamiales and substitution rates have an exceptional variability among genes of the A. reptans mitogenome (Fig. S1). Compared to the other Lamiales, H. palmeri diverges slowly from N. tabacum, with non-synonymous site divergence (d N ) ranging from 0.005 to 0.392 and synonymous site divergence (d S ) ranging from 0.008 to 1.098 (Fig. S1). Nonsynonymous and synonymous substitution rates strongly vary among genes in the mitogenome (Fig. 3). As with other Lamiales, genes coding for ATP synthase subunits and for ribosomal proteins in the H. palmeri mitogenome appear to have higher substitution rates, especially atp4, atp6 and atp9 (Figs. 3 andS1). This may be due to either episodic positive selection or a relaxed selective pressure compared to other protein-coding genes, but a larger sample of species is necessary to investigate in detail the past changes in evolutionary rates of these genes.

Biological explanations of such a variation in substitution rates across mitochondrial genes remain unknown (e.g. Mower et al., (Stamatakis, 2014) with a GTR + G model. We used a concatenated alignment of 28 mitochondrial protein-coding genes and 13 introns. We applied the rapid bootstrap algorithm with 1000 iterations. The phylogenetic tree was rooted with Nicotiana tabacum (Solanales).

2007).

A possible reason could be the co-evolution between the nuclear and the mitochondrial genomes. Enzyme complexes of oxidative phosphorylation (OXPHOS) and ribosomal protein subunits are encoded by genes located in both the nucleus and the mitochondria. In the case of cyto-nuclear co-evolution, coordinated substitutions between mitochondrial and nuclear proteins could lead to an increase in amino acid substitutions or an elevated d N /d S ratio [START_REF] Rand | Cytonuclear coevolution: the genomics of cooperation[END_REF][START_REF] Dowling | Evolutionary implications of non-neutral mitochondrial genetic variation[END_REF]. This co-evolution between the nucleus and the mitochondria could thus influence the evolutionary dynamics of the mitochondrial genome.

Conclusions and perspectives

Our approach shows that the assembly of mitogenomes or long mitochondrial sequences is feasible with shotgun data, even with lowquality DNA extracted from herbarium samples. The assembly of mitochondrial genomes from Illumina short sequence reads remains, however, a challenge due to the length of the mitogenome, plastid-derived regions and repetitive sequences. Indeed, the reconstruction of a master circle is not fully automatized and thus time-consuming (e.g. [START_REF] Cahill | Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies[END_REF][START_REF] Malé | Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family[END_REF].

The reconstruction of mitogenomes or partial mitochondrial sequences (i.e. genes and introns) could be an important step to develop mtDNA phylogenetics in Oleaceae, even from herbarium material. The possibility to include rare or extinct species in phylogenies thanks to museum specimens is especially relevant to the Oleaceae family, within which several species are rare and/or only known from herbaria (e.g. Hong-Wa and Besnard, 2014;Zedane et al., 2016). Mitochondrial markers are rarely used to reconstruct the evolutionary history of higher plants due to low substitution rate in coding sequences (Gualberto et al., 2014). In addition, phylogenies of plastid and mitochondrial DNAs may both reflect seed dispersal and be mostly congruent when they are inherited maternally (e.g. Olson and McCauley, 2000;Besnard et al., 2002). However, biparental inheritance or occasionally paternal transmission (i.e. paternal leakage) of organelles has been reported in some plant species [START_REF] Greiner | Why are most organelle genomes transmitted maternally?[END_REF]. In these cases, chloroplast and mitochondrial DNAs could reflect different evolutionary histories of plants and be complementary (e.g. Govindarajulu et al., 2015). For instance, such incongruence between chloroplast and mitochondrial phylogenies has been reported in Pinaceae, in which organelles have contrasting inheritance modes (Xiang et al., 2015). A comparison of chloroplast and mitochondrial phylogenies of Oleaceae may thus be useful to test for possible paternal leakage of organelles during Oleaceae evolution (e.g. Sodmergen et al., 1998;[START_REF] Li | Divergent potentials for cytoplasmic inheritance within the genus Syringa. A new trait associated with speciogenesis[END_REF]) and also to analyze potential heterogeneous patterns of nucleotide substitutions in plastomes and mitogenomes.

Furthermore, previous studies based on Restriction Fragment Length Polymorphisms (RFLPs) and full or partial mitochondrial DNA sequencing have shown that the mitogenome structure can vary even at the species level (e.g. Smith and Keeling, 2015). The study of this intraspecific variation has allowed detecting genes putatively involved in cytoplasmic male sterility (e.g. [START_REF] Liu | Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility (CMS) line and its maintainer line[END_REF]Mower et al., 2012a;[START_REF] Hiroshi | Complete mitochondrial genome sequence of black mustard (Brassica nigra; BB) and comparison with Brassica oleracea (CC) and Brassica carinata (BBCC)[END_REF][START_REF] Touzet | Cytoplasmic male sterility and mitochondrial metabolism in plants[END_REF]. In Oleaceae, three cytoplasmic lineages were identified with RFLPs in the Mediterranean olive tree (Besnard et al., 2000). One of these lineages (i.e. Lineage E3; formerly known as CCK-MCK) has been shown to be associated to a male sterility phenotype (ms2), for which a nucleo-cytoplasmic genetic determinism was demonstrated (Besnard et al., 2000). The comparative analysis of olive mitogenomes may thus be helpful to identify putative genes involved in this male sterility phenotype (C. Van

Abstract

The mitogenome is rarely used to reconstruct the evolutionary history of plants, contrary to nuclear and plastid markers. Here, we evaluate the usefulness of mitochondrial DNA for molecular evolutionary studies in Oleaceae, in which cases of cytoplasmic male sterility (CMS) and of potentially contrasted organelle inheritance are known. We compare the diversity and the evolution of mitochondrial and chloroplast genomes by focusing on the olive complex and related genera. Using high-throughput techniques, we reconstructed complete mitogenomes (ca. 0.7 Mb) and plastomes (ca. 156 kb) for six olive accessions and one Chionanthus. A highly variable organization of mitogenomes was observed at the species level. In olive, two specific chimeric genes were identified in the mitogenome of lineage E3 and may be involved in CMS. Plastid-derived regions (mtpt) were observed in all reconstructed mitogenomes. Through phylogenetic reconstruction, we demonstrate that multiple integrations of mtpt regions have occurred in Oleaceae, but mtpt regions shared by all members of the olive complex derive from a common ancestor. We then assembled 52 conserved mitochondrial gene regions and complete plastomes of ten additional accessions belonging to tribes Oleeae, Fontanesieae and Forsythieae.

Phylogenetic congruence between topologies based on mitochondrial regions and plastomes suggests a strong disequilibrium linkage between both organellar genomes. Finally, while phylogenetic reconstruction based on plastomes fails to resolve the evolutionary history of maternal olive lineages in the Mediterranean area, their phylogenetic relationships were successfully resolved with complete mitogenomes.

Overall, our study demonstrates the great potential of using mitochondrial DNA in plant phylogeographic and metagenomic studies. reconstructing phylogenies and/or species identification [START_REF] Coissac | From barcodes to genomes: Extending the concept of DNA barcoding[END_REF][START_REF] Hollingsworth | Selecting barcoding loci for plants: Evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants[END_REF][START_REF] Li | Plant DNA barcoding: From gene to genome[END_REF].

Unlike chloroplast and nuclear genes, polymorphism in the mitochondrial genome (hereafter mitogenome) is not frequently used to reconstruct phylogenies and phylogeographies or for DNA barcoding in higher plants, which stands in contrast with studies on animals (e.g., [START_REF] Donnelly | Reconstructing the plant mitochondrial genome for marker discovery: A case study using Pinus[END_REF][START_REF] Duminil | Mitochondrial genome and plant taxonomy[END_REF]Govindarajulu, Parks, Tennessen, Liston, & Ashman, 2015;Mower, Sloan, & Alverson, 2012;[START_REF] Qiu | Reconstructing the basal angiosperm phylogeny: Evaluating information content of mitochondrial genes[END_REF]. This is due to three main reasons: First, mitochondrial genes of plants usually evolve slowly compared to those of the plastome (three to four times slower) or the nuclear genome (~12 times slower; Wolfe, Li, & Sharp, 1987;Palmer & Herbon, 1988;[START_REF] Palmer | Mitochondrial DNA in plant systematics: Applications and limitations[END_REF]. The rate of nucleotide substitutions in coding regions of a plant's mitogenome has been estimated to be around 100 times lower than in animal mitogenomes and four to six times lower than in fungal mitogenomes [START_REF] Aguileta | High variability of mitochondrial gene order among fungi[END_REF][START_REF] Nabholz | The erratic mitochondrial clock: Variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals[END_REF]Wolfe et al., 1987).

However, studies have shown that substitution rates can be highly variable among mitochondrial genes and between species (Mower, Touzet, Gummow, Delph, & Palmer, 2007;Zhu, Guo, Jain, & Mower, 2014). Second, both organellar genomes (i.e., mitogenome and plastome) are usually inherited maternally (e.g., Corriveau & Coleman, 1988;Reboud & Zeyl, 1994). Consequently, mitochondrial DNA (mtDNA) and chloroplast or plastid DNA (ptDNA) polymorphisms are supposedly in strong linkage disequilibrium and may thus reveal similar phylogenetic patterns reflecting seed dispersal (e.g., [START_REF] Desplanque | The linkage disequilibrium between chloroplast DNA and mitochondrial DNA haplotypes in Beta vulgaris ssp. maritima (L.): The usefulness of both genomes for population genetic studies[END_REF]Olson & McCauley, 2000), making the combined analysis of both organellar genomes not necessarily useful (although potentially informative for demographic analyses). Differential inheritance of mitochondria and chloroplasts, however, can reveal divergent evolutionary histories of cytoplasmic genomes. Third, the complete assembly of plant mitogenomes remains challenging due to their complex and variable structure (Smith & Keeling, 2015). For example, in the angiosperm order Lamiales, only six complete mitogenomes versus 72 plastomes were available on GenBank as of April 2017.

Despite the above-mentioned features that discourage the analysis of mitogenomes, comparative studies of both organellar genomes should be relevant to reveal their potentially contrasted evolutionary histories and structural dynamics. First, although maternal transmission of organelles prevails in plants (e.g., Corriveau & Coleman, 1988;Reboud & Zeyl, 1994) [START_REF] Hao | Gorgeous mosaic of mitochondrial genes created by horizontal transfer and gene conversion[END_REF]Sloan & Wu, 2014). Although tRNAs of plastid origin appear to retain their functionality, most mtpt regions are noncoding or pseudogenes (Hao & Palmer, 2009;[START_REF] Notsu | The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: Frequent DNA sequence acquisition and loss during the evolution of flowering plants[END_REF], sometimes integrated in coding sequences of functional mitochondrial genes [START_REF] Wang | Plastid sequences contribute to some plant mitochondrial genes[END_REF]. In contrast, the transfer of mtDNA to the plastome has been reported in some rare cases only, such as in carrot (Iorizzo et al., 2012), milkweed [START_REF] Straub | Horizontal transfer of DNA from the mitochondrial to the plastid genome and its subsequent evolution in milkweeds (Apocynaceae)[END_REF]) and a few Poaceae [START_REF] Burke | Evolutionary relationships in panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae)[END_REF]Ma, Zhang, Guo, & Li, 2015).

The number of studies that compare the diversity of both organellar genomes at the family level remains limited (e.g., Smith & Keeling, 2015), and more investigations are necessary to understand the evolution of organellar genomes in various plant groups. Here, we report a study on the olive family (Lamiales: Oleaceae), which encompasses more than 600 species in 25 genera (Green 2004). The family has a worldwide distribution, occurring in tropical, subtropical and temperate climates [START_REF] Stevens | Angiosperm phylogeny website[END_REF]. Several species are of major economic interest, such as the Mediterranean olive (Olea europaea L.) and the ash tree (Fraxinus excelsior L.), while numerous other Oleaceae are widely used as ornamentals or fragrant species (i.e., the genera Chionanthus, Forsythia, Fraxinus, Jasminum, Ligustrum, Osmanthus and Syringa). Taxa of Oleaceae are currently classified in five tribes, namely Fontanesieae, Forsythieae, Jasmineae, Myxopyreae and

Oleeae (Wallander & Albert, 2000). Oleeae, including the olive, is the most speciose lineage, with four subtribes currently recognized:

Ligustrinae, Fraxininae, Schreberinae and Oleinae (Wallander & Albert, 2000). Phylogenetic trees of Oleaceae have been reconstructed with chloroplast and nuclear ribosomal DNA markers (e.g., Hong-Wa & Besnard, 2013;Wallander & Albert, 2000;Zedane et al., 2016).

Some nodes have yet to be resolved, while phylogenetic signals from nuclear and chloroplast DNA markers were partly incongruent. This calls for additional phylogenetic investigations into the evolutionary history of this plant family. In addition, while strict maternal inheritance and male sterility with a nucleo-cytoplasmic determinism have been reported in olive [START_REF] Besnard | Cytoplasmic male sterility in the olive (Olea europaea L.)[END_REF], potential paternal leakage of organelles has been suggested for two unrelated lineages [Jasminum spp. (Sodmergen et al., 1998), and

Ligustrinae (Liu, Cui, & Zhang, 2004)]. Oleaceae is thus an excellent study case to compare plastomes and mitogenomes, as their evolution may be potentially affected by contrasted inheritance modes of organelles and nucleo-cytoplasmic interactions.

In this study, we first aim to investigate the variation and evolutionary dynamics of mitogenome organization at the species level (i.e., the olive complex). We then evaluate the usefulness of mtDNA for molecular evolutionary studies at different taxonomic levels (from tribe to species). The organelle inheritance of accessions used here is assumed to be uniparental and maternal. As a consequence, we expected congruence between topologies deduced from mtDNA and ptDNA regions. To address these objectives, we develop a methodology to simultaneously characterize the diversity and evolution of plastomes and mitogenomes in Oleaceae. We first use high-throughput sequencing to assemble organellar genomes of Mediterranean olive lineages and another Oleinae species. The assembly of mitogenomes allows us to examine differences in size, gene content and structure of this organellar genome within the same species and between related Oleaceae genera. In olive, a CMS phenotype has been specifically associated to lineage E3 (Besnard et al., 2000). Here, we identify ORFs specific to this lineage as putatively determining the CMS. We then analyse ptDNA integration in mitogenomes of Oleinae through phylogenetic reconstruction from the plastid-derived sequences (mtpt regions). Finally, we compared nucleotide diversity and phylogenetic topologies based on conserved mtDNA regions and complete plastomes assembled from distantly related species of Oleaceae. The combined use of full plastomes and mitogenomes to resolve phylogenetic relationships between maternal lineages of the Mediterranean olive is also tested. The utility of the mitochondrial DNA in plant phylogeographic and metagenomic studies is briefly discussed.

| MATERIALS AND METHODS

| Plant material and DNA sequencing

We first aimed to investigate mitogenome variation at the species level in Olea europaea. We thus focused on the wild olive tree because cytoplasmic lineages have been previously described within the O. europaea complex (Besnard, Rubio de Casas, & Vargas, 2007). Two main clades have been identified: (i) the Mediterranean/North African clade (Cp-II) that includes lineages E1, E2, E3 and M, and (ii) the cuspidata clade (Cp-I) which extends from Southern Asia to Tropical Africa and includes lineages C1, C2 and A. Each lineage shows a well-delimited geographic distribution (Besnard et al., 2007). Sequence variation in the olive plastomes was not, however, sufficient to resolve relationships between lineages of clade Cp-II (Besnard et al., 2013). Chloroplasts and mitochondria are maternally inherited in olive, and as expected, the analysis of their polymorphisms allowed congruent identification of cytoplasmic lineages [START_REF] Besnard | Combination of chloroplast and mitochondrial DNA polymorphisms to study cytoplasm genetic differentiation in the olive complex (Olea europaea L.)[END_REF]. Interestingly, a CMS phenotype (ms2) is specifically associated with lineage E3 (formerly named CCK-MCK; Besnard et al., 2000). We here analysed six accessions of O. europaea to represent five cytoplasmic lineages of the olive complex ( To evaluate the phylogenetic utility of mtDNA on the family level, and especially in the olive subtribe (Oleinae), we then extended our sample to represent more distantly related lineages of Oleaceae (Table 1). In addition to Hesperelaea palmeri (for which organellar and 2012 (Table 1). The eight remaining samples were extracted from recent silica-dried leaves.

For all studied samples, total genomic DNA was extracted from ca. 5 mg of dried leaves. Each sample was ground in a 2-ml tube containing three tungsten beads with a TissueLyser (Qiagen Inc., Texas). The BioSprint 15 DNA Plant Kit (Qiagen Inc.) was then used to extract DNA, which was eluted in 200 ll AE buffer. Shotgun sequencing was performed using a genome skimming approach as described in Zedane et al. (2016). DNA extracted from herbarium specimens was highly fragmented and thus not sonicated before library construction. Sequencing libraries were multiplexed (24 per flow cell) and sequenced with an Illumina machine (i.e., HiSeq 2000, 2500 or 3000; Table S1). Duplicated reads were removed with Fas-tUniq (Xu et al., 2012), and overlapping paired-end reads were then merged using BBMerge [BBTOOLS implemented in GENEIOUS v. 9.0.5 (http://www.geneious.com; Kearse et al., 2012)].

| Assembly, annotation of organellar DNA and identification of mtpt regions

For all accessions, we first reconstructed full plastomes as described by Zedane et al. (2016). Read mapping against the obtained consensus sequence was also performed using GENEIOUS v. 9.0.5 (Biomatters Ltd., 2015) to check assembly quality and coverage depth. Then, we assembled full mitogenomes for the six O. europaea For the six olive accessions, the cytoplasmic DNA lineage and haplotypes (according to Besnard et al., 2007Besnard et al., , 2018) ) Accessions only used for the assembly of 52 conserved mitochondrial DNA regions (Table S6).

indels) and their lower sequencing depth compared to their plastid homologues (for the detailed procedure, see [START_REF] Van De Paer | Mitogenomics of Hesperelaea, an extinct genus of Oleaceae[END_REF]. The potential presence of another homologous nuclear copy does not interfere with mtpt assembly as coverage is much higher for the mitogenome compared to the nuclear genome (by at least 20 times; J.K. Olofsson et al. in preparation). Because no automated approach of full plant mitogenome assembly based on shotgun data is currently available, we then recovered selected mtDNA regions for our Oleaceae-wide sample. We focused on 52 conserved mtDNA regions (i.e., 36 protein-coding genes and 16 introns, for a total of ca. 55 kb) that were assembled for the remaining 10 species by mapping merged and unmerged reads against the annotated mitogenome of H. palmeri [KX545367] using GENEIOUS.

Annotation of plastomes was performed using GENEIOUS by transferring the annotations of an O. europaea plastome [FN996972]. In the same manner, we transferred the annotations of the H. palmeri mitogenome to annotate coding protein and rRNA mitochondrial genes and introns. Start and stop codons of protein-coding genes were manually adjusted. We identified tRNA genes in mitogenomes using ARAGORN [START_REF] Laslett | ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences[END_REF]. Within each complete mitogenome, we also identified repeats with a 40-bp minimum length and with at least 95% identity using RepeatFinder [START_REF] Volfovsky | A clustering method for repeat analysis in DNA sequences[END_REF]. Physical maps of mitogenomes were drawn with OGDRAW v.1.2 (Lohse, Drechsel, & Bock, 2007) and CIRCOS (Krzywinski et al., 2009).

We finally analysed the integration of plastid DNA in the mitogenomes (mtpt regions) of all O. europaea accessions, C. rupicolus and H. palmeri. We also analysed Boea hygrometrica (Lamiales: Gesneriaceae), for which both mitogenome and plastome are available on GenBank [NC_016741 and NC_016468, respectively]. For each mitogenome, we detected mtpt regions using BLASTN with the corresponding plastome as a query sequence. We retained mtpt regions with a 100-bp minimum length and at least 85% identity with a plastid region. We then mapped all mtpt regions detected in the nine accessions against the E1-e.4 plastome.

| Identification of mitochondrial genes specific to the E3 olive lineage

As mentioned above, one cytoplasmic lineage (E3) is associated with a type of CMS in the olive (mp2; Besnard et al., 2000). The responsible cytoplasmic gene or genes remain unknown, but are supposed to be located in the mitogenome (Besnard et al., 2000). To identify candidate mitochondrial genes that may determine this CMS, we first identified genomic regions specific to the E3.1 mitogenome using the progressiveMauve algorithm (Darling, Mau, & Perna, 2010). All ORFs (open reading frames) with a minimum length of 150 bp were identified within these genomic regions as done by Mower, Case, Floro, and Willis (2012). ORFs were then compared to the identified genes in the E3.1 mitogenome using BLASTN [START_REF] Altschul | Basic local alignment search tool[END_REF]) with a cut-off e-value of 0.001 to check for homology with known genes and detect chimeric genes.

2.4 | Nucleotide diversity within plastid and mitochondrial genomes of olive and Oleinae Nucleotide diversity (p; Nei & Li, 1979) was measured with MEGA [START_REF] Tamura | MEGA6: Molecular evolutionary genetics analysis version 6.0[END_REF] as an average number of substitutions per site between two homologous sequences. We computed p on plastid and mitochondrial sequence alignments, independently for protein-coding genes and noncoding regions. We also distinguished plastid regions from the single copies [i.e., long and short single copies (LSC and SSC, respectively)] and inverted repeats (IR). The standard error of p was estimated by bootstrapping on sequences (100 replicates). p was investigated at two taxonomical levels. First, we estimated p for the olive complex (species level), considering one member of each lineage (i.e., E1-e.4, E2.

| Phylogenetic analyses

Phylogenetic analyses were conducted to address three main issues. using MUSCLE (Edgar, 2004). Based on these sequence alignments, we then reconstructed nine maximum-likelihood (ML) phylogenetic trees using RAXML v. 8 (Stamatakis, 2014) on the CIPRES platform (Miller, Pfeiffer, & Schwartz, 2010). We applied the GTR + G model and the rapid bootstrap algorithm with 1,000 iterations. We used FigTree v.

1.4.2 (Rambaut, 2014) to visualize the ML trees.

Plastid and mitochondrial DNA phylogenies of Oleeae: We investigated phylogenetic relationships between 16 species representative of the main Oleeae lineages and the two Oleaceae species belonging to Fontanesieae and Forsythieae used as out-groups (Table 1). Two phylogenetic trees were inferred (i) from complete plastomes, and (ii) from mitochondrial protein-coding genes and introns. Complete plastomes of all 18 species were aligned using MUSCLE with default parameters. For the mitochondrial alignment, each assembled region (protein-coding gene or intron) was aligned individually using MUSCLE with default parameters. All aligned mitochondrial regions were then concatenated into one matrix. The alignments were manually refined, particularly in regions with short inversions (<100 bp). ML trees were reconstructed on both alignment data sets using RAXML as described above.

Phylogenetic relationships in the olive complex: lastly, phylogenetic trees were reconstructed for the olive complex using complete plastome and/or mitogenome sequences. First, we aligned the six complete O. europaea plastomes generated in this study, using MUS- CLE with default parameters. We also performed a second olive plastome alignment including seven additional O. europaea accessions from GenBank (Table S2) and O. woodiana as out-group to check the impact of plant sampling on the topology. Second, an alignment of the six O. europaea mitogenomes was constructed. Conserved regions between highly rearranged genomes were detected and aligned using the progressiveMauve algorithm (Darling et al., 2010).

Finally, the alignments of the six olive plastomes and mitogenomes generated in our study were also concatenated into one single matrix. ML phylogenetic trees were reconstructed on each data set using RAXML as described above.

| RESULTS

Shotgun sequencing produced between 3.2 and 58.4 million pairedend reads per accession (Table S1). As expected, the mean size of inserts was lower for DNA extracted from herbarium specimens (118 to 202 bp) than for DNA extracted from silica-dried samples (233 to 419 bp; Table S1). Using these data, we assembled 17 complete plastomes and seven complete mitogenomes (six for O. europaea). In addition, 52 conserved mitochondrial regions (genes and introns) were assembled for ten Oleaceae accessions. The lowest sequencing depth of mtDNA and ptDNA regions was observed in Schrebera arborea (i.e., 17.19 and 649, respectively; Table S1). In each analysed sample, the sequencing depth of the mitogenome was always lower than that of the plastome, with a ratio between 4% (Fo. fortunei) and 65%

(C. parkinsonii). Note that this ratio was higher for herbarium specimens (ranging from 10% to 65%; mean = 28.2%) than for silica-dried samples (ranging from 4% to 23%; mean = 10.6%), but we did not statistically test this difference due to the small sampling size.

| Mitogenome size and composition

The mitogenomes of O. europaea and C. rupicolus were assembled as single circular consensus sequences (i.e., master circle), with a length ranging from 667,135 (C. rupicolus) to 769,995 bp (O. e. europaea E3.1; Figure 1) and a GC content of 44.7%-44.8% (Table 2). While both mitogenomes belonging to lineage E1 (E1-l1.1 and E1-e.4) had a very similar structure, mitogenome organization was highly variable among olive cytoplasmic lineages (Figures S1 andS2).

Gene annotation with the H. palmeri mitogenome as reference identified 37 distinct protein-coding genes, three rRNA genes and 12

tRNA genes in all mitogenomes, except in the E3.1 olive mitogenome whose complete sdh3 gene was missing (Tables 2 andS3). A partially duplicated copy of ccmC was also detected in E2.1, E3.1, M-g1.3 and C2.13 olive mitogenomes (noted ᴪ-ccmC; Table S3). Several proteincoding and tRNA genes were found in duplicated regions and thus annotated two or three times (Table S3; Figures 1 andS1). Repeat content was highly variable among mitogenomes. While the mitogenome of C. rupicolus had only 3.85% repetitive sequences with no repeat >1 kb, the repeat content of E2.1 olive represented 20.38% of the mitogenome and contained two large repeats >10 kb (with one reaching ~25 kb; Figure S1, Tables 2 andS4).

The amount of mtpt regions in the mitogenomes of O. europaea was relatively constant between lineages (~4.7% of the mitogenome;

Table 3), but lower than in C. rupicolus (5.2%), H. palmeri (8.5%) and B. hygrometrica (10.1%). Four conserved plastid-derived genes (rpl23, petL, petN and psbD; Table S3) were additionally found in mtpt regions of O. europaea. petL was, however, annotated as a pseudogene in the E3.1 mitogenome (Figure 1) as its coding sequence was longer than expected due to a degenerated stop codon. We only found the plastid-derived gene petN in C. rupicolus (Table S3). We also detected ten and eight plastid-derived tRNA genes in the O. europaea and C. rupicolus mitogenomes, respectively (Table S3).

Three genomic regions, ranging from 1,453 to 5,278 bp, were found to be specific to the E3.1 mitogenome. Two chimeric ORFs [orf_E3a (upstream of cox3) and orf_E3b (upstream of atp6)] were detected within these regions (Figure S3). Both are chimeric genes since orf_E3a contains a fragment of sdh3 (271 bp; see above), while orf_E3b contains 1,225 bp of the plastid-derived gene rbcL. We also detected seven short ORFs of unidentified origin with length ranging from 162 to 270 bp (data not shown).

| Nucleotide diversity within organellar genomes among olive and Oleinae

A higher nucleotide diversity was observed in noncoding regions compared to protein-coding genes in ptDNA and mtDNA both within the olive complex and across Oleinae (Table 4). Within the plastome, lower nucleotide diversity in inverted repeats (IR) than in single copies was revealed for both coding and noncoding regions.

Globally, nucleotide diversity was lower in mtDNA than in ptDNA.

Within Oleinae, we estimated that mitochondrial sequences evolved three to five times slower than LSC and SSC plastid sequences (Table 4). However, mtpt regions of O. europaea showed higher nucleotide diversity (0.076, SE = 0.012) than their plastid homologous regions (0.024, SE = 0.009; Tables 4 andS5). In our study, we produced an alignment of 18 complete plastomes (134,130 bp; after excluding one IR). In this alignment, we identified 6,572 variable sites of which 1,735 were parsimony-informative. In addition, 52 conserved mitochondrial DNA regions were aligned for the same 18 taxa (Table S6). A concatenated alignment of 55,301 bp was obtained. In this mtDNA data set, we detected 776 variable sites of which 212 were parsimony-informative.

|

Topologies of both plastid and mitochondrial ML phylogenies were congruent, except for two nodes with low support (Figure 4).

The topology of the phylogeny derived from complete plastomes had >95% bootstrap support at most nodes (Figure 4a). Only intraspecific relationships between the olive lineages E1, E2 and M remained unresolved. The topology derived from mitochondrial regions was overall less supported, with bootstrap values inferior to 80% at seven nodes (Figure 4b). Both topologies differed in the placement of the clade including N. apetala and C. rupicolus that was strongly supported as sister to Olea (100%) in the ptDNA phylogeny

T A B L E 2
Comparison of genomic content among the mitogenomes of Olea europaea and Chionanthus rupicolus (sequence length in base pairs and per cent of the complete mitogenome sequence given in parentheses; GC content in per cent). We used the corresponding plastome of each accession as query to estimate the plastid-derived content of the mitogenomes 4b).

T A B L E 3

| Phylogenetic relationships between cytoplasmic lineages of the Mediterranean olive

We finally focused on the Mediterranean olive. Twenty-one parsimony-informative sites were detected in the plastome alignment including six olive accessions (out of a total of 130,242 bp; after excluding one IR; Table S7). These sites are distributed throughout the LSC and SSC of the plastome, in both coding and noncoding regions (12 and 9, respectively). Six of 12 substitutions located in plastid genes are nonsynonymous (Table S7). For the mitogenome, we aligned 26 conserved regions with a length ranging from 4,724 to 68,630 bp. We detected 156 parsimony-informative sites in the concatenated alignment (out of a total of 747,352 aligned nucleotides; Table S8). These sites are mainly located in the noncoding regions of the mitogenome (150 sites/156), including mtpt regions (7 sites). Of the six substitutions located in mitochondrial genes, five are nonsynonymous (Table S8).

The tree topology based on ptDNA was poorly resolved (as in the analysis based on the 18 Oleaceae accessions; see above), only supporting close relationships between the E1-l1 and E1-e sublineages (Figure 5a). Adding further O. europaea accessions from GenBank did not increase node support (Figure S5). In contrast, the topology of both phylogenies based on mtDNA and ptDNA + mtDNA was congruent and recovered lineage E2 as sister to E1, while lineage E3 was sister to other Mediterranean lineages with a strong support (94%-95%;

Figure 5b,c).

| DISCUSSION

Our study demonstrates the possibility of using genome skimming data to assemble both complete plastome and mitochondrial genomic regions, as already shown by [START_REF] Mal E | Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family[END_REF]. Assembly of full mitogenomes was possible but time-consuming, and we thus developed a methodology to recover selected, conserved mitochondrial regions (genes and introns) for phylogenetic reconstruction. Plastid-derived mitochondrial regions (mtpt) are a potential issue in reconstructing some genomic regions, but mtDNA sequences always had lower read coverage than ptDNA regions. However, the mitogenome/plastome sequencing depth ratio was highly variable and tended to be higher in herbarium specimens. The potential reasons for this pattern need to be investigated with a broader specimen sampling. It could be due to a generally variable ratio of plastids to mitochondria in our samples and/ or differential degradation of plastid and mitochondrial sequences,

T A B L E 4
Estimates of the nucleotide diversity (p in %; Nei & Li, 1979) among the olive complex and subtribe Oleinae for mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) regions. The number in parentheses indicates the number of accessions used to measure p.

First, p was estimated on five lineages of the olive complex (i.e., E1, E2, E3, M and C2) independently for protein-coding genes and noncoding regions, distinguishing regions in the single copies (SSC and LSC) and the inverted repeats (IR). It was also estimated for shared DNA regions [i.e., plastid-derived regions detected in the mitogenome (mtpt regions) and their homologous regions in the plastomes].

Second, p was calculated on protein-coding and noncoding regions at the subtribe level considering eight Oleinae species (i. F I G U R E 2 Position of each mtpt homologue within the E1-e.4 plastome. We mapped mtpt regions detected in the mitogenomes of all accessions of Olea europaea, Chionanthus rupicolus, Hesperelaea palmeri and Boea hygrometrica against the E1-e.4 plastome using GENEIOUS. The nine mtpt regions shared by all Oleeae are delimited with dotted grey lines and numbered from 1 to 9. The positions of the inverted repeat A (IR A ) and the small and long single copies (SSC and LSC, respectively) are indicated potentially inducing some sequencing bias (Bakker et al., 2016;Besnard et al., 2014;Staats et al., 2011).

| Structural diversity of mitogenomes in the olive complex

While complete plastid and nuclear genomes of the olive tree were available prior to our study [START_REF] Besnard | Genomic profiling of plastid DNA variation in the Mediterranean olive tree[END_REF][START_REF] Cruz | Genome sequence of the olive tree, Olea europaea[END_REF][START_REF] Mariotti | Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison[END_REF][START_REF] Unver | Genome of wild olive and the evolution of oil biosynthesis[END_REF], mitochondrial genomic resources of O. europaea were still lacking. We here present six newly assembled, complete mitogenomes of O. europaea. In accordance with previous results based on restriction fragment length polymorphisms (Besnard et al., 2002), we found no rearrangement within lineage E1

(sublineages e and l1) but a large variation in the mitogenome structure among cytoplasmic lineages of the olive tree (Figure S2). The 2). Lineage E3 has the largest mitogenome of the olive complex (Figure 1), with the highest content of noncoding regions and repeats >10 kb. Contrary to the other accessions, the E3.1 mitogenome has lost sdh3 (coding for a subunit of the succinate dehydrogenase), a gene known to have been recurrently transferred to the nuclear genome in higher plants [START_REF] R E F E R E N C E S Adams | Evolution of mitochondrial gene content: Gene loss and transfer to the nucleus[END_REF]. Interestingly, two chimeric ORFs (one including a sdh3 segment) were also found to be specific to the E3.1 mitogenome (Figures 1 andS3). Such genes, and particularly the chimeric ORF including a portion of sdh3, could be involved in the olive CMS (Besnard et al., 2000). Moreover, among all mitochondrial genes here assembled for the 18 Oleaceae accessions, sdh3 is the only gene missing in one accession. The CMS associated with lineage E3 could thus be associated with a dysfunction of sdh3, normally involved in the mitochondrial electron transport chain [START_REF] Huang | Succinate dehydrogenase: The complex roles of a simple enzyme[END_REF]. Furthermore, a sdh3 pseudogene has also been suggested as a candidate involved in a CMS type of Nicotiana tabacum (Zhang, Li, Jia, Li, & Feng, 2013). The chimeric ORF including a portion of sdh3 thus appears as a good candidate for CMS in olive, but testing this hypothesis requires further analyses, in particular of gene expression and its potential consequence for pollen development and abortion [START_REF] Hanson | Interactions of mitochondrial and nuclear genes that affect male gametophyte development[END_REF].

| Insertion of plastid sequences in mitogenomes of the Oleinae subtribe

A high proportion of Oleinae mitogenomes (ca. 5%-10%; was probably integrated before the divergence between Gesneriaceae and Oleaceae. Such recurrent integrations of plastid sequences in the mitogenome have also been reported in other plant families (Hao & Palmer, 2009;Sloan & Wu, 2014). The use of mtpt sequences for the reconstruction of plant phylogenies has thus to be taken with caution, and their orthology needs to be confirmed beforehand. Our phylogenies strongly suggest that all mtpt regions of the olive complex derived from a common ancestor, but this was not confirmed within the Oleinae subtribe for two mtpt regions (i.e., mtpt2 and mtpt9; Figure S4). Surprisingly, mtpt regions of the olive tree show higher nucleotide diversity than their plastid homologous regions, but only seven parsimony-informative sites were detected in those regions. This faster evolution in mtpt regions may at least partially reflect a relaxation of selective pressures on these sequences that mainly contain nonfunctional genes after their integration to the mitogenome (Sloan & Wu, 2014).

| Comparative phylogenetic reconstruction of

Oleeae based on plastid and mitochondrial genomes

Due to the difficulty in reconstructing full mitogenomes and the possibility that some mitochondrial noncoding regions may not be orthologous between distantly related taxa, we decided to reconstruct phylogenies of the Oleeae tribe using only conserved mitochondrial regions (i.e., protein-coding genes and introns). Fifty-two sequences (covering ca. 55 kb) were thus assembled from an annotated reference as reported in other studies (Govindarajulu et al., 2015;[START_REF] Mal E | Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family[END_REF][START_REF] Zhang | Congruent deep relationships in the grape family (Vitaceae) based on sequences of chloroplast genomes and mitochondrial genes via genome skimming[END_REF]. We then compared phylogenetic topologies based on conserved mitochondrial sequences and full plastomes and observed high congruence between these analyses, although some nodes were not resolved in the mtDNA tree (Figure 4). This suggests a strong linkage disequilibrium between DNA polymorphisms of both organellar genomes, as a consequence of their common maternal inheritance (Besnard et al., 2002;Liu et al., 2004). Our results also suggest that our ptDNA and mtDNA data may be appropriate for comparative phylogenetic studies on other Oleaceae lineages with potential biparental inheritance of organelles (Liu et al., 2004;Sodmergen et al., 1998). However, as similarly reported in other studies (Palmer & Herbon, 1988;Wolfe et al., 1987), such mitochondrial conserved regions evolved three to five times slower than plastid sequences in Oleinae and such a low variation could be insufficient to resolve certain nodes of the phylogeny (Figure 4b).

| Phylogenetic relationships between cytoplasmic olive lineages resolved using mitochondrial polymorphisms

Until now, phylogenetic relationships of Mediterranean plastid lineages have never been resolved despite repeated efforts (Besnard et al., 2002(Besnard et al., , 2007(Besnard et al., , 2011(Besnard et al., , 2013;;[START_REF] Qu Ezel | Systematics, ecology and phylogeographic significance of Olea europaea L. ssp. maroccana (Greuter & Burdet) P. Vargas et al., a relictual olive tree in south-west Morocco[END_REF], even using complete plastomes sequences. A rapid radiation of North African olive plastid lineages (E1, E2, E3, and M) in the Late Neogene may explain this difficulty (Besnard et al., 2013). Here, we were still unable to resolve phylogenetic relationships between the four Mediterranean cytoplasmic lineages based on new ptDNA sequences (Figures 5a andS5), due to the low number of informative sites in the plastome (Besnard et al., 2011). Yet, we resolved the phylogenetic tree (Figure 5b) using the complete mitogenome that contains 7.4 times more informative sites than the full plastome. This was unexpected, as the mitogenome is generally believed less informative than the plastome. In the tree topology, lineage E2 is sister

| CONCLUSION AN D PERSPECTIVES

Overall, our study confirms that the combined use of mtDNA and ptDNA is useful to investigate the evolutionary history of plants, opening new avenues of research in particular in the olive family. First, differential inheritance of organelles and nuclear-cytoplasmic incompatibilities have been reported in Oleaceae (Besnard et al., 2000;Liu et al., 2004;Sodmergen et al., 1998), and phylogenetic reconstruction with tests of selection may be performed on both organellar genomes to decipher the contrasted evolutionary histories and potential shifts in selective pressures on mitochondrial and/or chloroplast genes (e.g., Mensous et al., 2017;Zhu et al., 2014). Second, mtDNA polymorphisms may also be used in olive phylogeography. Here, the rapid radiation of maternal lineages of the Mediterranean olive was resolved for the first time with the use of mtDNA polymorphisms. This was potentially due to a higher number of selectively neutral informative sites in mtDNA (151 sites; when excluding the five nonsynonymous sites) than in ptDNA (15 sites; when excluding the six nonsynonymous sites), but a faster coalescence time of the mitogenome compared to the plastome (for instance due to a lower relative number of mitogenomes in reproductive cells) may also explain the better resolved topologies. Finally, the different genome compartments may be differently affected by postmortem damages, and their relative utility for ancient DNA analysis still needs to be tested. Our mitogenomic data are thus a new source of informative polymorphisms to investigate the spread of domesticated olive lineages (Besnard et al., 2013), in particular with metagenomic approaches to analyse subfossil remains from archaeological sites.

Introduction

The genome of eukaryotes is partitioned into different compartments in the cell. While the main part of the genome is located in the nucleus, some organelles (i.e. mitochondria and chloroplasts in plants) also contain their own genome, relic of bacterial endosymbioses (Keeling 2010;[START_REF] Martin | The origin of mitochondria[END_REF]Gray & Archibald 2012;Jensen & Leister 2014). Following the endosymbiosis events, mitochondrial genome (hereafter mitogenome) and chloroplastic genome (hereafter plastome) were drastically reduced and most of their genes were transferred to the nuclear genome [START_REF] Bock | Reconstructing evolution: gene transfer from plastids to the nucleus[END_REF]. Nuclear and organellar genomes have also co-evolved, especially due to their coordinated gene expression (mainly controlled by the nuclear genome) to ensure metabolic functions necessary for the proper functioning of the cell [START_REF] Greiner | Tuning a ménage à trois: co-evolution and co-adaptation of nuclear and organellar genomes in plants[END_REF]). Organellar genomes thus followed similar evolutionary pathways, for instance in their inheritance mode, and their strategies of gene expression and replication [START_REF] Greiner | Tuning a ménage à trois: co-evolution and co-adaptation of nuclear and organellar genomes in plants[END_REF]). An uniparental inheritance (usually maternal) also constrains the evolution of organellar genomes due to an absence of recombination. However, plastomes and mitogenomes of plants also generally show contrasted evolutionary rates and show distinct features particularly in their structural organization.

In flowering plants, the mitogenomes usually have a complex structure in subgenomic circles (i.e. multipartite structure; Gualberto et al. 2014). The numerous repeats [up to several kilobases (kb)] distributed in the mitogenome promote recombination events, leading to multiple and interchangeable configurations of the mitogenome. Moreover, mitogenomes of plants are very large and variable in size between species, or even at the intra-specific level. The size of angiosperm mitogenomes ranges from ~200 kb in Brassica rapa (Brassicaceae) to ~11 megabases (Mb) in Silene conica [Caryophyllaceae; Smith & Keeling (2015)]. These large variations of size are generally due to variable content of repeated sequences and noncoding regions in the mitogenome. The mitogenomes of plants contain approximately 30-37 protein-coding genes, 15-21 tRNA genes and three rRNA genes [START_REF] Clifton | Sequence and comparative analysis of the maize NB mitochondrial genome[END_REF][START_REF] Kubo | Male sterility-inducing mitochondrial genomes: how do they differ?[END_REF]. In contrast, the size, structure and organization of plastomes are typically very conserved in terms of size, structure and gene content. The typical plastome of angiosperms contains 66-82 protein-coding genes, 29-32 tRNA genes and four rRNA genes, and is usually represented as a circular molecule of ~150 kb in length (Jansen & Ruhlman 2012). Its structure is quadripartite including two long inverted, repeated regions (the Inverted Repeats, IR) separated by two single-copy regions [one small and one large region referred as SSC and LSC, respectively; [START_REF] Wicke | The evolution of the plastid chromosome in land plants: gene content, gene order, gene function[END_REF]]. Gene conversion between the IR contributes to the stability of the plastomes [START_REF] Khakhlova | Elimination of deleterious mutations in plastid genomes by gene conversion[END_REF].

In terms of sequence evolution, the rate of synonymous substitutions in mitochondrial genes is particularly low, being three to four times lower than in plant plastomes, 10-20 times lower than in plant nuclear genome and 50-100 times lower than in mammalian mitogenomes (Wolfe et al. 1987;Palmer & Herbon 1988). Deviations of these usual structural and evolutionary features have, however, been reported both in mitogenomes and plastomes. An extreme acceleration of the evolutionary rate has been described in organellar genomes of several lineages of flowering plants:

 Lineages of Silene (Sloan et al. 2012), Plantago (Cho et al. 2004), Ajuga (Zhu et al. 2014) and Geraniaceae [particularly Pelargonium (Parkinson et al. 2005;Park et al. 2017)] exhibit extreme accelerated rates of mitochondrial DNA (mtDNA) sequence evolution. In Geraniaceae, an increase of the substitution rate has been reported in at least two independent lineages, while reversions towards a low substitution rate have also occurred at least three times in the family (Parkinson et al. 2005). The acceleration of the substitution rate is usually homogeneous throughout the whole mitogenome, except for few cases such as the highly variable gene atp9 in most of Silene species [START_REF] Sloan | Phylogenetic analysis of mitochondrial substitution rate variation in the angiosperm tribe Sileneae[END_REF]).  Similarly, accelerations of the chloroplastic DNA (cpDNA) evolution have been reported in numerous lineages. Contrary to mitogenomes, only a portion of plastid genes exhibits high substitution rates. Genes coding for subunits of photosynthetic complexes (e.g. atp, pet, psa, psb, ndh, rbcL) et al. 2017;Park et al. 2017). In Lathyrus odoratus (Fabaceae), the evolutionary rate of ycf4 is also exceptionally high (Magee et al. 2010). These extreme accelerations of the substitution rates in ptDNA genes are usually correlated with genomic rearrangements of plastomes such as expansions/contractions of the IR, losses of genes and a high content of repeated sequences [START_REF] Jansen | Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns[END_REF][START_REF] Weng | Reconstruction of the ancestral plastid genome in Geraniaceae reveals a correlation between genome rearrangements, repeats and nucleotide substitution rates[END_REF]. First, large expansions of the IR have been reported, for instance, in plastomes of Pelargonium (Chumley et al. 2006), Plantago [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF], Campanulaceae (Cosner et al. 1997) and acacias (Mensous et al. 2017;Wang et al. 2017), while large contractions and even the complete loss of the IR have been described in Erodium [Geraniaceae, Guisinger et al. (2011)] and many Fabaceae [START_REF] Palmer | Chloroplast DNA evolution among legumes: loss of a large inverted repeat occurred prior to other sequence rearrangements[END_REF][START_REF] Lavin | Evolutionary significance of the loss of the chloroplast-DNA inverted repeat in the Leguminosae subfamily Papilionoideae[END_REF]. These large expansions and reductions of the IR resulted in the move of genes between the IR and the single-copy regions [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]. The synonymous substitution rate being ~3.4 fold lower in the IR compared to single-copy regions, the relocation of genes to the IR usually leads to a decrease of the substitution rate [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]. However, the lineages of Pelargonium, Plantago and Silene exhibit genes in the IR with a synonymous substitution rate higher (1.4-to 2.1-fold) than genes in single-copy regions [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]. Second, genes are often lost in lineages with accelerated evolutionary rate (for a review, see Jansen & Ruhlman 2012). Seven independent losses of accD are documented in the flowering plants, including lineages of Geraniaceae, Trachelium (Campanulaceae), Trifolium (Fabaceae) and Jasminum (Oleaceae). clpP has also been lost in several lineages such as Trachelium, Actinidiaceae or some Geraniaceae. Among the numerous gene losses reported in angiosperms, a gene relocation towards the nuclear genome has been demonstrated for rpl22 in pea [START_REF] Gantt | Transfer of rpl22 to the nucleus greatly preceded its loss from the chloroplast and involved the gain of an intron[END_REF], infA in Arabidopsis and soybean [START_REF] Millen | Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus[END_REF] It is generally acknowledged that the generation time (e.g. perennial versus annual plants), population size and speciation rate are involved in changes of the evolutionary rate [START_REF] Laroche | Molecular evolution of angiosperm mitochondrial introns and exons[END_REF][START_REF] Smith | Rates of molecular evolution are linked to life history in flowering plants[END_REF]. However, these factors cannot explain extreme accelerations of the substitution rate in organellar genomes. Localized hypermutation regions could be responsible of high variations in the synonymous rate (Parkinson et al. 2005;Magee et al. 2010;[START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]. Repeated DNA breakage and repair could lead to a hotspot of mutations (Guisinger et al. 2008(Guisinger et al. , 2011;;[START_REF] Yang | Hypermutability of damaged singlestrand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae[END_REF]. [START_REF] Sloan | Phylogenetic analysis of mitochondrial substitution rate variation in the angiosperm tribe Sileneae[END_REF] assumed that the extreme divergence of atp9 in the mitogenome of Silene could result from recombination between multiple copies of the gene, potentially associated with paternal leakage (i.e. occasional transmission of mitochondria via pollen). An association between accelerated substitution rates with large genomic rearrangements and potential biparental inheritance was also reported (Jansen & Ruhlman 2012).

Although the inheritance of organelles is predominantly maternal, paternal leakage has been observed in numerous lineages, both for mitochondria and/or chloroplasts (Corriveau & Coleman 1988;[START_REF] Zhang | Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species[END_REF]. The maternal inheritance of organelles could lead to the accumulation of the deleterious mutations (called Muller's ratchet) due to the absence of recombination [START_REF] Muller | The relation of recombination to mutational advance[END_REF]Greiner et al. 2014). It is assumed that paternal leakage could allow to counteract the Muller's ratchet (Greiner et al. 2014). The genomic consequences of the occasional transmission of organelles via pollen are, however, unclear. Paternal leakage could promote recombination between maternal and paternal organellar genomes (Greiner et al. 2014). While recombination between mitogenomes of plants have been demonstrated, recombination events between plastomes is less frequent, particularly due to the absence of chloroplastic fusion in the zygote (Greiner et al. 2014). Instead, gene conversion between cpDNA fragments could be a more likely process [START_REF] Goulding | Ebb and flow of the chloroplast inverted repeat[END_REF]. DNA repair processes via gene conversion could generate errors in DNA synthesis because the recombinational DNA repair by double-strand-break (DSB) may have an higher error rate than the other repair processes as shown, for instance, in yeast [START_REF] Strathern | DNA synthesis errors associated with double-strandbreak repair[END_REF]. In addition, the DSB of one IR followed by the recombinational repair against the complementary IR could result in large expansions of the IR [START_REF] Goulding | Ebb and flow of the chloroplast inverted repeat[END_REF].

A possible scenario of an accelerated substitution rate is that gene conversion by DSB between maternal and paternal organellar copies could generate errors in DNA synthesis. Moreover, the possibility of heteroplasmy with recombination and/or gene conversion between organelles of both parents could relax some constraints and, for instance, allow DNA replication enzymes to make more errors. Both these potentially error-prone processes should lead to an increase of the mutation rate. Several changes in selective pressures could result from a rise of the substitution rate: (i) a reinforcement of the purifying selection in order to eliminate deleterious mutations, (ii) a relaxation of the selection due to a new evolutionary context (i.e. organelle inheritance), and (iii) positive selection allowing deleterious mutations to be compensated by other mutations. This last process could provide the opportunity to reach new evolutionary optimums. Selection could operate in order to promote the organellar genomes compatible with the nuclear genome [START_REF] Weng | Reconstruction of the ancestral plastid genome in Geraniaceae reveals a correlation between genome rearrangements, repeats and nucleotide substitution rates[END_REF]. Biparental inheritance could allow to counteract the incompatibilities between the nuclear and organellar genomes [START_REF] Zhang | Why does biparental plastid inheritance revive in angiosperms[END_REF]). These expectations have, however, never been demonstrated and a first step to test these assumptions is to detect a correlation between paternal leakage and acceleration of the substitution rate.

Oleaceae is an excellent study system to investigate the link between paternal leakage and accelerations of the substitution rate with genomic rearrangements in organellar genomes, because both maternal inheritance and paternal leakage of organelles have been observed in this family. Maternal inheritance of both organellar genomes was documented in the olive complex (Olea europaea) based on the genetic characterization of cpDNA and mtDNA polymorphisms in progenies of controlled crosses (Besnard et al. 2000). In addition, Besnard et al. (2002) highlighted a strong linkage disequilibrium between these polymorphisms (despite intra-population diversity) also suggesting their strict maternal inheritance in the olive. A maternal transmission of chloroplasts and mitochondria has also been observed by microscopy in seven accessions of Syringa belonging to subgenus Syringa series Syringa and Pinnatifoliae (Corriveau & Coleman 1988;Liu et al. 2004). In contrast, potential paternal leakage has been reported in two lineages. Firstly, Liu et al. (2004) documented potential paternal leakage of the chloroplasts but a strict maternal inheritance of the mitochondria in 15 species of subtribe Ligustrinae (i.e. 12 Syringa species belonging to subgenus Ligustrina and subgenus Syringa series Villosae and Pubescentes, and three species of Ligustrum). These species belong to a monophyletic lineage of Ligustrinae meaning that the switch to potential paternal leakage has occurred in the ancestor of this lineage (Liu et al. 2004). Secondly, chloroplasts and mitochondria have also been observed in pollen grains of two distantly related species of Jasminum (J. officinale and J. nudiflorum) suggesting a paternal leakage of both organellar genomes in tribe Jasmineae (Sodmergen et al. 1998).

In this study, we investigated the genomic consequences of a paternal leakage in Oleaceae. We reconstructed phylogenies of 100 accessions covering the lineages of Oleaceae, based on complete plastomes and on coding and non-coding mtDNA regions. We detected changes in the evolutionary rate, both in cpDNA and mtDNA, by estimating rates of nonsynonymous and synonymous substitutions and testing the violation of the strict molecular clock. We then performed tests of positive selection in Jasmineae and a clade of Ligustrinae for each chloroplastic and mitochondrial genes, in order to test for changes of selective pressures.

Materials and methods

Taxon sampling

In this study, we analyzed 100 species representative of all tribes and sub-tribes of Oleaceae as defined by Wallander & Albert (2000). Firstly, we re-used 90 complete plastomes of Oleaceae generated by Olofsson et al. (in prep.). Compared to the taxon sampling used in Olofsson et al. (in prep.), we removed six accessions (Forestiera angustifolia, Nestegis cunninghamii, Olea europaea subsp. europaea E1-e.4, O. e. subsp. europaea E2.1, O. e. subsp. guanchica M-g1.3 and Phillyrea angustifolia) in order to have one accession per species. Secondly, we used the complete plastome [NC_015401] and mtDNA contigs of Fraxinus excelsior generated by Sollars et al. (2017). Lastly, we added nine species belonging to tribes Jasmineae, Forsythieae and Fontanesieae, including Nyctanthes arbor-tristis (tribe Myxopyreae) as an outgroup (Table 1). The position of tribe Myxopyreae as sister to all other Oleaceae tribes was reported by Wallander & Albert (2000) and confirmed by Zedane (2016). The nine additional samples were either collected from the field or herbarium collections. Organellar DNA sequencing and assembly Total genomic DNA of the nine species was extracted from ca. 5 mg of fresh or dried leaves. Each sample was ground in a 2-ml tube containing three tungsten beads with a TissueLyser (Qiagen Inc., Texas). The BioSprint 15 DNA Plant Kit (Qiagen Inc.) was then used to extract DNA, which was eluted in 200 µl of the AE buffer. We performed shotgun sequencing using a genome skimming approach as described in Zedane et al. (2016). Twenty four DNA libraries were multiplexed per flow cell and they were sequenced with an Illumina machine (i.e. HiSeq 2000, 2500 or 3000).

We reconstructed full plastomes of the nine species as described in Zedane et al. (2016). A verification of the assembly was performed by mapping all paired-reads against the obtained consensus sequence using GENEIOUS V.9.0.5 (Kearse et al. 2012) to check assembly quality and coverage depth. We annotated plastomes using GENEIOUS by transferring the annotations of an O. europaea plastome [FN996972]. Start and stop codons of protein-coding genes were manually adjusted. Then, we assembled 52 mtDNA regions (i.e. 36 protein-coding genes and 16 introns) for each of the 100 accessions. For each accession, we mapped all paired-end reads against the annotated mitogenome of H. palmeri [KX545367] using GENEIOUS following the method described in [START_REF] Van De Paer | Prospects on the evolutionary mitogenomics of plants: a case study on the olive family (Oleaceae)[END_REF]. To retrieve mtDNA regions of F. excelsior, we mapped assembled mtDNA genes and introns of F. angustifolia against the 26 mtDNA contigs of the library BATG-0.5-CLCbioSSPACE generated by Sollars et al. (2017) .

Phylogenetic analyses

We performed two phylogenetic analyses based on the complete plastomes and on the concatenation of the 52 newly assembled mtDNA regions. We aligned the plastomes and mtDNA regions using MUSCLE (Edgar 2004) with default parameters. Maximum likelihood (ML) analyses were then performed using RAXML (Stamatakis 2014) in the CIPRES platform (Miller et al. 2010). We applied the GTR+G model and the rapid bootstrap algorithm with 1,000 iterations.

Estimation of substitution rates and GC content

We extracted nucleotide sequences for all protein-genes from each plastome. Each chloroplastic and mitochondrial gene was aligned individually using MUSCLE with default parameters. The gene alignments were manually refined taking into account their amino-acid translation. We calculated non-synonymous and synonymous substitution rates (d N and d S ) using codeml implemented in PAML V.4.9 (Yang 2007) for (i) each chloroplastic and mitochondrial gene individually, and (ii) each concatenated dataset of genes classified per functional category. We estimated d N and d S for each accession against the outgroup (Nyctanthes).

As a paternal leakage was detected in Jasmineae (Sodmergen et al. 1998) and a monophyletic lineage of Ligustrinae [hereafter 'LVP': Ligustrum spp., plus Syringa subgenus Syringa series Villosae (Sy. yunnanensis) and Pubescentes (Sy. microphylla); Liu et al. 2004],

we tested for a difference in the substitution rates between these two clades and the other accessions of the tree. To compare the synonymous and the non-synonymous substitution rates between Jasmineae, LVP and the other accessions of Oleaceae (noted in the analyses 'Jasmineae', 'LVP' and 'Others', respectively), we performed a phylogenetic ANOVA between the three groups for each gene and each functional category with the function phylANOVA in the R package phytools [START_REF] Garland | Phylogenetic analysis of covariance by computer simulation[END_REF][START_REF] Revell | phytools: an R package for phylogenetic comparative biology (and other things)[END_REF]. We used the previous phylogenetic topology based on the complete plastomes to perform the analyses. We assigned Syringa vulgaris to 'Others' as strict maternal inheritance of chloroplasts was observed in this species (Corriveau & Coleman 1998;Liu et al. 2004).

We estimated the GC content [GC] on six datasets: (i) the complete plastomes and the concatenated coding and non-coding mtDNA regions, (ii) the concatenated chloroplastic and mitochondrial coding genes, separately, and (iii) the third position of codons of chloroplastic and mitochondrial genes, separately. For each of these three cases, we performed the estimations on (i) all 100 species of Oleaceae, (ii) the Oleaceae species without Jasmineae and LVP, (iii) the Jasmineae, and (iv) the LVP clade.

Models of molecular clock

In order to highlight different evolutionary rates in cpDNA and mtDNA between both clades Jasmineae and LVP and the other accessions of Oleaceae, we compared models of molecular clock using baseml implemented in PAML. First, we tested the violation of the strict molecular clock among the Oleaceae lineages without clades Jasmineae and LVP. We compared for each gene the strict molecular clock, which assumes a single rate for all lineages in the tree (noted clock1), and the relaxed molecular clock, which allows all branches to evolve independently in the tree (noted clock0). Second, we tested the assumption of ratechange events across both clades Jasmineae and LVP in the Oleaceae tree. We compared the strict and relaxed molecular clocks tested on the whole Oleaceae tree with the local molecular clock, which allows a distinct evolutionary rate to some lineages while all the other lineages evolved with a same evolutionary rate (noted clock2). To test the local molecular clock model, we assigned a priori changes of evolutionary rates in the Oleaceae tree: (i) on Jasmineae (noted clock2_Jasmineae), (ii) on the LVP clade (noted clock2_LVP), and (iii) on both Jasmineae and the LVP clade (noted clock2_Jasmineae_LVP). The molecular clock model that fits the data best was obtained given the lowest absolute Akaike Information Criterion (AIC). We considered that models were not significantly different if the difference between their AIC was inferior to six (ΔAIC < 6). We performed these analyses using the previous phylogenetic topology based on complete plastomes. To test the relaxed molecular clock (i.e. clock0), we used the unrooted cpDNA tree.

Positive selection tests

We analyzed changes of selective pressures in Jasmineae and LVP for each chloroplastic and mitochondrial gene. We performed the analyses with codeml implemented in PAML and using the phylogenetic topology based on complete plastomes. For each gene, we tested three codon models, which use the non-synonymous versus synonymous ratio (ω = d N / d S ; omega). The ratio ω allows to evaluate the selective pressures at the protein level, indicating purifying (ω < 1), relaxed (ω = 1) or positive (ω > 1) selection. The first model M1a is a site model allowing ω to vary among codons (model = 0, NSSites = 1). In this model, codons are either under relaxed selection (ω = 1) or under purifying selection (ω < 1; Yang et al. 2000). Second, the model A is a branch-site model, which allows ω to vary among both codons and branches of the phylogenetic tree (model = 2, NSSites = 2, fix_omega = 0; Zhang et al. 2005). Branch-site models allow distinct evolutionary rates along particular branches (called foreground branches) while a constant rate is assumed for all other branches of the tree (called background branches). The branch-site models need to assign a priori foreground branches for which positive selection is expected. In the model A, codons can be attributed to four types of selection: (i) purifying selection on the whole tree (ω < 1), (ii) relaxed selection on the whole tree (ω = 1), (iii) purifying selection on background branches (ω 0 < 1) and positive or relaxed selection on foreground branches (ω 2 ≥ 1), and (iv) relaxed selection on background branches (ω 1 = 1) and positive or relaxed selection on foreground branches (ω 2 ≥ 1). The last model A' is the same as model A, except that positive selection on foreground branches is replaced by relaxed selection (ω 2 = 1; model = 2, NSSites = 2, fix_omega = 1, omega = 1; Zhang et al. 2005). Individually for each lineage (i.e. Jasmineae and LVP), we tested two hypotheses: (i) positive selection operates on the internal branch ancestral to the clade (hypothesis H1; Fig. 1); and (ii) positive selection operates on all branches of the clade (hypothesis H2; Fig. 1). We removed the LVP clade from the phylogenetic topology to perform selection tests on Jasmineae; and similarly, we removed Jasmineae to perform selection tests on LVP. To identify the model that fits data best, we choose for each lineage the model with the lowest AIC between the five models (model M1a; models A and A' under the hypothesis H1; models A and A' under the hypothesis H2). We considered that the models were not significantly different if the difference between their AIC was inferior to six (ΔAIC < 6).

Results

Content and structure of organellar genomes First, we sequenced and assembled complete plastomes of nine accessions of Oleaceae. All the newly assembled plastomes and those generated by Olofsson et al. (in prep.) contained 80 protein-coding genes, except for those of Jasminum. As previously reported in J. officinale and J. nudiflorum (Lee et al. 2007), accD was absent from the four Jasminum plastomes that we reconstructed. Lastly, we assembled between 34 and 36 mtDNA protein-coding genes and between 14 and 16 mtDNA introns (Table S1). We excluded the mtDNA regions with a too low depth coverage, which did not allow us to reconstructed the complete mtDNA sequence.

The GC content was ~37% and ~46% across the complete plastomes and the concatenated mtDNA fragments, respectively; and was ~29% and ~37% at the third position of codons of concatenated chloroplastic and mitochondrial genes, respectively (Table 2). We did not detect any differences of the GC content between the Jasmineae and LVP clades and the other accessions of Oleaceae, whatever in cpDNA or mtDNA (Table 2).

Table 2. The GC contents [GC] estimated on all Oleaceae species (noted 'All'), on Oleaceae species without the Jasmineae and LVP clades (noted 'Others'), on the Jasmineae clade (noted 'Jasmineae'), on the LVP clade (Ligustrinae excluding Sy. vulgaris). We estimated GC contents on several datasets of cpDNA and mtDNA.

We observed rearrangements in plastomes of Jasmineae and Ligustrinae. The four Jasminum are highly rearranged with large inversions and multiple insertions/deletions, as previously described in other Jasminum species by Lee et al. (2007). In particular, we observed expansion or contraction of the Inverted Repeat (IR; Fig. S1). The IR of J. polyanthum exhibits both an extension of 4,662 bp into the SSC resulting in two complete copies of ycf1 in IR and a large reduction of 18,889 bp at the LSC/IR boundary, leading to the incorporation of 13 genes (including seven protein-coding genes, five tRNA genes and one rRNA gene) in the LSC (Fig. S1A). In J. kitchingii and J. fluminense, we also detected an expansion of the IR (of 4,695 bp and 4,708 bp, respectively) with a shift of ycf1 from the SSC to the IR (Fig. S1A). In Ligustrinae, we observed an extension (of ~6.5 kb) of the IR in all studied species, except in Sy. vulgaris that is sister to the LVP clade (Fig. S1B). This extension involved a shift of ycf1, rps15 and ndhH from the SSC to the IR. We also detected a IR extension of 918 bp at the IR/SSC boundary in Priogymnanthus hasslerianus (Oleinae).

Phylogenetic analyses

We performed an alignment of the 100 complete plastomes (179,063 bp, after excluding one IR), of which 12,718 (~7%) out of 24,550 (~14%) variable sites are parsimony-informative.

In addition, we aligned the 52 coding and non-coding mtDNA regions (58,585 bp). We detected 1,024 (~2%) parsimony-informative substitutions out of 2,095 (~4%) variable sites.

Parsimony tree topologies based on cpDNA and mtDNA were mostly congruent at the genus level, except for the slowly supported nodes (< 70%; Fig. 2). The cpDNA topology was strongly supported with > 95% bootstrap values at most nodes (Fig. 2A). Only phylogenetic relationships between closely related species were slowly supported (< 70%) in five cases. In contrast, the mtDNA topology was overall weakly supported, with a lot of bootstrap values inferior to 70% (Fig. 2B). In the cpDNA phylogeny, we observed long branches for Jasmineae and the LVP clade. The liana species C. macrobotrys has also evolved more rapidly than the other tree species of Oleinae. In the mtDNA phylogeny, long branches were observed for Jasmineae and for a pair of Noronhia species (N. intermedia and N. clarinerva).

Estimation of sequence divergence

We estimated non-synonymous and synonymous rates (d N and d S ) on the 80 cpDNA proteincoding genes (Fig. S2A). We found a significant difference between 'Others' and 'Jasmineae' and/or 'LVP' for d N in 35 genes and for d S in 17 genes (P < 0.05). These genes are distributed throughout the plastome and do not belong to a particular functional group. It appears that d N does not differ significantly between the three groups for the genes belonging to the functional categories atp, pet, psa and psb (except for atpA and psbB). A significant increase of d N and d S was detected between 'Jasmineae' and 'Others' for 34 genes (out of 35) and 13 genes (out of 17), respectively (P < 0.05). Similarly, a significant increase of d N and d S was detected between 'LVP' and 'Others' for 15 genes (out of 35) and 8 genes (out of 17), respectively (P < 0.05). Among these genes, d N was higher in both 'Jasmineae' and 'LVP' than 'Others' for 13 genes (out of 35; P < 0.05), while d S was higher in both 'Jasmineae' and 'LVP' than 'Others' for 5 genes (out of 17; P < 0.05). An extreme increase of d N and d S was observed on clpP in both 'Jasmineae' and 'LVP' compared to 'Others', and particularly between 'Jasmineae' ( N = 0.4852; S = 0.3070) and 'Others' ( N 0.0047); S = 0.0859). Similarly, we estimated d N and d S on the 36 mtDNA genes and we only detected a significant increase of d S between 'Jasmineae' and 'Others' in mttB (Fig. S2B).

We defined eight functional categories for chloroplastic genes [ATP synthase (atp), NADH dehydrogenase (ndh), cytochrome b 6 f (pet), photosystem I (psa), photosystem II (psb), RNA polymerase (rpo), and small and large ribosomal subunits (rps and rpl)] and seven functional categories for mitochondrial genes [ATP synthase (atp), cytochrome c maturation (ccm), cytochrome c oxydase (cox), NADH dehydrogenase (nad), succinate dehydrogenase (sdh), and small and large ribosomal subunits (rps and rpl)]. We detected a significant increase of d N and d S between 'Jasmineae' and 'Others' for five and six functional categories of chloroplastic genes, respectively (Fig. S3A). Similarly, we found a significant increase of d N and d S between 'LVP' and 'Others' for three and two functional categories of chloroplastic genes, respectively. No significant difference has been detected between 'Jasmineae', 'LVP' and 'Others' for functional categories of mitochondrial genes (Fig. S3B).

Models of molecular clocks

The test of strict molecular clock violation among the Oleaceae lineages without Jasmineae and LVP revealed that the hypothesis of a strict molecular clock (i.e. a constant evolutionary rate among lineages of the tree) is validated for all chloroplastic genes, except for two genes (rpoC2 and ycf1; Table S2.A). The assumption of a relaxed molecular clock, which assumes more than a single rate on the whole tree, fitted better to these two genes than the strict molecular clock. All the mitochondrial genes validated the assumption of a single rate among the lineages of the tree, except atp1 and atp6, which followed the hypothesis of a relaxed molecular clock among the Oleaceae lineages without Jasmineae and LVP (Table S2.B). We performed the same test for atp1 and atp6 after removing N. intermedia and N. clarinerva (for which an acceleration of the evolutionary rate occurred), but we also found that the relaxed molecular clock fitted better to these two genes.

By testing the assumption of more than a single rate across the Oleaceae tree including Jasmineae and LVP, we found that the local molecular clock fitted to 54 (71%) cpDNA genes (Table S3.A). We detected 23 genes for which a distinct rate is assumed for both clades Jasmineae and LVP, 29 genes for which a distinct rate is assumed to Jasmineae (or at least to Jasmineae, in the case of a non-significant difference between clock2_Jasmineae_LVP and clock2_Jasmineae) and two genes (accD and ycf15) for which a distinct rate is assumed to LVP (or at least to LVP, in the case of a non-significant difference between clock2_Jasmineae_LVP and clock2_LVP). We found that four genes followed the relaxed molecular clock. The molecular clock models were not significantly different for the other genes and did not allow us to conclude. For mitochondrial genes, only mttB followed the hypothesis of a distinct rate for both Jasmineae and LVP and three genes (cox1, cytB and rps3) followed the hypothesis of a distinct rate for Jasmineae (or at least for Jasmineae; Table S3.B). We found that the relaxed molecular clock fitted to atp1 and atp6. The molecular clock models were not significantly different for the other genes.

Positive selection

We tested the occurrence of positive selection in Jasmineae and LVP for the 80 chloroplastic genes (Table S4.A). The model A under the hypothesis H2, which assumes that positive selection occurred on all branches of the clade (i.e. Jasmineae or LVP), was significantly better than other models for nine genes (clpP, rbcL, rpoB, rpoC1, rps7, rps11, rps12, rps18 and ycf1) in Jasmineae (Tables 3 andS4.A). The model A under the hypothesis H1, which assumes that positive selection occurred on the internal branch of the clade, was significantly better than other models for atpF in Jasmineae and for matK in LVP (Tables 3 andS4.A). We also found that models A under both hypotheses H1 and H2 were not significantly different from each other for ycf2 in Jasmineae but were significantly better than the other models. We thus could assume that positive selection occurred at least on the internal branch of Jasmineae for this gene. The models were not significantly different for the other genes, which did not allow us to conclude. We performed similar analyses for the 36 mitochondrial genes (Table S4.B). Model A under the hypothesis H2 was significantly better than other models for cytB and nad5 in Jasmineae (Tables 3 andS4.B). We also found that models A under both hypotheses H1 and H2 were not significantly different from each other for ccmFc and rps3 in Jasmineae but were significantly better than the other models. The models for the other genes were not significantly different.

Discussion

Plastomes showing deep rearrangements and faster evolution have been reported in angiosperm species with paternal leakage [START_REF] Jansen | Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns[END_REF]Jansen & Ruhlman 2012). To test for possible switches in the evolutionary trajectory linked to potential biparental inheritance of organelles, the Oleaceae family is an interesting case study, since both strict maternal inheritance and paternal leakage of chloroplasts and/or mitochondria have been detected in this tree lineage. Here, the structural features and substitution rates were compared in plastomes and mitochondrial genes between lineages with an a priori strict inheritance of organelles (that however needs to be documented in most species) and two lineages (Jasmineae and the clade LVP of Ligustrinae) with potential paternal leakage. We also tested the changes in selective pressures occurring in these two latter lineages.

Variation of the structural features in plastomes of Jasmineae and a clade of Ligustrinae

As already reported by Lee et al. (2007), the plastomes of Jasmineae are highly rearranged with large inversions and multiple indels. In our study, an extension of the IR into the SSC (~5 kb) was detected in three species out four, as similarly revealed in J. nudiflorum (Lee et al. 2007). In addition, both an extension of the IR into the SSC and a large contraction (~19 kb) of the IR at the LSC/IR boundary were detected in the plastome of one species (J. polyanthum). Plastomes of J. fluminense and J. kitchingii sharing the IR extension of J. polyanthum, we assume that this extension is ancestral to the divergence of these three species. Interestingly, the LVP clade of Ligustrinae with potential paternal leakage of chloroplasts also exhibits an extension (~6 kb) of the IR into the SSC. The expansions of the IR in Jasmineae and Ligustrinae are not of the same extent and should thus be independent events. A third lineage showing a shorter extension (~1 kb) of the IR into the SSC was also detected in Priogymnanthus (Oleinae). While most small IR extensions (< 100bp) in land plants involve a shift of the IR/LSC boundary [START_REF] Goulding | Ebb and flow of the chloroplast inverted repeat[END_REF][START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF], both IR extension events in Oleaceae consist in an extension of the IR into the SSC. An extension of the IR with a shift of the IR/SSC boundary has also been described in acacias (Mensous et al. 2017;Wang et al. 2017), Plantago [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF], Trachelium (Cosner et al. 1997) and Silene (Sloan & Wu 2014) and successive extensions and contractions of the IR occurred in Pelargonium (Chumley et al. 2006) and Apiales (Downie & Jansen 2015). Here, it is noticeable that the two lineages with possible paternal leakage (Jasmineae and the clade LVP of Ligustrinae) also show deep structural reorganizations of plastomes suggesting that transitory heteroplasmy have favored plastomic rearrangements. [START_REF] Goulding | Ebb and flow of the chloroplast inverted repeat[END_REF] proposed a model involving a double-strand break (DSB) to explain an IR expansion at the boundary IR/SSC in Nicotiana. The DSB of one IR would be followed by strand invasion and gene conversion, which could lead to large expansions of the IR. The different features of the IR expansions observed in angiosperms could, however, result from various mechanisms still unknown [START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF].

The gene content was conserved among plastomes of Oleaceae except accD that is absent from our Jasmineae plastomes as previously reported by Lee et al. (2007) in this lineage. This gene encodes for the chloroplastic subunit of the acetyl-CoA carboxylase (ACCase), which is an essential enzyme implicated in the fatty acid synthesis (Magnusson et al. 1993). Plants contain two ACCase forms: an eukaryotic form and a prokaryotic form. While genes encoding for the eukaryotic-type ACCase are located in the nuclear genome, the prokaryotic-type ACCase comprises four subunits encoded by three nuclear genes (accA, accB and accC) and accD in the plastome [START_REF] Wakil | Fatty acid synthesis and its regulation[END_REF][START_REF] Magnuson | Regulation of fatty acid synthesis[END_REF][START_REF] Kode | The tobacco plastid accD gene is essential and is required for leaf development[END_REF]. The latter gene has been lost in multiple lineages of angiosperms (Jansen & Ruhlman 2012), but its functional transfer towards the nuclear genome has been demonstrated in Campanulaceae [START_REF] Rousseau-Gueutin | Potential functional replacement of the plastidic acetyl-CoA carboxylase subunit (accD) gene by recent transfers to the nucleus in some angiosperm lineages[END_REF] and Trifolium (Magee et al. 2010).

Acceleration of the evolutionary rate of chloroplastic genes in Jasmineae and the LVP clade of Ligustrinae

While the substitution rate of chloroplastic genes is usually slow with a low variation among lineages, our study reveals an acceleration of the evolutionary rate in plastomes of Jasmineae and the clade LVP of Ligustrinae. Again, the acceleration of the evolutionary rate in Ligustrinae occurred after the divergence from Sy. vulgaris, thus in apparent link to genomic reorganizations and potential paternal leakage mentioned above for this lineage. We thus hypothesize that transient heteroplasmy with possible gene conversion/recombination could make more allowable DNA polymerase errors and in turn change the selective pressures on some chloroplastic genes. While an acceleration of the non-synonymous substitution rate (d N ) is detected for more than one third (42%) of chloroplastic genes in Jasmineae, this d N acceleration is less pronounced in Ligustrinae (concerning 19% of chloroplastic genes). According the phylogeny, the event responsible of the acceleration of the evolutionary rate seems to be more ancient in Jasmineae than in the clade LVP of Ligustrinae, which could explain the lower number of genes exhibiting a significant change of d N in Ligustrinae. A similar increase of d N in chloroplastic genes has been described in Geraniaceae (Guisinger et al. 2008;Park et al. 2017), Silene (Sloan et al. 2012) and acacias (Mensous et al. 2017). This acceleration of d N could reflect a relaxation of the purifying selection or adaptive selection for chloroplastic genes which are involved in various functions, but most affected genes are not involved in the photosynthesis metabolism. Considering that the reading frame of fast evolving genes is probably maintained over a few millions of years in Jasmineae and the clade LVP of Ligustrinae, we consider that genes are still functional. One process could result in such deep changes of selective patterns. The acceleration of the mutation rate could lead to the fixation of slightly deleterious mutations that need to be compensated by other mutations (i.e. involving epistatic interactions) positively selected (e.g. "Red Queen" hypothesis). In this case, evidence of positive selection should be detected.

Positive selection in chloroplastic genes

Ten genes were significantly affected by positive selection in Jasmineae. These genes include ycf1, ycf2, and clpP which have also evolved quickly in multiple lineages [START_REF] Haberle | Extensive rearrangements in the chloroplast genome of Trachelium caeruleum are associated with repeats and tRNA genes[END_REF]Sloan et al. 2012;[START_REF] Gurdon | Two distinct plastid genome configurations and unprecedented intraspecies length variation in the accD coding region in Medicago truncatula[END_REF][START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]Mensous et al. 2017;Park et al. 2017). The exact function of the highly diverging ycf1 and ycf2 remains however unclear, but they are essential for the cell survival, being probably involved in the cellular metabolism or in cellular structure [START_REF] Drescher | The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes[END_REF]. More interestingly, clpP (also known as clpP1) exhibits an extreme acceleration of d N and d S in Jasmineae (and also, at a lower extent, in the LVP clade). This gene encodes for a serine protease, which is a subunit of the Clp protease [START_REF] Maurizi | ClpP represents a unique family of serine proteases[END_REF]). The chloroplastic gene clpP has an essential function in the cell survival and growth [START_REF] Ramundo | Conditional depletion of the Chlamydomonas chloroplast ClpP protease activates nuclear genes involved in autophagy and plastid protein quality control[END_REF]. A link between the paternal leakage and a rapid evolution of clpP (including a loss in Actinidia) seems to occur (Jansen & Ruhlman 2012;Wang et al. 2016), but it has been shown that the gene expression levels of plastid-targeted Clp proteins are very low or absent in the mature pollen suggesting that clpP does not determine the potential paternal inheritance of chloroplasts [START_REF] Olinares | The Clp protease system; a central component of the chloroplast protease network[END_REF]. A possible experiment to test a possible role of clpP in the plastid paternal leakage should be to compare the expression levels of the gene at different stages of the pollen development between lineages with differential inheritance mode of chloroplasts (maternal vs. biparental).

Slow evolution of mitochondrial genes

The presence of mitochondria in the mature pollen of Jasmineae suggested a potential paternal leakage of mitochondria in this lineage (Sodmergen et al. 1998). However, our study does not reveal clear evidence for evolutionary trajectory changes of mitochondrial genes in Jasmineae. Although a change of the global substitution rate occurred for three mtDNA genes (cox1, cytB, rps3) in Jasmineae and for mttB in Jasmineae and the LVP clade of Ligustrinae, their d N and d S are too low to conclude to a significant acceleration of the evolutionary rate for these genes. Only mttB exhibits a significant increase of d S in Jasmineae. The function of mttB is unclear and it probably encodes for a protein translocase (Mower et al. 2012). Four genes (ccmFc, cytB, nad5 and rps3) are, however, detected to be under positive selection in Jasmineae. Besides this, the phylogenetic reconstruction of the Oleaceae based on coding and non-coding mtDNA regions also revealed long branches for a pair of Noronhia species (N. intermedia and N. clarinerva). These two species were sampled in the same forest patch, located in a diversity hotspot of genus Noronhia in northern Madagascar, where tens of species co-occur (Hong-Wa 2016). We can just hypothesize that frequent hybridizations between sympatric species may influence some nucleo-cytoplasmic genome interactions and in turn could result in an accelerated divergence rates of mitochondrial genes.

Conclusions and perspectives

Our study highlights an acceleration of the evolutionary rate in the plastomes of Jasmineae and a clade of Ligustrinae, in which potential paternal leakage occurs. Most chloroplastic genes of Jasmineae are highly divergent. Contrary to plastomes, the mitogenome of both lineages evolves slowly and does not seem to experience a significant increase of the evolutionary rate. Our results add evidences of a link between paternal leakage and genomic rearrangements with a faster evolution of chloroplastic genes (particularly accD, clpP, ycf1 and ycf2). However, the cause and effect relationship between these processes are still unclear. More investigations are necessary to define the role of gene conversion between maternal and paternal copies of organellar genomes on the substitution rate change and on genomic rearrangement in these lineages. To detect adaptive selection on genes, further analyses could be also considered using mutation-selection models (Rodrigue & Lartillot 2016). In addition, the mechanisms of chloroplast degradation in the case of a maternal inheritance are poorly understood in plants. The confirmation by controlled crosses of a real paternal inheritance of organelles are thus necessary in the cases where we suspect a paternal leakage only based on microscopic observations. Also, the detection of potential paternal leakage of organellar genomes should be investigated in a larger taxonomic sampling in Oleaceae. Last, the interactions between the nuclear and organellar genomes remain little known. With the expansion of new NGS technologies producing long reads with high sequencing coverage, we can also imagine that more nuclear genome will be soon sequenced, and that the study of the interactions between genomes will be more accessible.

DISCUSSION ET PERSPECTIVES

Durant ce travail de thèse, j'ai pu aborder différents aspects sur la diversité et l'évolution des génomes cytoplasmiques. Tout d'abord, l'assemblage des tout premiers mitogénomes d'Oleaceae nous a permis d'étudier leur diversité structurelle mais aussi leur origine complexe, notamment en relation avec des transferts récurrents de régions plastidiques. Pour la première fois, des séquences mitochondriales et plastidiques ont été assemblées sur un large échantillonnage d'Oleaceae, ce qui nous permet désormais d'analyser l'histoire évolutive parallèle de ces génomes cytoplasmiques ouvrant de nombreuses perspectives de recherches, aussi bien appliquées que fondamentales. Nous pouvons spéculer que les approches développées dans ce travail vont fortement influencer les travaux sur la taxonomie et la biogéographie des Oleaceae, d'une part, et d'autre part, nos recherches devraient aussi stimuler de nouvelles recherches en biologie moléculaire et fonctionnelle des gènes cytoplasmiques chez les plantes en général.

I/ Mitogénome et phylogénomique des plantes 1. Reconstruction d'un mitogénome complet de plante à partir d'ADN ancien

Tout d'abord, la reconstruction du mitogénome complet du genre éteint Hesperelaea (CHAPITRE I) ouvre de nouvelles perspectives dans l'analyse d'ADN provenant d'échantillons muséologiques pour étudier l'histoire évolutive de groupes de plantes. A partir d'un spécimen d'herbier récolté en 1875, nous avons reconstruit le mitogénome d'H. palmeri (~658 kb) en utilisant des données de type "shotgun". L'utilisation de telles données génomiques peut permettre d'éclaircir le statut taxonomique d'un spécimen et de résoudre ses relations phylogénétiques avec d'autres taxa (Bakker et al. 2016 ;Zedane et al. 2016). Par ailleurs, l'intégration d'espèces ou genres rares ou éteints a une importance majeure dans les études biogéographiques de groupes de plantes. Afin d'expliquer la distribution géographique actuelle des espèces, un échantillonnage exhaustif des différents clades du groupe de plantes étudié est généralement nécessaire pour inférer des hypothèses de dispersion, de temps de divergence et d'aires ancestrales de distribution des espèces. L'intégration dans la phylogénie d'espèces rares issues d'herbiers peut également être utilisée dans l'étude de traits fonctionnels ou de traits d'histoire de vie. Par exemple, l'inclusion de spécimens d'herbier de Sartidia dans l'étude phylogénétique des Poaceae a permis de révéler deux transitions indépendantes de la photosynthèse C 3 vers C 4 chez la sous famille des Aristidoideae et même d'identifier quelques bases génétiques associées à ces transitions (Christin et al. 2008 ;Besnard et al. 2014). Aussi, l'utilisation d'ADN provenant d'échantillons d'herbier peut permettre d'analyser les variations de fréquences de certains allèles dans un passé récent. Par exemple, la substitution responsable de la résistance aux herbicides chez l'ACCase de certaines graminées a été identifiée sur un spécimen d'herbier récolté en 1888, i.e. avant l'utilisation d'herbicides dans l'agriculture intensive moderne [START_REF] Délye | DNA analysis of herbarium specimens of the grass weed Alopecurus myosuroides reveals herbicide pesistance pre-dated herbicides[END_REF].

La reconstruction de mitogénomes ou de plastomes complets d'échantillons d'herbiers dépend en majeure partie de la qualité de l'ADN et de son degré de dégradation. Un facteur important pour limiter la dégradation de l'ADN est d'optimiser le conditionnement de l'échantillon en herbier. La prolifération de bactéries et de champignons peut fortement contaminer l'échantillon. Pour éviter de telles contaminations, il est souvent essentiel de soumettre l'échantillon à une exposition à la chaleur ou au froid au moment de sa fixation afin d'éliminer les champignons contaminants et les arthropodes (Bakker et al. 2016). Une fois fixés, les échantillons sont protégés des rayons ultra-violets et conservés à des températures modérées et sous de faibles niveaux d'humidité (Staats et al. 2011). Les échantillons subissent généralement des cycles de refroidissement à -20 °C tous les deux ans (Staats et al. 2011). Au moment de la fixation, les échantillons provenant de régions tropicales humides nécessitent en particulier une exposition à la chaleur plus élevée afin d'éviter le développement de moisissure (Bakker et al. 2016). Néanmoins, l'exposition d'échantillons à de fortes températures (60-70°C) est susceptible de provoquer des cassures double brin de l'ADN, résultant à la fragmentation de l'ADN (Bakker et al. 2016). Staats et al. (2011) ont montré que les niveaux de fragmentation de l'ADN sont équivalents entre l'ADN nucléaire et l'ADN des organites. En particulier, il n'y a pas de différences en termes de fragmentation entre l'ADN mitochondrial et l'ADN chloroplastique. Néanmoins, l'ADN chloroplastique d'herbier présente des niveaux élevés de transitions C→T/G→A comparé à l'ADN chloroplastique frais (Staats et al. 2011). Ces transitions semblent être en partie dues à la désamination hydrolytique de la cytosine en uracile, possiblement liée à l'humidité ambiante (Staats et al. 2011). A la différence de l'ADN chloroplastique, l'ADN mitochondrial n'est pas soumis à de telles transitions et pourrait ainsi constituer une ressource de plus grand intérêt dans les études d'ADN ancien, par exemple comme nous l'avons mentionné dans le chapitre II pour des analyses archéo-génétiques de restes d'olivier.

Assemblage de mitogénomes de plantes

Dans le CHAPITRE II, nous avons ensuite reconstruit le mitogénome complet de Chionanthus rupicolus et de six accessions d'Olea europaea. Notre étude a confirmé que les mitogénomes de plantes sont très variables en termes de structure à l'échelle interspécifique, et surtout entre lignées maternelles d'une même espèce. Les mitogénomes des différentes lignées d'O. europaea présentent de larges réarrangements génomiques (déjà mis en évidence par la méthode RFLP ; Besnard et al. 2000), dus à la dynamique structurelle inhérente aux mitogénomes de plantes. Par ailleurs, les mitogénomes d'O. europaea sont variables en termes de contenu en régions non-codantes. En particulier, le contenu en régions répétées est largement variable entre les mitogénomes des différentes lignées d'olivier. Les répétitions contenues dans le mitogénome sont responsables de la dynamique structurelle des mitogénomes de plantes. Notre étude a permis d'élargir nos connaissances quant à la structure des mitogénomes, et en particulier au degré de variabilité structurelle des mitogénomes entre lignées maternelles d'une même espèce. De plus, les huit mitogénomes complets reconstruits dans les CHAPITRES I et II constituent des ressources génomiques d'intérêt, pouvant servir de références pour reconstruire les mitogénomes ou des régions mitochondriales d'espèces proches. Enfin, la reconstruction des mitogénomes des différentes lignées d'O. europaea nous a permis d'identifier deux gènes chimériques potentiellement associés à un type de stérilité mâle cytoplasmique chez l'olivier. Besnard et al. (2000) ont mis en évidence dans des croisements contrôlés une stérilité mâle cytoplasmique (CMS) liée à une lignée maternelle d'olivier (appelée E3, ou MCK). Néanmoins, aucun gène mitochondrial n'avait été jusqu'alors identifié comme étant associé à la CMS. Dans notre étude, nous avons identifié deux cadres de lecture ouverts (ORFs) potentiellement liés à cette CMS par comparaison de mitogénomes. Ces deux ORFs sont chimériques, l'une contenant une portion de sdh3, l'autre une portion de rbcL dérivé du plastome. Bien que l'identification de ces ORFs par comparaison de mitogénomes est insuffisante pour véritablement confirmer que ces gènes sont associés à la CMS chez la lignée E3, plusieurs éléments nous laissent à penser que l'ORF chimérique portant une partie de sdh3 est un candidat intéressant pour la CMS. D'une part, parmi les gènes classiquement retrouvés dans la plupart des mitogénomes de Lamiales, sdh3 est le seul gène qui soit absent, et en particulier dans le mitogénome de la lignée E3, ce qui laisse à penser que la CMS pourrait être liée au dysfonctionnement de ce gène. D'autre part, un pseudogène de sdh3 est également suspecté comme un candidat impliqué dans une CMS chez Nicotiana tabacum (Zhang et al. 2013). Pour tester notre hypothèse, d'autres analyses sont bien évidemment nécessaires. On pourrait envisager d'effectuer une PCR quantitative pour mesurer le niveau d'expression des gènes chimériques de la lignée E3. D'autre part, on pourrait introduire un gène fonctionnel de sdh3 dans le mitogénome afin de détecter d'éventuelles conséquences sur la production de pollen (e.g. "complémentation" de la mutation), ce qui nécessiterait de maîtriser des technologies de modification génétique chez l'olivier.

La reconstruction de mitogénomes de plantes présente donc un intérêt majeur pour mieux comprendre et caractériser la diversité structurelle des mitogénomes, mais aussi pour identifier des gènes cytoplasmiques associés à des traits fonctionnels tels que la CMS. Néanmoins, la reconstruction de mitogénomes de plantes reste laborieuse. Jusqu'à aujourd'hui, aucune méthode automatisée et standardisée n'a été développée pour assembler et résoudre les complexités structurelles des mitogénomes de plantes. Des logiciels spécialisés dans l'assemblage de génomes d'organites tels que l'assembleur développé par [START_REF] Coissac | .04: A de novo assembler dedicated to organelle genome assembling[END_REF] arrêtent l'assemblage du mitogénome au niveau des régions répétées et des zones dérivées du plastome. Pour chacun des mitogénomes reconstruits dans notre étude, nous avons dû assembler manuellement ces régions en comparant avec les séquences homologues du plastome. La résolution des régions dérivées du plastome (dites mtpt) se base sur une faible divergence entre la région mtpt et la région plastidique homologue. La résolution de ces régions mtpt s'effectue alors en identifiant les reads présentant les substitutions en plus faible couverture, le mitogénome étant généralement représenté plus faiblement que le plastome dans le pool de reads généré par "genome skimming". Aujourd'hui, la reconstruction de mitogénomes complets de plantes peut être envisagée dans des études se limitant à quelques accessions mais est inadaptée pour des études à plus large échantillonnage taxonomique.

Néanmoins, le développement de technologies de séquençage à haut débit générant des reads toujours plus longs pourrait faciliter l'assemblage des mitogénomes de plantes dans les années futures (sur matériel frais). Le séquençage PacBio présente déjà des performances intéressantes pour résoudre les problématiques rencontrées dans l'assemblage des mitogénomes. Le séquençage PacBio est considéré comme une méthode de séquençage en temps réel, qui permet de générer des reads beaucoup plus longs avec des runs plus rapides que les autres méthodes de séquençage [START_REF] Rhoads | PacBio sequencing and its applications[END_REF]. Cependant, le séquençage PacBio génère un taux d'erreurs important et le coût par base reste élevé. On pourrait néanmoins envisager de combiner le séquençage de type "genome skimming" et le séquençage PacBio (ou une autre méthodologie générant des longs reads) pour résoudre les difficultés d'assemblage du mitogénome des plantes.

Origines des régions non codantes dans les mitogénomes de plantes

Les mitogénomes reconstruits dans notre étude contiennent environ 90% de régions intergéniques non codantes dont des séquences répétées (entre ~5 et 20% du mitogénome). Cependant, l'origine de ~70% du mitogénome reste inconnue. Dans le CHAPITRE II, nous avons mis en évidence des intégrations récurrentes de régions d'origine chloroplastique dans le mitogénome (mtpt), ce qui rejoint les résultats d'études précédentes [START_REF] Wang | Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA[END_REF]Sloan & Wu 2014). Afin d'identifier les régions mtpt dans les mitogénomes, nous avons fixé un seuil minimum de similarité avec le plastome de 85%. Cependant, ce seuil permet d'identifier uniquement des événements d'intégration relativement récents de régions mtpt. Il est donc possible qu'une partie du mitogénome soit issue d'événements d'intégration de régions chloroplastiques beaucoup plus anciens, qui seraient désormais plus difficiles à détecter du fait d'une plus grande divergence avec le plastome. De la même façon, on peut supposer qu'il y a eu des intégrations de séquences d'origine nucléaire ou des événements de transferts horizontaux (e.g. fusions de mitogénomes) avec d'autres plantes comme déjà révélés chez Amborella trichopoda [START_REF] Bergthorsson | Massive horizontal transfer of mitochondrial genes from diverse land plant donors to the basal angiosperm Amborella[END_REF][START_REF] Rice | Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella[END_REF]. On peut également envisager qu'une partie des régions non codantes du mitogénome de plantes dérive de l'ADN de la bactérie endosymbiotique à l'origine de la mitochondrie. La longue divergence de ces séquences depuis ces événements d'endosymbiose rend désormais leur identification impossible. Enfin, les séquences répétées du mitogénome entraînent de nombreux événements de recombinaison au sein du mitogénome des plantes. Ces événements de recombinaison peuvent être à l'origine de duplications. Le mitogénome des plantes est donc potentiellement composé d'anciennes duplications ayant divergé au cours du temps.

Phylogénie à partir de données mitogénomiques.

Dans le CHAPITRE II, nous avons montré que l'utilisation des mitogénomes complets peut permettre de résoudre les relations phylogénétiques entre les lignées maternelles d'O. europaea, jusqu'alors non résolues à partir d'ADN chloroplastique. Bien que l'ADN mitochondrial évolue plus lentement que l'ADN chloroplastique, sa taille est beaucoup plus grande (~700 kb vs. ~150 kb) et peut donc contenir plus de sites informatifs pour résoudre certaines relations phylogénétiques. Néanmoins, l'utilisation de mitogénomes complets dans des études phylogénétiques nécessite une analyse préliminaire pour vérifier l'orthologie des régions mtpt présentes dans les mitogénomes. Notre étude a montré que l'orthologie des régions mtpt est vérifiée entre les lignées d'O. europaea, mais plusieurs événements d'intégration d'ADN chloroplastique dans les mitogénomes semblent avoir eu lieu à l'échelle de la tribu des Oleeae. Si il y a eu plusieurs événements d'intégration d'ADN chloroplastique, les régions mtpt ne sont pas nécessairement orthologues. Dans ce cas, il convient d'éliminer les régions mtpt de l'alignement avant toute reconstruction phylogénétique. Notre étude a également montré que la reconstruction phylogénétique basée sur des régions mitochondriales codantes et non-codantes relativement conservées confirme la topologie des Oleeae obtenue à partir de plastomes complets. La comparaison des topologies de reconstructions phylogénétiques basées sur l'ADN chloroplastique et mitochondrial présente surtout un intérêt majeur dans le cas d'une évolution contrastée du plastome et du mitogénome, comme c'est le cas chez les Oleaceae.

II/ Conséquences génomiques d'un "paternal leakage" des organites

Dans le CHAPITRE III, nous avons discuté d'un lien potentiel entre le passage occasionnel de chloroplastes dans le pollen ("paternal leakage") et de larges réarrangements du plastome associés à une accélération du taux d'évolution chez certains gènes chloroplastiques au sein des Jasmineae et d'un clade de Ligustrinae. Un tel lien a par ailleurs été détecté chez d'autres groupes de plantes tels que des lignées d'acacias (Mensous et al. 2017 ;Wang et al. 2017), de Silene (Sloan et al. 2012), de Campanulaceae (Cosner et al. 1997(Cosner et al. , 2004)), de Geraniaceae (Parkinson et al. 2005 ;Park et al. 2017) ou de Plantago (Cho et al. 2004). En dépit de ces observations similaires chez des groupes de plantes éloignés, la relation de cause à effet entre "paternal leakage" et réarrangements génomiques associés à une accélération du taux d'évolution reste mal comprise.

Lien entre "paternal leakage" et recombinaison

Bien que la transmission des organites est uniparentale et maternelle chez la plupart des plantes, la transmission occasionnelle d'organites par le pollen ("paternal leakage") a été observée indépendamment chez de nombreuses lignées de plantes (cf. Introduction). Une des raisons avancée pour expliquer le "paternal leakage" est l'évitement de l'accumulation de mutations délétères liée à l'absence de recombinaison (Greiner et al. 2014). En permettant la recombinaison (ou conversion génique) entre les copies maternelles et paternelles des organites, le "paternal leakage" pourrait contrecarrer le cliquet de Muller. Cependant, aucune preuve évidente de recombinaison entre les copies maternelles et paternelles des plastomes n'a été démontrée jusque maintenant. Le taux de GC ne varie pas entre les lignées à transmission maternelle des organites et celles présentant du "paternal leakage", suggérant peu de recombinaison [qui, dans le cas contraire, se traduirait par une augmentation du taux de GC; [START_REF] Marais | Biased gene conversion: implications for genome and sex evolution[END_REF]]. Cependant, les événements de recombinaison suite à du "paternal leakage" sont vraisemblablement des événements rares.

Identification des modes de transmission des organites au sein des Oleaceae

Dans notre étude, il serait nécessaire de déterminer le mode de transmission des organites chez davantage d'espèces. Nos conclusions seraient d'autant plus robustes si nous démontrons une transmission maternelle stricte des organites dans les différents clades d'Oleaceae, mis à part les Jasmineae et un clade de Ligustrinae pour lesquels on observe une accélération du taux d'évolution du plastome. Par ailleurs, il serait nécessaire d'effectuer des croisements contrôlés pour confirmer que la descendance peut hériter véritablement de la copie paternelle du plastome. En effet, le chloroplaste paternel peut être transmis mais pas forcément hérité. Le mode de dégradation des organites est peu documenté chez les plantes. En revanche, il a été montré que les mécanismes de dégradation des mitochondries dans le monde animal sont variés et peuvent intervenir avant ou après fécondation. Chez les mammifères par exemple, la copie paternelle du mitogénome entre dans le cytoplasme de l'oocyte après fécondation, avant d'être dégradée dans un second temps [START_REF] Sato | Maternal inheritance of mitochondrial DNA by diverse mechanisms to eliminate paternal mitochondrial DNA[END_REF].

Estimation de la fréquence du "paternal leakage" chez le troène en conditions semi-contrôlées

Bien que je n'ai pas présenté dans ce manuscrit cette expérimentation, nous avions envisagé de détecter du "paternal leakage" par croisements semi-contrôlés chez Ligustrum vulgare. Pierre Saumitou-Laprade et Philippe Vernet du laboratoire Evo-Eco-Paléo de Lille ont récolté des individus de L. vulgare provenant de huit populations naturelles relativement distantes en Europe. Cinq populations provenaient de localités situées dans le nord de la France (parfois distantes de plusieurs centaines de kilomètres), une population était située dans les Cévennes, une autre à Grenoble et une dernière était localisée en Allemagne. Ces différents individus ont été disposés en jardin expérimental et sans contrôle de la pollinisation. L'idée de notre expérimentation était d'identifier un individu ayant produit des graines dont le chlorotype était unique. Dans un tel cas, il serait possible de détecter dans les descendances un variant chloroplastique d'origine paternelle. Nous avons choisi 48 individus qui présentaient des génotypes différents (d'après des données microsatellites nucléaires), provenant des différentes populations. Nous avons séquencé deux fragments d'environ 600-700 bp d'ADN chloroplastique (un au niveau d'accD et un autre au niveau de clpP) présentant une grande variabilité interspécifique, et génotypé trois longs microsatellites [no 19, 22, 51 ;Besnard et al. (2011)]. Nous n'avons détecté aucune variabilité entre les individus de L. vulgare, ce qui ne nous a pas permis d'aller plus loin dans cette étude. En revanche, L. ovalifolium présentait de nombreux polymorphismes par rapport à L. vulgare. On suppose que ces espèces puissent s'hybrider. Une expérimentation qui serait à envisager serait donc de pulvériser du pollen de L. vulgare sur des individus de L. ovalifolium et de récolter les graines à l'automne. Si on détecte de l'hétéroplasmie chez les hybrides liée à la présence du chlorotype de L. vulgare, cela permettrait de confirmer sans ambiguïté qu'il existe du "paternal leakage".

Autres mécanismes que le "paternal leakage" pour limiter l'accumulation de mutations délétères

D'autres mécanismes que le "paternal leakage" pourraient permettre de limiter le fardeau de mutations délétères. Les travaux récents de Radzvilavicius et al. (2017) soulignent que l'hypothèse selon laquelle le "paternal leakage" permettrait de limiter l'accumulation de mutations délétères via de la recombinaison repose essentiellement sur des modèles théoriques de génétique des populations basés sur l'ADN nucléaire [START_REF] Felsenstein | The evolutionary advantage of recombination[END_REF][START_REF] Charlesworth | Mutation accumulation in finite outbreeding and inbreeding populations[END_REF]. Or, [START_REF] Radzvilavicius | Mitigating mitochondrial genome erosion without recombination[END_REF] remettent en cause l'application de telles hypothèses pour l'ADN mitochondrial. [START_REF] Radzvilavicius | Mitigating mitochondrial genome erosion without recombination[END_REF] précisent que, à la différence du génome nucléaire, plusieurs copies du mitogénome se situent dans un nucléoïde mitochondrial, que plusieurs nucléoïdes mitochondriaux se situent dans une mitochondrie et que plusieurs mitochondries se situent dans une cellule. Dans ce cas, la sélection contre les mutations délétères agit essentiellement sur les mutations qui ont un effet délétère sur la fitness de la cellule, i.e. au niveau du groupe de copies de mitogénomes dans la cellule. Or, à chaque division cellulaire, le groupe de copies de mitogénomes dans une cellule change du fait de la ségrégation stochastique. Une cellule fille n'aura pas le même groupe de copies de mitogénomes que la cellule mère, ce qui a pour conséquence d'augmenter la variance inter-cellulaire et donc de diluer le fardeau de mutations délétères. Une autre explication du maintien du "paternal leakage" chez certaines lignées de plantes à fleurs pourrait être liée à de la sélection adaptative.

"Paternal leakage" et évolution adaptative

Une de nos hypothèses pour expliquer l'augmentation du nombre de substitutions nonsynonymes dans des gènes plastidiques de Jasmineae et d'un clade de Ligustrinae est liée à une évolution adaptative. Comme exposé dans l'introduction du CHAPITRE III, la possibilité de recombinaison pourrait permettre à la sélection d'être plus efficace et en conséquence de relâcher la pression de sélection visant à préserver l'intégrité maximale de la séquence plastidique. Une augmentation du taux de mutation pourrait mener à une apparition plus fréquente de mutations délétères, mais en outre d'autres mutations qui les compenseraient pourraient aussi être positivement sélectionnées. Cette évolution accélérée avec la possibilité de mutations compensatoires pourrait offrir l'opportunité d'atteindre de nouveaux optimums adaptatifs. Afin de tester l'hypothèse selon laquelle le "paternal leakage" pourrait promouvoir l'évolution adaptative, nous envisageons, avec la collaboration d'Hervé Philippe, d'utiliser le modèle de mutation-sélection développé par Rodrigue & Lartillot (2016). Dans les modèles habituels tels que ceux utilisés par PAML, la sélection positive est détectée si ω > 1. Or, l'évolution adaptative peut opérer et se traduire par une augmentation du nombre de substitutions non-synonymes, sans pour autant que ω soit supérieur à 1. Dans leur modèle, Rodrigue & Lartillot (2016) définissent un modèle neutre qui infère un profil d'acides aminés optimal pour chaque codon. Ils introduisent un nouveau paramètre, ω*, qui absorbe toute déviation à ce modèle neutre. De l'évolution adaptative peut alors être détectée si ω* > 1.

CONCLUSION GENERALE

Ce projet de thèse a permis d'ouvrir de nouvelles perspectives dans l'utilisation des données mitogénomiques et chloroplastiques. Nous avons montré que la reconstruction de gènes ou de génomes d'organites à partir d'ADN ancien provenant de spécimens d'herbiers est possible, ce qui permettra d'inclure de nombreuses espèces rares dans les futurs travaux phylogénétiques des systématiciens et biogéographes. Par ailleurs, nous avons montré que l'utilisation des données mitogénomiques peut permettre de résoudre certaines relations phylogénétiques et ne reflète pas forcément la même histoire évolutive que l'ADN chloroplastique, signifiant que ces données pourraient être complémentaires dans certaines études. Enfin, nous avons mis en évidence un possible lien entre "paternal leakage" et réarrangements génomiques associés à une accélération du taux d'évolution. Néanmoins, la relation de cause à effet reste mal comprise. Ce travail de thèse pose une base pour de futures études sur les conséquences génomiques d'un "paternal leakage" et sur les interactions nucléo-cytoplasmiques. S1. List of repeat pairs predicted using VMATCH (Kurtz, 2016) in the mitogenome of Hesperelaea palmeri. We retained repeats with a 40-bp minimum length and with at least 95% of identity. (D: direct orientation; P: palindromic orientation) * Start codon is given in brackets for each protein-coding gene of Hesperelaea: Note that we reported 39 protein-coding genes with the standard ATG start codon. The ACG codon could be an alternative start codon in four protein-coding genes (atp6, nad4L, rps4 and rps10), as reported by [START_REF] Giegé | RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs[END_REF], [START_REF] Picardi | Large-scale detection and analysis of RNA editing in grape mtDNA by RNA deep-sequencing[END_REF] and Mower et al. (2012b). The codon initiating translation in sdh4 and rpl16 genes is GTG, as already suggested in other plant mitogenomes [START_REF] Bock | Rps3 and rpl16 genes do not overlap in Oenothera mitochondria: GTG as a potential translation initiation codon in plant mitochondria?[END_REF]. The ATG start codon is also not conserved in mttB and the TTG codon was an alternative start codon, as annotated in the mitogenome of Salvia miltiorrhiza (Mower et al., 2012a).

Table S4. List of ORFs detected with GENEIOUS v. 9.0.5 (Biomatters Ltd., 2015) in the mitogenome of Hesperelaea palmeri. We retained ORFs with a minimum length of 300 bp. a Average coverage depth of mitogenomes was estimated excluding mtpt regions; b N50 was calculated on single-copy mitochondrial contigs (excluding mtpt regions) after the initial step of the mtDNA assembly. This statistic defines the assembly quality in terms of contiguity. The N50 length (in bp) is defined as the shortest sequence length at 50% of the genome. The number in brackets corresponds to the number of contigs superior or equal to N50; c Note that the percentage of merged reads is higher in the four herbarium accessions (>64% of paired-end reads) than in DNA extracted from fresh material (<30%). This is due to the more fragmented DNA usually extracted from herbarium specimens. Table S2. List of individuals from GenBank used to reconstruct the phylogenetic tree in Fig. S5. Olive plastid DNA lineage and haplotypes are given (according to Besnard et al. 2007Besnard et al. , 2018)).

Species and origin of individuals GenBank no Plastid lineage, and haplotype

Olea europaea L. subsp. europaea -Manzanilla de Sevilla, Spain

+ + + + + + Pseudogenes ᴪ-ccmC - - 2+ 2+ 2+ + - orf_E3a - - - 2+ - - - orf_E3b - - - + - - -
a Two copies of exons 4 and 5; b Two copies of exon 4; * Chloroplast-derived protein-coding and tRNA genes Table S5. Estimates of the nucleotide diversity (π in %; Nei & Li 1979) among the olive complex for the nine mtpt regions (Fig. 2) and their ptDNA homologous regions. The standard error of π was estimated by bootstrapping on sequences (100 replicates).

C T A A C A T G G T T T G C G T T G G T O. e. europaea E3.1 A A T A A A A T G G T G T G C G T T G G T O. e. guanchica M-g1.3 A C T A A A C T G G G G T G C G T T T G T O. e. europaea E2.1 A A T A A A C T G G G T T G A G T T G G T O. e. europaea E1-e.4 G A G G G C C G A A G T G A A A A A T C A O. e. laperrinei E1.l1.1 G C G G G C C G A A G T G A C A A A T C A Gene location rpoC2 psbG ndhC petA rpl14 rps3 rps3 ndhF ndhF ndhD ycf1 ycf1
Position in codon 2 nd 3 rd 3 rd 3 rd 3 rd 1 st 3 rd 2 nd 2 nd 3 rd 2 nd 3 rd Non syn. changes

L/F L/F N/H W/L A/V T/S
Table S8. Nucleotide polymorphisms at the 156 parsimony informative sites among the mitogenomes of the olive complex. Sites are numbered according to their position in the mitogenome of O. e. laperrinei E1-l1.1. When the site is located in a coding sequence, the gene name, the position of the substitution in the codon and the amino-acid change for non-synonymous substitutions are given. We also indicate sites that were detected in mtpt regions.

G G T C -* A G T C G G G G A G T C A G C A G A G C T T C G T O. e. europaea E3.1 T T T C A C T T C - - G G A G T A A G C A G A A C T A A G A O. e. guanchica M-g1.3 G G T C A A T T G G G G G A G T C A G G A G A A G T T C T A O. e. europaea E2.1 T T T A A C G A G G A - - G G T A A T G C A G T C T A A T T O. e. europaea E1-e.4 G T G A T C G A C T A A T G C A C C T C C A G T G G A C T A O. e. laperrinei E1-l1.1 G T G A T C G A C T A A T G C A C C T C C A G T G G A C T A Gene location cox3 Position in codon 1 st
Non syn. changes L/I * "-" means that the region is missing

mtpt mtpt O. e. cuspidata C2.13 C A G T A T C A A C C A A T A T T T - T A C T G G A T C C G O. e. europaea E3.1 A A G G T G C C A A C A A T A T T T A T A C T G T A T G C T O. e. guanchica M-g1.3 A A G G A G A C A C C A C C G T T - A T A C T T G C T G C G O. e. europaea E2.1 A A G T A T A A G A C A A C G T G T A T A C G T G C T G C T O. e. europaea E1-e.4 A C T G A G C A G A A C C C G A G C C G C A G G T A G G A T O. e. laperrinei E1-l1.1 C C T G T G C A G A A C A C G A G C C G C A G G G A G C A T
G T C T A G T G G T C C G T C T T C T C T G A A C G T A T T O. e. europaea E3.1 G T A T C G G G G T C A G T C T T C T C T G C A A G T A T G O. e. guanchica M-g1.3 G T A T A G T T T T C A G T C T T A T A A G C A C G T C T T O. e. europaea E2.1 G T A T A C G - T T C C G T C T - - T A A G C T A T T C T G O. e. europaea E1-e.4 T G A C C C G T T A A A T A A C C A G C A T A T A T G C G G O. e. laperrinei E1.l1.1 T G C C A C G T T A A C T A A C C C G C A T C T A T G C G G
Gene location Position in codon Non syn. changes (Krzywinski et al. 2009). Both phylogenetic trees were reconstructed with RAXML V.8 (Stamatakis 2014) using a GTR + G model, applying the rapid bootstrap algorithm with 1,000 iterations. We rooted the phylogenetic tree at midpoint. S5. ML phylogenetic tree of the olive complex based on complete plastomes. We reconstructed a phylogenetic tree based on plastomes of the six newly sequenced accessions and eight Olea accessions from GenBank. O. woodiana is used as outgroup to root the tree. The phylogenetic tree was obtained using RAXML V.8 (Stamatakis 2014) with a GTR + G model. We applied the rapid bootstrap algorithm with 1,000 iterations.

G T T G A C G T C G G G T C C A G C A G G C C C A A G C T O. e. europaea E3.1 C C T A G C C G T C G G G T C C A G C A G G C C A C A G C G O. e. guanchica M-g1.3 C G T A T C G C - - G G G G A C T T C A G G C A C C T T A G O. e. europaea E2.1 A C T T T C C G G C T T G G A A T T C A T T A A A A T T A T O. e. europaea E1-e.4 A C A T T A C G G T G T T G C A T T A C T T A A A C T T A G O. e. laperrinei E1.l1.1 A C A T T C G C G T T T T G A A T T A C T T A A A C T T A G
G C T C T G A C C G A A C A C T C G T G C G A A A C A C O. e. europaea E3.1 A G A G C T G A C A T T C C A C T C G T G A G A C A C A G O. e. guanchica M-g1.3 A G C G A T T C C A G A A C A C T C G G A C G A C A A - - O. e. europaea E2.1 A G A T A T G A A C G A A C A C T A G T A C G T A T A A C O. e. europaea E1-e.4 C C A T A G T C A C T T C A G T A A T T A T T T A T A C G O. e. laperrinei E1.l1.1 C C A T A G T C A C G T C A G T A A T G A T T T A T A C G
Table S2. Results of the strict molecular clock violation tests for cpDNA (Table S2.A) and mtDNA (Table S2.B) genes. We performed these tests on Oleaceae accessions without clades Jasmineae and LVP, except for Sy. vulgaris. To determine the model which fitted better to data, we compared AIC between the model of strict molecular clock (noted clock_1) and the model of relaxed molecular clock (noted clock_0). We considered that models were not significantly different if the difference between their AIC was inferior to six (ΔAIC<6). For each gene, the model with the lowest AIC value was indicated in bold and highlighted in gray.

Table S2.A Table S3. Results of local molecular clock tests for cpDNA (Table S3.A) and mtDNA (Table S3.B) genes. We performed these tests on all 100 Oleaceae accessions. To determine the model which fitted better to data, we compared AIC between the model of strict molecular clock (noted clock_1), the model of relaxed molecular clock (noted clock_0) and the three models of local molecular clock which assume a distinct evolutionary rate (i) in both clades Jasmineae and LVP (clock2_Jasmineae_ LVP), (ii) in clade Jasmineae only (clock2_Jasmineae), (iii) in clade LVP only (clock2_ LVP). For each gene, models with the lowest AIC value was indicated in bold and highlighted in gray. We considered that models were not significantly different if the difference between their AIC was inferior to six (ΔAIC<6).

Table S3.A Fig. S1. Physical maps of the inverted repeats (IR1 and IR2) and the small single copy (SSC) of the plastomes of Nyctanthes arbor-tristis, Forsythia intermedia and species belonging to clades Jasmineae (Figure S1.A) and Ligustrinae (Figure S1.B). These maps were drawn with OGDRAW (Lohse et al. 2007). On the right, IR and SSC sizes are given in bp for each species.

Introduction

Acacias (Fabaceae, Mimosoideae) represent a major group of woody legumes in arid and subarid habitats of tropical and subtropical regions, particularly in the Southern hemisphere [START_REF] Lewis | Legumes of the world. Royal Botanic Gardens, Kew L o h s e M , D r e c h s e l O , K a h l a u S , B o c k R ( 2 0 1 3 ) OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[END_REF]. They are highly diversified with ca. 1350-1450 species around the world and ca. 130-150 species in Africa [START_REF] Ross | An analysis of the African Acacia species: their distribution, possible origins and relationships[END_REF][START_REF] Maslin | Overview of the generic status of Acacia (Leguminosae: Mimosoideae)[END_REF][START_REF] Lewis | Legumes of the world. Royal Botanic Gardens, Kew L o h s e M , D r e c h s e l O , K a h l a u S , B o c k R ( 2 0 1 3 ) OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[END_REF]. Acacias are an important source of wood (for fuel and lumber), fibers (to produce ropes), or medicines for human populations and of forage for wild and domestic animals (Le [START_REF] Floc | Acacia raddiana, un arbre des zones arides à multiples usages[END_REF][START_REF] Lewis | Legumes of the world. Royal Botanic Gardens, Kew L o h s e M , D r e c h s e l O , K a h l a u S , B o c k R ( 2 0 1 3 ) OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[END_REF][START_REF] Hobbs | Acacia trees on the cultural landscapes of the Red Sea Hills[END_REF]. In addition, they can be used to fight against the desertification Mohamed Mensous and Céline Van de Paer contributed equally to this work.

Communicated by G. G. Vendramin

Electronic supplementary material The online version of this article (doi:10.1007/s11295-017-1131-2) contains supplementary material, which is available to authorized users. and soil erosion, for instance in the Sahelo-Saharan region where Vachellia and Senegalia spp. have been planted on a large scale as part of the BGreat Green Wall^initiative (FAO 2014). Their ability to fix nitrogen, thanks to rhizobia in root nodules, can also favor the growth of other plants, especially for cereal crops in traditional agrosystems [START_REF] Payne | Crop diversification in the Sahel through use of environmental changes near Faidherbia albida (Del) A Chev[END_REF]. In semi-desert conditions, acacias often dominate tree communities and several species can occur in sympatry, but taxon identification based on morphological criteria can be challenging, especially on saplings [START_REF] Brenan | The present state of taxonomy of four species of Acacia (A. albida, A. senegal, A. nilotica, A. tortilis). FAO UN, Rome Bukhari YM (1997) Cytoevolution of taxa in Acacia and Prosopis (Mimosaceae)[END_REF][START_REF] Doran | Guide des semences d'acacias des zones sèches: Récolte, extraction, nettoyage, conservation et traitement des graines d'acacias des zones sèches[END_REF]. A few species are widespread in the Central Saharan mountains. While some taxa are frequent such as Vachellia tortilis subsp. raddiana and Vachellia flava, others (as Vachellia nilotica, Faidherbia albida, and Senegalia laeta) have a very fragmented distribution (Table S1). In addition, the various uses of these woody species by autochthonous people strongly threaten the long persistence of some populations that may be locally endangered [START_REF] Quézel | La végétation du Sahara: du Tchad à la Mauritanie[END_REF][START_REF] Quézel | High mountains of the Central Sahara: dispersal, speciation, origin and conservation of the flora[END_REF][START_REF] Sahki | Guide des principaux arbres et arbustes du Sahara Central (Ahaggar et Tassili)[END_REF]CSFD 2004;[START_REF] Chenoune | La flore et la végétation du Hoggar[END_REF][START_REF] Baâli-Cherif | Etude des populations d'olivier de Laperrine (Olea europaea ssp. laperrinei) du Sahara Central Algérien (Hoggar et Tassili): aspects biologiques et caractérisation moléculaire[END_REF]. The inventory of such populations and the characterization of their genetic diversity are necessary to assist their conservation (FAO 1996). Knowledge on the population dynamic (e.g., turnover, life span of individuals and importance of clonal growth, seedversus pollen-mediated gene flow) is also essential for defining optimal strategies of management [START_REF] Borgel | Diversité génétique des acacias sahéliens: exploitation par les voies clonales et sexuée[END_REF]).

To date, population genetic studies have been performed only on a few African acacia species [e.g., F. albida [START_REF] Joly | Population-genetics of an African acacia, Acacia albida: 1. Genetic diversity of populations from West Africa[END_REF][START_REF] Harris | Isozyme variation in Faidherbia albida (Leguminosae, Mimosoideae)[END_REF], Senegalia mellifera [START_REF] Guajardo | Landscape genetics of the key African acacia species Senegalia mellifera (Vahl)-the importance of the Kenyan Rift Valley[END_REF], Senegalia senegal [START_REF] Odee | Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion[END_REF][START_REF] Assoumane | Highlighting the occurrence of tetraploidy in Acacia senegal (L.) Willd. and genetic variation patterns in its natural range revealed by DNA microsatellite markers[END_REF]]. Such studies also allowed unraveling phylogeographic patterns [START_REF] Guajardo | Landscape genetics of the key African acacia species Senegalia mellifera (Vahl)-the importance of the Kenyan Rift Valley[END_REF][START_REF] Odee | Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion[END_REF]) that can bring important insights for land managers by depicting the recent history of species, identifying barriers to dispersal and distinguishing main gene pools with putative hotspots of diversity (e.g., Besnard et al. 2007;Migliore et al. 2012). A prerequisite step to such studies is the development of molecular markers based on genomic sequences. Nuclear microsatellite markers have been developed on a few African acacia species, including V. tortilis [START_REF] Winters | Characterizations of microsatellite DNA markers for Acacia tortilis[END_REF][START_REF] Osmondi | Isolation and characterization of microsatellite markers for Acacia tortilis (Forsk.) Hayne[END_REF] and S. senegal [START_REF] Assoumane | Isolation and characterization of microsatellite markers for Acacia senegal (L.) Willd., a multipurpose arid and semi-arid tree[END_REF]). However, one major limitation to these approaches is related to the existence of variable ploidy levels among and within species (Bukhari 1997;[START_REF] Chevalier | Diversité génétique des acacias[END_REF][START_REF] Assoumane | Highlighting the occurrence of tetraploidy in Acacia senegal (L.) Willd. and genetic variation patterns in its natural range revealed by DNA microsatellite markers[END_REF][START_REF] Odee | Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion[END_REF][START_REF] Odee | Rangewide ploidy variation and evolution in Acacia senegal: a north-south divide?[END_REF]; Table S1). Consequently, phylogeographic patterns were also investigated using the plastid DNA variation that is haploid whatever the ploidy level (e.g., in Senegalia spp.; [START_REF] Guajardo | Landscape genetics of the key African acacia species Senegalia mellifera (Vahl)-the importance of the Kenyan Rift Valley[END_REF][START_REF] Odee | Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion[END_REF]. In these studies, microsatellite length variation (i.e., variable number of repeats in mono-nucleotide stretches) was a source of valuable information, as such loci are usually considered as the fastest evolving regions of the plastid genome (hereafter plastome) [START_REF] Powell | Polymorphic simple-sequence repeat regions in chloroplast genomes: applications to the population genetics of pines[END_REF][START_REF] Weising | A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms[END_REF]. Full plastomes of mimosoid legumes have also been recently released, mainly for Australian species [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF][START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF][START_REF] Williams | Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia[END_REF] and represent a valuable resource to develop new markers.

Plastids (e.g., chloroplasts) are usually maternally inherited in angiosperms (Corriveau and Coleman 1988). In mimosoids, maternal inheritance (with potential rare paternal leakage) has been reported for two species (i.e., Acacia decurrens and Acacia mearnsii) based on cytological observations of pollen grains (Reboud and Zeyl 1994). Polymorphism of the plastome can thus be useful to follow seed dispersal. The plastome is also more prone to stochastic events (i.e., genetic drift) because its effective population size is half that of diploid genomes, while its dispersal is limited due to seeds generally dispersed at shorter distance than pollen [START_REF] Petit | Finite island model for organelle and nuclear genes in plants[END_REF]. For these two reasons, polymorphism of plastomes allows revealing pronounced geographic structure that is suitable for phylogeographic analyses [START_REF] Petit | Finite island model for organelle and nuclear genes in plants[END_REF]. Furthermore, both a high number of copies per cell and the haploid nature of the plastome (due to mono-parental inheritance) facilitate its sequencing and analysis, even on poorly preserved DNAs (e.g., herbarium and forensic analyses; Besnard and Bervillé 2002;[START_REF] Pérez-Jiménez | Varietal tracing of virgin olive oils based on plastid DNA variation profiling[END_REF]. Although plastid DNA markers offer many advantages for population genetic and phylogenetic analyses, they may, however, show some limitations because phylogenetic patterns are not reflecting the whole history of a plant lineage, especially when hybridizations and cytoplasmic captures have occurred [START_REF] Harrison | Hybridization, introgression, and the nature of species boundaries[END_REF]. In addition, structural rearrangements [inverted repeat (IR) extension] and a faster evolution of some genes (in particular clpP) have been revealed in the plastomes of Acacia ligulata and Inga leiocalycina [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF][START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF]. The reasons for this phenomenon still remain unclear, and additional investigations are necessary to understand the contrasted evolutionary trajectories of plastid genes in mimosoid plastomes.

In the present study, we investigated the plastome diversity of six mimosoid species from the Central Sahara (i.e., Vachellia spp., S. laeta, and F. albida) in order to evaluate its utility for phylogenetic and population genetic analyses. We first sequenced complete plastomes (and nuclear ribosomal DNA clusters) using a genome skimming strategy (Straub et al. 2012). Based on these data, phylogenies were reconstructed. Structural and evolutionary changes were detected in plastomes of a mono-phyletic lineage (tribe Ingeae + Acacia sensu stricto), and several fast-evolving genes under positive selection were identified. Finally, plastid microsatellite markers were developed and tested on populations of the six South Algerian species. Perspectives on the genetic characterization of mimosoid species from Central Africa are finally proposed.

Material and methods

Plant sampling

The six species of Mimosoideae occurring in the Hoggar range were sampled in the Tamanrasset district (Fig. S1). According to taxonomical and phylogenetic studies, these taxa belong to three different lineages (e.g., [START_REF] Lewis | Legumes of the world. Royal Botanic Gardens, Kew L o h s e M , D r e c h s e l O , K a h l a u S , B o c k R ( 2 0 1 3 ) OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[END_REF][START_REF] Maslin | Generic and subgeneric names in Acacia following retypification of the genus[END_REF][START_REF] Murphy | A review of the classification of Acacia (Leguminosae, Mimosoideae)[END_REF][START_REF] Seigler | New combinations in the genus Senegalia (Fabaceae: Mimosoideae)[END_REF][START_REF] Bouchenak-Khelladi | The evolutionary history and biogeography of Mimosoideae (Leguminosae): an emphasis in African acacias[END_REF][START_REF] Kyalangalilwa | Phylogenetic position and revised classification of Acacia s.l. (Fabaceae: Mimosoideae) in Africa, including new combinations in Vachellia and Senegalia[END_REF] S1). Variable ploidy levels were also reported, especially in V. tortilis and V. nilotica [i.e., 2n = 26 (2x) to 104 (8x); Table S1]. Note that S. laeta was reported as triploid (3x = 39), and possibly issued from an interspecific hybridization [START_REF] Giffard | Les gommiers: Acacia senegal Willd., Acacia laeta R. Br[END_REF][START_REF] Amin | Acacia laeta (R. Br.) ex. Benth., considered as a species of hybrid origin[END_REF][START_REF] Ross | A conspectus of the African Acacia species[END_REF][START_REF] Chevalier | Diversité génétique des acacias[END_REF].

We sampled between 14 (S. laeta) and 42 mature individuals (V. seyal) per species (Table 1). For Vachellia spp. and S. laeta, only apparently unconnected ramets distant at a minimum of 10 m from each other were sampled to avoid the analysis of clones. While V. tortilis and V. flava are relatively frequent in the study area, the other species are present at a few locations (Fig. S1). Individuals of each species were collected on different sites (in wadis), except for V. nilotica that was found at only one site (BAmezgin^) in the southern part of the study area. For F. albida, we collected all ramets (18) that we found in the study area. Four isolated trees were sampled for this taxon. At BIn Zbib,^some of the 10 collected ramets were relatively close from each other (<5 m), and clones are possibly present at this locality.

Plastome and nuclear ribosomal DNA sequencing

For all samples, DNA extractions were performed from 20 mg of leaves (dried in silica gel) with the Biosprint method (Qiagen Inc.). To generate complete plastomes (chloroplast DNA (cpDNA)) and nearly complete nuclear ribosomal DNA units (nrDNA) of each species, we used the genome skimming approach (i.e., shotgun; Straub et al. 2012). Two individuals of V. tortilis were analyzed (haplotypes 302 and 303; see the BResults^section), while one tree accession was used for the five other species. Five hundred nanograms of double-stranded DNA (according to the NanoDrop quantification) were used for shotgun sequencing with the Illumina technology (HiSeq 2000, Illumina Inc.). The library was constructed using the Illumina TruSeq DNA Sample Prep v.2 Kit following the instructions of the supplier. Purified fragments were Atailed and ligated to sequencing indexed adapters. The seven libraries were multiplexed with 17 other libraries (generated in other projects). The pool of libraries was then hybridized to the HiSeq 2000 flow cell using the Illumina TruSeq PE Cluster Kit v.3. Bridge amplification was performed to generate clusters, and paired-end reads of 100 nucleotides were collected on the HiSeq 2000 sequencer using the Illumina TruSeq SBS Kit v.3 (200 cycles). Complete chloroplast sequences and nuclear ribosomal units were assembled using the approach described by Besnard et al. (2013). Finally, reads were mapped onto the different sequences obtained with Geneious v.9.1.2 (Kearse et al. 2012) to assess the depth of sequencing of all investigated regions. Gene annotation of plastomes was performed after their alignment (see below) using Geneious by transferring the annotations of the Leucaena trichandra plastome (NC_028733.1). Physical maps of plastomes were drawn with OGDRAW (Lohse et al. 2013).

Phylogenetic reconstructions and analysis of evolutionary rates among protein-coding genes

A phylogenetic tree based on the complete plastomes was reconstructed (for phylogenetic analyses based on the nrDNA cluster, see Supplementary Methods). Three plastome sequences were downloaded from GenBank. First, a cpDNA sequence of L. trichandra (NC_028733.1; [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF] was used as outgroup to root the tree, since this taxon belongs to tribe Mimoseae that is sister to a clade encompassing all Mimosoideae species from the Hoggar mountains [START_REF] Bouchenak-Khelladi | The evolutionary history and biogeography of Mimosoideae (Leguminosae): an emphasis in African acacias[END_REF]. Then, the plastome of Acacia ligulata (NC_026134; [START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF] and Inga leiocalycina (NC_028732; [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF] were also added to our alignment as two additional members of a lineage that encompasses tribe Ingeae and Acacia s.s. [START_REF] Bouchenak-Khelladi | The evolutionary history and biogeography of Mimosoideae (Leguminosae): an emphasis in African acacias[END_REF]. Sequences were aligned with the application Mega v.7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF]. The alignment was manually refined, and the second inverted repeat (IR) was removed. Tree inference was made under a maximum likelihood criterion, using RAxML v.8 (Stamatakis 2014) in the CIPRES platform. The alignment was divided into six partitions: (i) protein-coding genes of the large and the single-copy regions (LSC and SSC, respectively), (ii) non-coding regions of LSC and SSC, (iii) protein-coding genes of the IR, (iv) non-coding regions of the IR, (v) tRNA genes, and (vi) rRNA genes. We applied the GTR + G model for each partition and we used the rapid bootstrap algorithm with 1000 iterations. The phylogenetic tree was visualized using FigTree v1.4.2 (Rambaut 2014).

The evolutionary rates of each plastid protein-coding genes were then estimated and compared between species. We performed the analysis with one individual per species, using accession BNez19^to represent V. tortilis. Sequence alignment of each gene was individually done using Muscle (Edgar 2004) and refined manually taking into account their amino acid translation. Non-synonymous and synonymous substitution rates (d N and d S ) were calculated on all concatenated protein-coding genes and independently for each gene using the software codeml implemented in PAML V.4.8 (Yang 2007). We estimated d N and d S for each species against the outgroup species L. trichandra.

An IR extension in tribe Ingeae + Acacia s.s. was associated to an accelerated evolutionary rate, in particular on clpP [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF][START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF]; see the BResultsŝ ection). We analyzed changes of selective pressures in tribe Ingeae + Acacia s.s for genes in which we previously detected an acceleration of substitution rates in I. leiocalycina, F. albida, and A. ligulata compared to other species. This analysis was performed across branches of the phylogenetic tree using codeml in PAML. The phylogenetic topology based on complete plastomes was used. On each gene, we tested three codon models, which used the non-synonymous versus synonymous substitution rate ratio (d N /d S, denoted ω). The ω ratio is used to evaluate selective pressures at the protein level, with ω < 1, =1, or >1, indicating purifying selection, neutral selection, or positive selection, respectively. The first model, M1a, is a site model which allows ω to vary among sites. This model allows sites to be either under neutral selection (ω = 1) or under purifying selection (ω < 1; Yang et al. 2000). The second model, A, is a branch-site model that allows ω to vary among both sites and branches of the phylogenetic tree (Zhang et al. 2005). Branch-site models allow different ω for branches and need to assign foreground branches on which positive selection can potentially operate. In this model, sites can be attributed to four types of selection: (i) purifying selection on the whole tree (ω < 1), (ii) neutral selection on the whole tree (ω = 1), (iii) purifying selection on background branches (ω 0 < 1) and positive or neutral selection on foreground branches (ω 2 ≥ 1), and (iv) neutral selection on background branches (ω 1 = 1) and positive or neutral selection on foreground branches (ω 2 ≥ 1). The last model, A′, is the same as model A, except that positive selection on foreground branches is replaced by neutral selection (ω 2 = 1; Zhang et al. 2005). In this study, we tested two hypotheses (Fig. S2): (i) positive selection occurred on the internal branch ancestral to I. leiocalycina, A. ligulata, and F. albida, on which the IR extension has occurred (hypothesis H1) and (ii) positive selection occurred on all branches of the clade including I. leiocalycina, A. ligulata, and F. albida, after the IR The seven individuals analyzed with a genome skimming (GS) strategy are also indicated extension (hypothesis H2). For each hypothesis, we performed two likelihood ratio tests (LRTs) to compare models. As model A is nested into both models M1a and A′, we can compare model A versus model M1a and model A versus model A′. To choose the hypothesis (H1 or H2) that fits the data best, we compared the best model of each hypothesis using the Akaike Information Criterion (AIC). In the case of non-significantly different models under a given hypothesis, we compared the AIC of the three models M1a, A, and A′ with that of the best model of the other hypothesis. Given the small number of species in our analysis, we also performed simulations to evaluate the frequency of the type-I error (i.e., the incorrect rejection of a true null hypothesis H0). For each gene, we generated 1000 simulated data under the null hypothesis, i.e., assuming two classes of sites [either under neutral selection (ω = 1) or under purifying selection (ω < 1)], using the software evolverNSsites in PAML. We used the value of ω and the proportion of sites attributed to each class estimated under model M1a with the real dataset. We then tested the three models M1a, A, and A′ under hypotheses H1 and H2 for each simulated data. In order to estimate the proportion of false positives, we estimated LRT relative to χ 2 for each simulated data to compare (i) models A with M1a and (ii) models A with A′.

Characterization of cpDNA microsatellite polymorphisms in mimosoid populations

Based on six complete plastome sequences of acacias from the Hoggar (one for each studied species), we targeted regions that show microsatellite motif(s) (i.e., mono-nucleotide stretches) in the six investigated species. We identified 73 regions with at least one mono-nucleotide stretch (min. 8 repeats) in all species. After preliminary tests, 21 loci (all located in intergenic spacers) were finally selected for their polymorphism and reliable amplification (Tables 2 andS2). These loci were hereafter referred to chloroplast simple sequence repeats (cpSSRs). The PCR primers were designed in flanking regions to specifically amplify short segments (inferior to 265 bp; Table 2). For locus multiplexing, the annealing temperature of all these primers needed to be similar, while the size of PCR products of each locus should be as different as possible. Finally, these primers were also designed to allow amplification of short DNA segments for the characterization of degraded DNAs. All primer pairs and specific characteristics of generated fragments are given in Tables 2 andS2. To reduce time and costs of the PCR characterization, we used the method described by [START_REF] Schuelke | An economic method for the fluorescent labelling of PCR fragments[END_REF]. For each locus, an 18bp tail of M13 was added on the forward primer (Table 2). Additionally, the 5′ end of the reverse primer was also tagged with the sequence GTGTCTT to minimize band stuttering (Table 2). Three or four loci were simultaneously amplified in the same reaction (PCR multiplexes defined in Table 2).

Each PCR reaction (25 μl) contained 10 ng DNA template, 1× reaction buffer, 5 mM MgCl 2 , 0.2 mM dNTPs, 0.2 μmol of one universal fluorescent-labeled M13(-21) primer (5′-TGTA AAACGACGGCCAGT-3′, labeled with one of the three following fluorochromes: YAK, 6-FAM or AT550), 0.04 μmol of the reverse primer, 0.01 μmol of the forward primer, and 0.5 U of Taq DNA polymerase (Promega). The reaction mixtures were incubated in a T1 thermocycler for 2 min at 95 °C, followed by 28 cycles of 30 s at 95 °C, 30 s at 57 °C, and 1 min at 72 °C, and then by 8 cycles of 30 s at 95 °C, 30 s at 51.5 °C, and 1 min at 72 °C. The last cycle was followed by a 20-min extension at 72 °C. The PCR products labeled with a fluorochrome were mixed together with GeneScan-600 LIZ as internal standard to run all loci at the same time. They were separated on an ABI Prism 3730 DNA Analyzer (Applied Biosystems), and the size of amplified fragments was determined with Geneious.

Based on fragment genotyping (cpSSRs), we defined a cpDNA haplotype for each individual (i.e., combination of alleles at the 21 loci). The probability that two individuals taken at random display a different haplotype was computed as D = 1 -Σ p i 2 , where p i is the frequency of the haplotype i. Haplotype richness was computed with FSTAT [START_REF] Goudet | FSTAT (version 1.2): a computer program to calculate Fstatistics[END_REF]. The relationships among cpDNA haplotypes were then visualized by constructing a reduced median network implemented in the application Network v.5 [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF]. cpSSRs are fast-evolving parts of the plastome, and thus, generally not informative when comparing too distant lineages. Such markers have proven to be valuable tools at the population level, and eventually at the species complex level (e.g., Olea europaea; Besnard et al. 2011). Consequently, we decided to reconstruct haplotype networks separately on Vachellia spp. and on F. albida (+ S. laeta that was used as outgroup). Multistate microsatellites were treated as ordered alleles and coded by the number of repeated motifs for each allele (e.g., number of nucleotides in mono-nucleotide stretches; see also Besnard et al. 2011). Basically, this coding strategy assumes that variation at cpSSRs is mainly due to single-step mutations (e.g., Besnard et al. 2011). For haplotypes whose plastome is not available, we checked length polymorphisms when superior to 2 bp compared to related haplotypes. These fragments were sequenced with the Sanger method as described in Besnard et al. (2011). For loci combining microsatellite motifs and non-SSR indels either in Vachellia spp. or in F. albida (i.e., loci 3 and 14), we separately coded the two types of characters based on available sequences. The presence or absence of such indels was coded as 1 and 0, respectively. In addition, size homoplasy is frequent on plastid microsatellites [START_REF] Powell | Polymorphic simple-sequence repeat regions in chloroplast genomes: applications to the population genetics of pines[END_REF][START_REF] Navascués | Chloroplast microsatellites: measures of genetic diversity and the effect of homoplasy[END_REF]. To downweight the impact of the most variable (and thus the most homoplasic) markers in the network reconstruction, we arbitrarily assigned four classes of weight to character changes according to the number of alleles at a locus [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF]): weight of 10 for loci with two alleles, 5 for loci with three alleles, 3 for loci with four alleles, and 1 for loci with five alleles and more.

Results

Assembly of plastomes and nuclear ribosomal DNA clusters

We first generated between 7,298,994 and 41,826,997 pairedend reads for the seven tree accessions analyzed by shotgun (Table S3). These data allowed us to assemble the plastome and nuclear ribosomal cluster for at least one individual per species. The sequencing depth of these genomic regions (Table S3) was relatively high, from 194 to 4257× for the plastome and from 235 to 10,064× for the ribosomal cluster, allowing their perfect assembly.

The plastome length varies from 162,754 bp (S. laeta) to 175,675 bp (F. albida; Table S3). The two V. tortilis plastomes are very similar, and differ by only three nucleotidic substitutions and one nucleotide indel in a poly-T stretch (corresponding to cpSSR locus 10; see below). The plastome of Faidherbia is longer than this of other acacia species by ca. 10-13 kb. This difference in the plastome length is mainly due Three or four loci were simultaneously amplified in six multiplexes (a to f, considering non-overlapping allele size range for a given fluorochrome). Additional loci features (microsatellite motifs and their genome position) are given in Table S2 a M13-tail on each forward primer: tgtaaaacgacggccagt to an extension of the IR (Fig. S3) that notably encompasses the whole coding sequence of genes that are usually located in the SSC (ycf1, rps15, ndhH, ndhA, ndhI, ndhG, ndhE, psaC and ndhD;[START_REF] Xu | Dynamics of chloroplast genomes in green plants[END_REF], as similarly reported in I. leiocalycina and A. ligulata [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF][START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF]. As expected, plastomes are AT-rich regions (GC content = 35.3 to 35.8%; Table S3). In contrast, the nrDNA clusters are GC-rich regions (GC content = 57.1 to 59.1%; Table S3). The assembled nrDNA regions are 6401 to 8104 bp long and comprise 5′ and 3′ external transcribed sequences (ETS), the 18S gene, the internal transcribed spacer 1 (ITS-1), the 5.8S gene, the internal transcribed spacer 2 (ITS-2), and the 26S gene. The internal gene spacer (IGS), which includes ETS, was not completely assembled due to the presence of repeated and inverted regions that render its assembly very challenging. Interestingly, two divergent units (i.e., 98.3% of homology on the whole sequence) were separated in S. laeta, the major and minor units representing approximately 65 and 35% of nrDNA reads, respectively (Table S3).

Phylogenetic topologies and molecular evolution of plastid protein coding genes

Phylogenetic relationships based on plastomes and nrDNA are mostly congruent (Figs. 1 and S4). In both phylogenetic trees, relationships between Vachellia spp. are fully congruent. This genus is sister to a clade including Senegalia, Inga, and Acacia s.s. While the placement of S. laeta as sister to a paraphyletic tribe Ingeae and Acacia s.s. (F. albida, I. leiocalycina, and A. ligulata) is strongly supported, the placement of L. trichandra differs in the two phylogenies (Figs. 1 andS4). In the plastome phylogenetic tree, branches of F. albida, I. leiocalycina, and A. ligulata are longer than those of other species, suggesting a faster evolution of the plastome for these taxa (Fig. 1).

Non-synonymous and synonymous substitution rates (d N and d S ) were estimated on 78 plastid protein-coding genes (Figs. 2 and S5). These rates were computed for each species against the outgroup species L. trichandra. Both nonsynonymous and synonymous substitution rates are globally higher in F. albida, I. leiocalycina, and A. ligulata (0.0137 > d N > 0.0315; 0.0300 > d S > 0.0455) than in S. laeta and Vachellia spp. (0.0073 > d N > 0.0077; 0.0258 > d S > 0.0272; Fig. S5). As shown by [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF], clpP has quickly evolved in A. ligulata and I. leiocalycina (Fig. 2). A similar pattern is here detected in F. albida. For these three species that belong to a monophyletic lineage, we found an accelerated evolutionary rate for 24 genes compared with the other species (Fig. S5; Tables 3 andS4). Interestingly, an IR extension is observed in plastomes of F. albida, I. leiocalycina, and A. ligulata (see above). Changes of selective pressure during or after this IR extension were then tested with codon models on the 24 genes mentioned above. Under hypothesis H1, assuming positive selection on the internal branch ancestral to F. albida, I. leiocalycina, and A. ligulata, model A is significantly better than both null models for clpP, ycf1, and ycf2 (models A and M1a are marginally different for ycf1, and ycf2; Table 3), while the three models are not significantly different for the other genes (Table S4). Under hypothesis H2, assuming positive selection on all branches of the clade including F. albida, I. leiocalycina, and A. ligulata, model A is significantly better than both null models for accD, clpP, rps2, rps3, ycf1, ycf2, and ycf4 (models A and M1a are marginally different for rps2; Table 3), while the three models are not significantly different for the other genes (Table S4). Using sequence simulations, we did not detect any inflation of false positives for accD, clpP, rps2, rps3, ycf1, ycf2, and ycf4 under H1 and H2 (Table S5) and type-I errors may thus not affect our analyses. Based on AIC comparison, hypothesis H2 fits to our data (Stamatakis 2014). Main lineages (i.e., tribes and genera) usually recognized are indicated (see [START_REF] Lewis | Legumes of the world. Royal Botanic Gardens, Kew L o h s e M , D r e c h s e l O , K a h l a u S , B o c k R ( 2 0 1 3 ) OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets[END_REF][START_REF] Maslin | Generic and subgeneric names in Acacia following retypification of the genus[END_REF][START_REF] Murphy | A review of the classification of Acacia (Leguminosae, Mimosoideae)[END_REF][START_REF] Seigler | New combinations in the genus Senegalia (Fabaceae: Mimosoideae)[END_REF][START_REF] Bouchenak-Khelladi | The evolutionary history and biogeography of Mimosoideae (Leguminosae): an emphasis in African acacias[END_REF][START_REF] Kyalangalilwa | Phylogenetic position and revised classification of Acacia s.l. (Fabaceae: Mimosoideae) in Africa, including new combinations in Vachellia and Senegalia[END_REF]. The red star indicates the internal branch ancestral to I. leiocalycina, A. ligulata, and F. albida, on which the IR extension has occurred (Fig. S3) significantly better than hypothesis H1 for clpP, ycf1 and ycf2 (Table 4). Because models M1a, A, and A′ are not significantly different under hypothesis H1 for accD, rps2, rps3, and ycf4, we compared the AIC of these three models with that of the best model of hypothesis H2. We also found that hypothesis H2 fits to our data significantly better than hypothesis H1. Hypothesis H2, assuming positive selection on all branches of the clade, is thus the best-fit hypothesis for , clpP, rps2, rps3, ycf1, ycf2, and ycf4. Under this hypothesis, model A detected between 0.4 and 10.6% of sites under positive selection for ycf2 and clpP, respectively (posterior probability >0.95; Table S6).

Development of plastid microsatellites and inter-versus intra-specific variation

All 21 cpSSR loci rendered length polymorphism at the interspecific level, while 18 were variable at the intra-specific level in at least one species (Tables 5 and6). The combination of alleles of these 21 loci allows us to define a haplotype for each individual (Tables 6 andS7). Each haplotype is unique to a species. No variation was found in three taxa: V. flava, V. seyal, and S. laeta. In contrast, we detected diversity (D = 0.51 to 0.78) within the three other species, with three haplotypes in populations of V. tortilis and F. albida and five haplotypes in the population of V. nilotica. The haplotype network for Vachellia species reveals two small clusters of related haplotypes for V. tortilis and V. nilotica (Fig. 3). All haplotypes of V. nilotica are, however, not closely related, since one divergent haplotype (no 201) is detected in three individuals. The distribution of haplotypes in V. tortilis does not show obvious geographic structure in our study area since the three haplotypes are detected in all locations with more than two samples (Fig. 3). Although our F. albida sampling is limited, the distribution of haplotypes seems not random in this species and no diversity is found within populations (Fig. S6).

Discussion

On the phylogenetic relationships between Central Saharan mimosoids

In our study, we generated new genomic resources for African mimosoid species, in particular the first complete plastomes and long sequences of the nrDNA cluster. Such data are potentially useful to resolve relationships in the Mimosoideae [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF][START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF][START_REF] Williams | Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia[END_REF], for which taxon sampling in phylogenetic works is not yet comprehensive compared to other legumes of the subfamily Papilionoideae [START_REF] Schwarz | Plastid genome sequences of legumes reveal parallel inversions and multiple losses of rps16 in papilionoids[END_REF]. Based on our complete plastomes, and as expected from [START_REF] Luckow | A phylogenetic analysis of Mimosoideae (Leguminosae) based on chloroplast DNA sequence[END_REF][START_REF] Bouchenak-Khelladi | The evolutionary history and biogeography of Mimosoideae (Leguminosae): an emphasis in African acacias[END_REF][START_REF] Kyalangalilwa | Phylogenetic position and revised classification of Acacia s.l. (Fabaceae: Mimosoideae) in Africa, including new combinations in Vachellia and Senegalia[END_REF], S. laeta is sister to a paraphyletic tribe Ingeae (F. albida and I. leiocalycina) and Acacia s.s. (A. ligulata), while Vachellia spp. is sister to this clade. The topology based on nrDNA is mostly congruent, but Leucaena shows an unexpected position (as sister to Acacia s.s.), possibly due to a too rapid and/or heterogeneous evolution of the ITS marker (e.g., saturated phylogenetic signal and long branch attraction For each gene, we compared the best-fit model of both hypotheses. For each gene, the AIC value indicating the best-fit hypothesis is in italics lnL log-likelihood, Npar number of parameters in the model 1.00 0.000 Allspecies 5 6 7 3 5 6 7 3 6 7 6 4 5 5 6 4 5 7 3 7 4 14 --N h number of haplotypes (combination of polymorphism from the 21 cpSSR loci) per species and among species, R S haplotype richness (calculated for 14 individuals), D probability that two individuals show a different allele within a species evolutionary rates also need to be taken into consideration in phylogenetic analyses of mimosoids, especially on genes under positive selection.

Utility of cpSSRs for phylogeographic reconstructions and population genetic analyses

Although our sampling is on a limited geographic area around Tamanrasset, most of our cpSSRs (18/21) revealed intraspecific polymorphism in at least one species. In addition, haplotypes (multiloci profiles) are found to be unique to a taxon, demonstrating the potential utility of our markers to identify species at this local scale. We also suggest our markers to be used on a larger sampling of populations (and species) to reconstruct phylogeographies at the continental scale. Such genetic analyses may also be valuable to identify gene pools and assist taxonomical revisions (e.g., [START_REF] Taylor | Species limits in Vachellia (Acacia) karroo (Mimosoideae: Leguminoseae): evidence from automated ISSR DNA Bfingerprinting^[END_REF][START_REF] Binks | Genetic entities and hybridisation within the Acacia microbotrya species complex in Western Australia[END_REF]. Knowledge on the distribution of plastid haplotypes is potentially useful to develop tests of provenances and to trace the spread of lineages (for instance in invasive acacia species; [START_REF] Thompson | A tree well travelled: global genetic structure of the invasive tree Acacia saligna[END_REF]. Based on released plastomes, other cpSSR loci could also be developed but many of them are not conserved among mimosoid genera and can not be used for comparative analyses at large taxonomical scale.

Our set of cpSSR markers, which is expected universal for the African acacias, may allow direct comparisons of diversity patterns on a mostly maternally inherited haploid genome, even on species complexes with distinct ploidy levels [e.g., Vachellia spp. (Bukhari 1997;[START_REF] Ferchichi Ouarda | Diversity analysis of Acacia tortilis (Forsk.) Hayne ssp. raddiana (Savi) Brenan (Mimosaceae) using phenotypic traits, chromosome counting and DNA content approaches[END_REF]); Senegalia spp. [START_REF] Assoumane | Highlighting the occurrence of tetraploidy in Acacia senegal (L.) Willd. and genetic variation patterns in its natural range revealed by DNA microsatellite markers[END_REF][START_REF] Odee | Rangewide ploidy variation and evolution in Acacia senegal: a north-south divide?[END_REF]]. In our study, only three species harbor several haplotypes, and the highest diversity is detected in V. nilotica (D = 0.78) for which only one population was sampled (Fig. 3; Table 5). In contrast, its most closely species, V. flava, does not show any variation among two populations. The reason for such a difference remains unclear but could be due to distinct colonization histories and/or demographic processes. The occurrence of phylogenetically divergent chlorotypes in the V. nilotica population also suggests a possible event of hybridization/ admixture between differentiated taxa/populations. The combined use of our cpSSR markers with bi-parentally inherited markers may help separating the contribution of pollen-and seed-mediated gene flows and unraveling such admixture events in species complexes [Vachellia spp. (Ali andQaiser 1980, 1992); Senegalia spp. [START_REF] Odee | Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion[END_REF]]. While the nuclear genome analysis is limited by variable ploidy levels among and within species, the use of nuclear markers also remains essential to investigate the reproductive strategies of mimosoid species by assessing the importance of asexual assessed with a reduced median network [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF]. The data matrix used for the network analysis is given in Table S8. The network without downweigthing character changes according to the number of alleles per locus is given in Fig. S7

Fig. 1

 1 Fig. 1 Représentation schématique de l'arbre de la vie illustrant les relations phylogénétiques des trois domaines (Bacteria, Archaea et Eucaryota). Les événements d'endosymbioses à l'origine des chloroplastes et des mitochondries sont représentés par une flèche verte et une flèche bleue, respectivement. [Figure extraite de Delsuc et al. (2005)]

Fig. 2

 2 Fig. 2 Schéma des endosymbioses primaires et secondaires des chloroplastes. L'endosymbiose primaire implique un hôte eucaryote et une cyanobactérie photosynthétique et est à l'origine de trois lignées : les glaucophytes (Glaucophyta), les algues rouges (Rhodophyta) et les algues vertes (Chlorophyta, à partir desquelles les plantes terrestres ont dérivé). L'endosymbiose secondaire implique un hôte eucaryote et une algue eucaryote photosynthétique (une algue rouge ou une algue verte). [Figure extraite de Bölter & Soll (2016)]

Fig. 3

 3 Fig. 3 Structure classique d'un plastome avec deux "Inverted Repeats" (IR A et IR B ) séparées par une petite (SSC) et une grande (LSC) régions en simple copie. [Figure extraite de Khan et al. (2010)]

Fig. 4

 4 Fig. 4 Structure classique des mitogénomes de plantes en sous-molécules circulaires (structure multipartite). Les longues régions répétées (ici, représentées par des flèches rouges et bleues) favorisent la recombinaison intra-moléculaire, ce qui génère une structure du mitogénome en sous-cercles d'ADN. [Figure extraite de Gualberto et al. (2014)]

Fig. 5

 5 Fig. 5 Contexte écologique de transferts horizontaux d'ADN chez Amborella : A et B. Branches d'Amborella couvertes de plantes grimpantes (flèches vertes) et d'épiphytes, incluant des mousses, des hépatiques, des fougères et des angiospermes ; C à F. Feuilles et branches d'Amborella couvertes de lichens (C et F), d'hépatites (D) et de mousses (E). [Figure extraite de Rice et al. (2013)]

Fig. 6

 6 Fig. 6 Hypothèse consensus des pressions de sélection entraînant l'apparition et la perte répétées de la transmission uniparentale maternelle des organites chez les plantes à fleurs. [Figure extraite Greiner et al. (2014)]

Fig. 7

 7 Fig. 7 Principe du séquençage par la technologie Illumina. A. Après fractionnement du génome en petits fragments d'ADN, chaque fragment (préalablement dénaturé en simple brin) fixe ses extrémités sur un support solide en s'hybridant avec des amorces, pour former un pont. Le brin complémentaire de chacun des fragments est alors synthétisé. B. La synthèse du brin complémentaire s'effectue en incorporant dans la séquence un des quatre nucléotides associés à des fluorochromes réversibles. Le fluorochrome associé à un nucléotide émet un signal lumineux, qui est directement détecté par le séquenceur et traduit en nucléotide. Le fluorochrome est ensuite clivé et la synthèse du fragment peut continuer. Au cycle suivant, un des quatre nouveaux nucléotides associés à des fluorochromes est incorporé à la séquence et le deuxième nucléotide est lu par le séquenceur. Les cycles de séquençage sont ainsi répétés, jusqu'à la synthèse complète du brin complémentaire au fragment séquencé. [Figure extraite de Metzker (2009)]

Fig. 8

 8 Fig. 8 Exemple d'un alignement de séquences de six taxons. Chaque colonne contient les différents états de caractères du site homologue. L'ajout d'espaces dans les séquences permet d'aligner les sites homologues des différentes séquences.

Fig. 9

 9 Fig. 9 Aperçu de la diversité florale chez la famille des Oleaceae. a. Noronhia densiflora; b. Fraxinus ornus; c. Jasminum nudiflorum; d. Ligustrum vulgaris; e. Forsythia × intermedia; f. Syringa vulgaris. [a. : photographie de C. Birkinshaw extraite de Hong-Wa (2016); b. à f. : photographies libres de droits*]

Fig. 10

 10 Fig. 10 Arbre phylogénétique de la famille des Oleaceae reconstruite par Wallander & Albert (2000), basé sur deux régions chloroplastiques non-codantes (l'intron de rps16 et l'espace inter-génique trnL-trnF). [Figure extraite de Wallander & Albert (2000)]

  descendants d'un ancêtre commun Groupe polyphylétique : ne comprend pas tous les descendants d'un ancêtre commun Groupe paraphylétique : comprend tous les descendants d'un ancêtre commun sauf une lignée (cas spécifique d'un groupe polyphylétique)

Fig. 9 .

 9 Fraxinus ornus : Amada44 [cc-by-2.0]; Jasminum nudiflorum : FlickrLickr[cc- by-2.0]; Ligustrum vulgaris : redagainPatti [cc-by-nd-2.0]; Forsythia × intermedia : Dan Smith [cc-by-sa-2.0]; Syringa vulgaris : pixel2013 [cc0 1.0].

Fig. 2 .

 2 Fig.2. Best likelihood phylogenetic tree of the Lamiales obtained with RAxML v.8(Stamatakis, 2014) with a GTR + G model. We used a concatenated alignment of 28 mitochondrial protein-coding genes and 13 introns. We applied the rapid bootstrap algorithm with 1000 iterations. The phylogenetic tree was rooted with Nicotiana tabacum (Solanales).

Fig. 3 .

 3 Fig.3. Substitution rates among 27 protein-coding genes in Hesperelaea palmeri. For each protein-coding gene, non-synonymous (d N ) and synonymous (d S ) rates were calculated against Nicotiana tabacum using PAML(Yang, 2007). The value over each bar corresponds to the d N /d S ratio (ω) for each gene. For a comparative analysis among Lamiales species, see Fig.S1in Supplementary material.

  K E Y W O R D Scytoplasmic male sterility, genome skimming, metagenomics, mitogenome, mtpt, Olea europaea, Oleinae, phylogeny, plastome1 | INTRODUCTIONMolecular phylogenetics is a fundamental tool to study the evolutionary history of living organisms, the assembly of communities and the evolution of genes and genomes (e.g.,[START_REF] Avise | Phylogeography: Retrospect and prospect[END_REF][START_REF] Paterson | Insights from the comparison of plant genome sequences[END_REF][START_REF] Pillar | A framework for metacommunity analysis of phylogenetic structure[END_REF]. Different genomic regions can be used for phylogenetic reconstruction and/or DNA barcoding-based identification, but these often produce conflicting signals as a consequence of incomplete ancestral lineage sorting and reticulate evolution (including hybridization and horizontal gene transfer), as well as other more complex processes such as molecular convergence (due to selection or mutational biases) and heterogeneity of evolutionary rates among lineages (e.g., Christin, Besnard, Edwards, & Salamin, 2012; Delsuc, Brinkmann, & Philippe, 2005; Wendel & Doyle 1998). Confronting phylogenetic signals from cytoplasmic and nuclear genomic compartments is thus essential to understand the evolutionary history of species and their genomes. In plants, while ribosomal DNA has been extensively used as a nuclear phylogenetic marker (e.g., Alvarez & Wendel, 2003; Nieto Feliner & Rossell o, 2007), plastid genes, and since recently, full chloroplast genomes (hereafter plastomes), are now the markers of choice for

  genomes have been already released on GenBank; Van de Paer, Hong-Wa, Jeziorski, & Besnard, 2016; Zedane et al., 2016), we included nine species representative of different Oleeae lineages, plus one representative of two other Oleaceae tribes: Fontanesia fortunei (Fontanesieae) and Forsythia 9 intermedia (Forsythieae). Four Oleeae samples were herbarium specimens collected between 2001

  accessions and Chionanthus rupicolus in order to investigate their structural organization and evolution within the olive species and in related genera (i.e., Chionanthus and Hesperelaea). Mitogenomes have only in part or not at all been assembled in current Oleaceae genome-sequencing projects[START_REF] Cruz | Genome sequence of the olive tree, Olea europaea[END_REF] Sollars et al., 2017;[START_REF] Unver | Genome of wild olive and the evolution of oil biosynthesis[END_REF] due to their structural complexity, especially the presence of mtpt regions. Here, we followed the procedure described by[START_REF] Van De Paer | Mitogenomics of Hesperelaea, an extinct genus of Oleaceae[END_REF] to assemble complete mitogenomes. The H. palmeri mitogenome [KX545367] was initially used as a reference to generate mitochondrial DNA contigs that were then used to assemble complete mitogenomes. In this process, mtpt regions were manually resolved by identifying mitochondrial reads based on polymorphisms (i.e., single nucleotide polymorphisms and T A B L E 1 List of plant accessions analysed in our study. GenBank accession number of complete plastomes and mitogenomes is indicated.

First, we investigated

  the evolution and origin of mtDNA regions showing high homology with plastomes (mtpt regions) in the olive subtribe (Oleinae). Second, we tested for the congruence of phylogenetic signal (i.e., topology and node support) from mitochondrial and chloroplast DNA at the tribe level (Oleeae). Finally, phylogenetic relationships between cytoplasmic lineages of the Mediterranean olive were investigated for the first time based on both full plastomes and full mitogenomes. Plastid integration in mitogenomes of the olive subtribe (Oleinae): Phylogenetic signal from mtpt regions (detected in all Oleinae) and their plastid homologues may inform on approximately when plastid regions were integrated into the mitogenome. We thus investigated the relative placement of each mtpt region and its plastid homologue in phylogenies of Oleeae with Boea hygrometrica as out-group. The nine mtpt regions shared by the eight accessions of Oleinae (see results) were individually aligned with their homologous plastid regions (extracted from the plastome of 18 Oleaceae and Boea hygrometrica)

  Integration of mtpt regions in the olive mitogenome The total length of mtpt regions ranged from 31,905 (in O. e. ssp. cuspidata) to 55,513 bp (in B. hygrometrica), covering 20.6%-35.2%of the plastome (Figure2). These regions had origins in both single copies (LSC and SSC) and the IR. Most boundaries of mtpt regions were shared between O. europaea accessions, while the boundaries of mtpt regions of H. palmeri and C. rupicolus were partially shared with those of O. europaea (Figure2). In contrast, the mtpt regions detected in B. hygrometrica were differentially distributed along the E1-e.4 plastome compared to Oleaceae accessions (Figure2). We identified nine mtpt regions shared by all studied Oleeae accessions with length ranging from 495 to 3,198 bp (Figure2). Six of these mtpt regions were also partly present in the mitogenome of B. hygrometrica (regions 1, 2, 3, 4, 6 and 9; Figure2). ML phylogenies were reconstructed from each mtpt segment and their homologous regions extracted from the plastomes of B. hygrometrica and Oleaceae (Figures3 and S4). In the nine topologies, mtpt sequencesF I G U R E 1Master circle of olive mitogenome E3.1 (associated with CMS;Besnard et al., 2000). The outer circle represents the gene content, with protein-coding genes, rRNA and tRNA genes. The inner circle represents repeated sequences with a minimum length of 100 bp in the mitogenome. The large repeats >10 kb are represented in light blue colour. The maps were performed with OGDRAW v1.2(Lohse et al., 2007) and CIRCOS(Krzywinski et al., 2009) of olive accessions clustered together, suggesting they have derived from a common ancestor. In contrast, the phylogenetic placement of Oleinae mtpt sequences relative to their homologous plastid regions was not congruent between segments (Figures3 and S4), indicating that they were not all integrated into the mitogenome at the same time: (i) first, one topology (mtpt6) supported the distinction of mtpt sequences of B. hygrometrica and Oleaceae from their plastid homologous regions (Figure3b). This suggests that this mtpt region integrated in the mitogenome before the divergence between Gesneriaceae and Oleaceae; (ii) second, three topologies (regions corresponding to mtpt1, mtpt3 and mtpt8) showed an mtpt clade of Oleinae sister to plastid homologous regions of Oleeae + Fontanesieae + Forsythieae, supporting the integration of these mtpt regions before the divergence of these three tribes; (iii) third, three topologies (regions corresponding to mtpt4, mtpt5 and mtpt7) showed an mtpt clade of Oleinae sister to plastid homologous regions of Oleinae, supporting the integration of these mtpt regions before the diversification of the investigated taxa of Oleinae (but after the divergence of subtribe Oleeae; Figure 3a); and finally (iv) two topologies (regions corresponding to mtpt2 and mtpt9) did not support a common origin of all mtpt regions of Oleinae, suggesting potential multiple integrations of these mtpt regions in the mitogenome, but this remains tentative due to the low node support. 3.4 | Reconstructing the phylogeny of Oleeae from plastid and mitochondrial DNA data

O

  . e. cuspidata C2.13-mtptDNA O. e. guanchica M-g1.3-mtptDNA O. e. europaea E3.1-mtptDNA O. e. europaea E2.1-mtptDNA O. e. europaea E1-e.4-mtptDNA O. e. laperrinei E1-l1.1-mtptDNA Hesperelaea palmeri-mtptDNA Chionanthus rupicolus-mtptDNA 82 O. e. cuspidata C2.13-ptDNA O. e. europaea E2.1-ptDNA O. e. europaea E3.1-ptDNA O. e. guanchica M-g1.3-ptDNA O. e. europaea E1-e.4-ptDNA O. e. laperrinei E1-l1.1. guanchica M-g1.3-mtptDNA O. e. europaea E3.1-mtptDNA O. e. europaea E2.1-mtptDNA O. e. europaea E1-e.4-mtptDNA O. e. laperrinei E1-l1.1-mtptDNA Syringa vulgaris-ptDNA O. e. cuspidata C2.13-ptDNA O. e. europaea E2.1-ptDNA O. e. europaea E3.1-ptDNA O. e. guanchica M-g1.3-ptDNA O. e. europaea E1-e.4-ptDNA O. e. laperrinei E1-l1.1Maximum-likelihood phylogenetic trees reconstructed on two mtpt segments [(a) mtpt4 and (b) mtpt6] and their homologous plastid regions extracted from plastomes of Boea hygrometrica (Gesneriaceae), 16 accessions belonging to tribe Oleeae and two accessions belonging to Fontanesieae and Forsythieae. The mtpt regions are represented in red and their corresponding homologous plastid sequences in green. Both phylogenetic trees were reconstructed with RAXML V.8 (Stamatakis, 2014) using a GTR + G model, applying the rapid bootstrap algorithm with 1,000 iterations. We rooted the trees at mid-point variation in size among mitogenomes is mainly explained by different proportions of repeated sequences (Table

  Maximum-likelihood phylogenetic trees of the Oleeae obtained with RAxML v.8(Stamatakis, 2014) with the GTR + G model. We reconstructed two phylogenetic trees of Oleeae using Fontanesia fortunei and Forsythia 9 intermedia to root the trees: (a) based on complete plastomes, and (b) based on 52 mitochondrial protein-coding genes and introns VAN DE PAER ET AL.| 11[START_REF] Unseld | The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides[END_REF]. These regions cover a large part (>20%) of the plastome (Figure2). While most mtpt regions show the same boundaries between the six olive accessions, nine mtpt regions are shared between Olea, Hesperelaea and Chionanthus (Figure2). Phylogenies individually reconstructed from each mtpt and their homologous regions support several rounds of integration of plastid sequences in ancestral mitogenomes of Oleinae (Figures3 and S4). One of them (mtpt6, which includes a putative functional tRNA gene, trnS-GGA)

  Maximum-likelihood phylogenetic trees of the olive complex obtained with RAXML V.8(Stamatakis, 2014) with a GTR + G model. We applied the rapid bootstrap algorithm with 1,000 iterations. We reconstructed three phylogenetic trees of the six newly sequenced accessions of O. europaea based on (a) complete plastomes, (b) conserved mitochondrial regions and (c) the concatenation of complete plastomes and conserved mitochondrial regions. We used O. e. cuspidata (lineage C2) as outgroup to root the trees

  to E1 as reported by M edail et al. (2001) based on the analysis of restriction fragment length polymorphisms of the mtDNA. The position of lineage E3 as sister to all North African lineages is also strongly supported. Overall, the topology indicates that Mediterranean olive lineages may have rapidly diversified in the Western Mediterranean Basin first (lineages E2, E3 and M, including Macaronesia) before colonizing the Eastern Mediterranean and Saharan Africa (lineage E1). Although other species complexes share a circum-Mediterranean distribution (e.g., Laurus, Myrtus, Phillyrea, Pinus,Quercus), such a colonization from west to east during the Late Neogene has never been suggested until now for other tree species based on phylogeographic reconstructions[START_REF] Migliore | From Mediterranean shores to central Saharan mountains: Key phylogeographical insights from the genus Myrtus[END_REF][START_REF] Nieto Feliner | Patterns and processes in plant phylogeography in the Mediterranean Basin[END_REF][START_REF] Rodr Iguez-S Anchez | Late Neogene history of the laurel tree (Laurus L., Lauraceae) based on phylogeographical analyses of Mediterranean and Macaronesian populations[END_REF][START_REF] Thompson | Plant evolution in the Mediterranean[END_REF].

  .D.P. and G.B. are members of the Laboratoire Evolution & Diversit e Biologique (EDB) part of the LABEX TULIP managed by the Agence Nationale de la Recherche (ANR-10-LABX-0041). We also acknowledge an Investissement d'Avenir grant from the Agence Nationale de la Recherche (CEBA: ANR-10-LABX-25-01). C.V.D.P. and G.B. were funded by the Regional Council Midi-Pyr en ees (AAP 13053637, 2014-EDB-UT3-DOCT). This work was performed in collaboration with the GeT core facility, Toulouse, France (http://get.genotoul.fr), and was supported by France G enomique National infrastructure, funded as part of "Investissement d'avenir" program managed by Agence Nationale pour la Recherche (contract ANR-10-INBS-09). This study also received support from the PhyloAlps project, and we thank H. Holota and A. Iribar. L. Csiba and E. Kepos (Jodrell Laboratory) provided DNA extracts from accessions of the living collection of Royal Botanic Gardens (Kew, Richmond). We are also grateful to D. Stadie (Eisleben), M.S. Vorontsova and D. Goyder (Royal Botanical Gardens, Kew), T. Haevermans and M. Gaudeul (Mus eum National d'Histoire Naturelle de Paris), H. Esser (Munich Botanical Gardens), J. Razanatsoa (Parc de Tsimbazaza, Antananarivo), P. Saumitou-Laprade and P. Vernet (Evo-Eco-Pal eo Lille), T. Josseberger (Botanische G€ arten der Universit€ at Bonn) for providing samples for our study. We thank Freek T. Bakker, Pascal-Antoine Christin, Jan Hackel, Jill K. Olofsson and Herv e Philippe for helpful suggestions.VAN DE PAER ET AL. | 13 DATA ACCESS IBI LITY In the NCBI nucleotide database, all newly assembled chloroplast genomes, mitogenomes and mitochondrial DNA phylogenetic markers are available under the accession numbers provided in Tables 1 and S6. AUTHOR' S CONTRIBUTIONS C.V.D.P. and G.B. designed the study. O.B. and G.B. generated the sequence data. C.V.D.P. and G.B. analysed the data, interpreted the results and wrote the manuscript. O R C I D C eline Van de Paer http://orcid.org/0000-0003-2207-3721

Fig. 1 .

 1 Fig. 1. Alternative hypotheses used for positive selection tests. For the two tested hypotheses, foreground branches of the phylogenetic tree are indicated in red. In hypothesis H1, positive selection has occurred only on the internal branch ancestral to the Jasmineae and LVP clades; while in hypothesis H2, positive selection has occurred on all branches of the Jasmineae and LVP clades.

Fig. 2 .

 2 Fig. 2. ML phylogenetic trees of the Oleaceae obtained with RAxML v.8 (Stamatakis 2014) with the GTR + G model. We reconstructed cpDNA and mtDNA phylogenetic trees based on complete plastomes (Fig. 2.A) and on the concatenated non-coding and coding mtDNA regions (Fig. 2.B), respectively. Bootstrap supports between 70% and 99% are represented with a black point and those inferior to 70% are represented with a gray point. Bootstrap values equal to 100% are not represented on phylogenies.

Fig. 2 .

 2 Fig. 2. End

Figure S1 .

 S1 Figure S1. Substitution rates among 27 protein-coding genes in five Lamiales species. For each protein-coding gene, non-synonymous (d N ) and synonymous (d S ) divergence rates were calculated against Nicotiana tabacum using PAML (Yang, 2007). The star symbol (*) indicates d N /d S ratio (ω) superior to 1. (Aju: Ajuga reptans; Boea: Boea hygrometrica; Hes: Hesperelaea palmeri; Mim: Mimulus guttatus; Salv: Salvia miltiorrhiza)

FN996972

  Olea europaea subsp. maroccana (Greuter & Burdet) P. Vargas et al. Olea woodiana Knobl. subsp. woodiana -A.Costa 02 [MPU], South Africa FN998901 -

Fig. S1 .

 S1 Fig. S1.Master circles of the seven reconstructed mitogenomes of Olea europaea (six accessions) and Chionanthus rupicolus. The outer circle represents the gene content, with protein-coding genes, rRNA and tRNA genes. The inner circle represents repeated sequences with a minimum length of 100 bp in the mitogenome. The large repeats >10 kb are represented in light blue color. The maps were performed with OGDRAW v1.2(Lohse et al. 2007) and CIRCOS(Krzywinski et al. 2009).

Fig

  Fig. S1. Continued

Fig. S3 .

 S3 Fig. S3. Chimeric ORFs (orf_E3a and orf_E3b) specific to the E3.1 mitogenome. The number of bases of the known gene detected in the chimeric ORF is indicated. We represented the genes closest to each ORF, with the distance in bp between them.

Fig. S4 .

 S4 Fig. S4. ML phylogenetic trees reconstructed on nine mtpt segments and their homologous plastid regions extracted from plastomes of Boea hygrometrica, 16 accessions belonging to tribe Oleeae, and two accessions belonging to Fontanesieae and Forsythieae. The mtpt regions are represented in red and their corresponding homologous plastid sequences in green.

Fig

  Fig. S4. Continued

Fig

  Fig. S1. End.

Fig

  Fig.S2.A Mean substitutions rates among the 80 cpDNA genes for clade Jasmineae (noted 'Jasmineae'), clade (noted 'LVP') and for Oleaceae species without clades Jasmineae and LVP (noted 'Others'). For each gene, non-synonymous (d N ) and synonymous (d S ) were calculated against the outgroup species (Nyctanthes arbortristis) using PAML v.4(Yang 2007). The significant differences between groups are indicated by Latin letters for d N and by Greek letters for d S .

Fig

  Fig. S2.B Mean substitutions rates among the 36 mtDNA genes for clade Jasmineae (noted 'Jasmineae'), clade (noted 'LVP') and for Oleaceae species without clades Jasmineae and LVP (noted 'Others'). For each gene, non-synonymous (d N ) and synonymous (d S ) were calculated against the outgroup species (Nyctanthes arbortristis) using PAML v.4 (Yang 2007). The significant differences between groups are indicated by Latin letters for d N and by Greek letters for d S .

Fig

  Fig.S3.A Mean substitutions rates among the eight cpDNA functional categories for clade Jasmineae (noted 'Jasmineae'), clade LVP and for Oleaceae species without clades Jasmineae and LVP (noted 'Other'). For each functional category, non-synonymous (d N ) and synonymous (d S ) were calculated against the outgroup species (Nyctanthes arbortristis) using PAML v.4(Yang 2007). The significant differences between groups are indicated by Latin letters for d N and by Greek letters for d S .

Fig

  Fig.S3.B Mean substitutions rates among the seven mtDNA functional categories for clade Jasmineae (noted 'Jasmineae'), clade LVP and for Oleaceae species without clades Jasmineae and LVP (noted 'Other'). For each functional category, non-synonymous (d N ) and synonymous (d S ) were calculated against the outgroup species (Nyctanthes arbortristis) using PAML v.4(Yang 2007). The significant differences between groups are indicated by Latin letters for d N and by Greek letters for d S .

Fig. 1

 1 Fig.1Phylogenetic reconstruction of Mimosoideae based on complete plastomes. Tree inference was made under a maximum likelihood criterion, using RAxML v.8(Stamatakis 2014). Main lineages (i.e., tribes and genera) usually recognized are indicated (seeLewis et al.

Fig. 2

 2 Fig. 2 Substitution rates among genes accD, clpP, rps2, rps3, ycf1, ycf2, and ycf4 of the eight acacia species belonging to Ingeae, Vachellia, Senegalia, and Acacia s.s. For each protein-coding gene, nonsynonymous (d N ) and synonymous (d S ) rates were calculated against

Fig. 3

 3 Fig. 3 Plastid diversity assessed with cpSSRs in Vachellia spp. a Distribution of haplotypes in locations sampled around Tamanrasset. b Phylogenetic relationships between plastid haplotypes of Vachellia spp.assessed with a reduced median network[START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF]. The data

  et Forestiera, Priogymnanthus et Hesperelaea (Zedane et al. 2016). Ces différentes études ont montré que certains genres (Olea, Chionanthus, Syringa ou Osmanthus) ne sont pas des groupes monophylétiques et nécessiteraient une révision taxonomique. Par ailleurs, des genres tels que Nestegis, Notelaea, Haenianthus ou Picconia ne sont que très peu étudiés. Les travaux de thèse de Zedane (2016) ont permis d'éclairer les relations phylogénétiques au sein des différents genres de la tribu des Oleeae. Zedane et ses collaborateurs ont reconstruit une phylogénie chloroplastique des Oleeae basée sur les plastomes complets de 82 accessions couvrant l'ensemble des genres d'Oleeae. La phylogénie

  American Journal of Botany, 87, 1827-1841. Wang D, Wu YW, Shih ACC et al. (2007) Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA. Molecular Biology and Evolution, 24, 2040-2048.

	Chez les plantes à fleurs, les chloroplastes et les mitochondries sont les seuls organites à (CHAPITRE III). A partir de croisements ou d'observations au microscope à fluorescence, du and research implications for a theory of the Sanger F, Nicklen S, Coulson A (1977) DNA Xia J, Lu J, Wang ZX et al. (2013) Pollen limitation Références origin and evolution of microbial, plant and sequencing with chain-terminating inhibitors. and Allee effect related to population size and "paternal leakeage" a été décrit chez quelques espèces. Il est supposé que le "paternal Adams KL, Palmer JD (2003) Evolution of protein import research from infancy to future animal cells on the precambrian Earth. Yale Proceedings of the National Academy of sex ratio in the endangered Ottelia acuminata leakeage" entraînerait de la recombinaison sexuelle, ce qui permettrait de réduire le fardeau de mitochondrial gene content: gene loss and challenges. Molecular Plant, 9, 798-812. University Press. Sciences of the United States of America, 74, (Hydrocharitaceae): implications for
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[START_REF] Besnard | Combination of chloroplast and mitochondrial DNA polymorphisms to study cytoplasm genetic differentiation in the olive complex (Olea europaea L.)[END_REF]

Combination of chloroplast and mitochondrial DNA polymorphisms to study cytoplasm genetic differentiation in the olive complex (Olea europaea L.). Theor. Appl. Genet. 105, 139-144. Besnard, G.,

Christin, P.A., Malé, P.J., Coissac, E., Ralimanana, H., Vorontsova, M.S., 2013.

Phylogenomics and taxonomy of Lecomtelleae (Poaceae), an isolated, early diverging panicoid tribe from Madagascar. Ann. Bot. 112, 1057-1066.

Table 1 ;

 1 Besnard et al.,

	2007; Besnard, Terral, & Cornille, 2018): three accessions of subsp. eu-
	ropaea ["Stavrovouni Monastery 11" (Cp-II, lineage E1, sublineage e,
	haplotype 4; hereafter E1-e.4), "Vall ee du Fango 5" (Cp-II, lineage E2,
	haplotype 1; hereafter E2.1), "Oeiras 1" (Cp-II, lineage E3, haplotype 1;
	hereafter E3.1)], one of subsp. laperrinei ("Adjelella 10"; Cp-II, lineage

E1, sublineage l1, haplotype 1; hereafter E1-l1.1), one of subsp. guanchica ("La Gomera 10"; Cp-II, lineage M; sublineage g1, haplotype 3; hereafter M-g1.3) and one of subsp. cuspidata ("Menagesha Forest 14"; Cp-I, lineage C2, haplotype 13; hereafter C2.13).

  1, E3.1, M-g1.3 and C2.13). p of mtpt regions (that are considered orthologous in O. europaea; see results) and their plastid homologue regions was also compared. We then calculated p for protein-coding and noncoding regions at the subtribe level considering eight Oleinae

species (i.e., C. parkinsonii, C. rupicolus, For. isabelae, H. palmeri, Ne. apetala, No. lowryi, O. exasperata and O. e. europaea E1-e.4).

  Comparison of plastid-derived regions (mtpt) content among the mitogenomes of Olea europaea, Chionanthus rupicolus, Hesperelaea palmeri and Boea hygrometrica (number of mtpt regions, total length in base pairs, accumulative length of mtpt regions against length of the mitogenome and the plastome, in per cent)

				Amount	Amount
				of the	of the
		Number	Total	mitogenome	plastome
	Taxon	of mtpt	length	(%)	(%)
	Boea hygrometrica	57	55,513 10.1	35.2
	Hesperelaea palmeri but not resolved in the mtDNA phylogeny. The placement of 20 55,761 8.5 35.8
	Chionanthus rupicolus Mediterranean olive lineages (E1, E2, E3 and M) was not resolved 22 34,905 5.2 27.0 either (bs < 80%) in the mtDNA phylogeny (Figure
	O. e. cuspidata C2.13	18	31,905	4.5	20.6
	O. e. europaea E3.1	23	37,560	4.9	29.1
	O. e. guanchica M-g1.3 21	32,545	4.7	25.2
	O. e. europaea E2.1	21	33,211	4.5	25.7
	O. e. europaea E1-e.4	20	33,696	4.7	21.8
	O. e. laperrinei E1-l1.1	20	33,698	4.7	26.1

e. cuspidata C2.13 O. e. europaea E3.1 O. e. guanchica M-g1.3 O. e. europaea E2.1 O. e. europaea E1-e.4 O. e. laperrinei E1-l1.1

  e., C. parkinsonii, C. rupicolus, For. isabelae, H. palmeri, Ne. apetala, No. lowryi, O. exasperata and O. e. europaea E1-e.4). The standard error of p was estimated by bootstrapping on sequences (100 replicates)

			p
	Clade	DNA region type	mtDNA		ptDNA
	Olea	Protein-coding genes	0.022 AE 0.003 0.035 AE 0.005
	europaea
	(5)
		Protein-coding genes-IR -		0.00 AE 0.00
		Noncoding regions	0.041 AE 0.001 0.088 AE 0.008
		Noncoding regions-IR	-		0.021 AE 0.010
		Shared DNA regions	0.076 AE 0.012 0.024 AE 0.009
		(mtpt)
	Oleinae	Protein-coding genes	0.159 AE 0.009 0.532 AE 0.021
	(8)
		Protein-coding genes-IR -		0.124 AE 0.017
		Noncoding regions	0.188 AE 0.014 0.936 AE 0.026
		Noncoding regions-IR	-		0.133 AE 0.022
					LSC			IR A	SSC
	B. hygrometrica
		H. palmeri
	C. rupicolus
	O. 1	2	3	4	5	6	7 8	9

Table 3

 3

) consists of plastid-derived sequences (mtpt) as similarly reported in numerous plants (e.g.,

[START_REF] Alverson | Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)[END_REF][START_REF] Marienfeld | The mitochondrial genome of Arabidopsis is composed of both native and immigrant information[END_REF]

  , rpl32 in poplar[START_REF] Cusack | When gene marriages don't work out: divorce by subfunctionalization[END_REF][START_REF] Ueda | Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus[END_REF] and accD in Trifolium and Campanulaceae(Magee et al. 2010;[START_REF] Rousseau-Gueutin | Potential functional replacement of the plastidic acetyl-CoA carboxylase subunit (accD) gene by recent transfers to the nucleus in some angiosperm lineages[END_REF]. Lastly, plastomes of lineages with an extreme substitution rate acceleration (e.g. Geraniaceae, Campanulaceae, Plantago) appear to contain more repeated regions than those of other angiosperms and to be highly rearranged [with multiple indels, inversions and gene duplications;Cosner et al. (1997Cosner et al. ( , 2004));Chumley et al. (2006);Lee et al.

(2007)

;

Guisinger et al. (2011)

;

[START_REF] Weng | Reconstruction of the ancestral plastid genome in Geraniaceae reveals a correlation between genome rearrangements, repeats and nucleotide substitution rates[END_REF]

;

[START_REF] Zhu | Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates[END_REF]

].

Table 1 .

 1 List of plant accessions sequenced in our study.

	Tribe, subtribe	Species, and name and origin of analyzed individuals
	Jasmineae	Jasminum fluminense Vell.
		-O. Maurin 273 [PET], South Africa
		Jasminum fruticans L.
		-CEFE (J. Thompson), G. Besnard 2016, France
		Jasminum kitchingii Baker
		-F. Rakotonasolo, sn [TAN], Madagascar
		Jasminum polyanthum French.
		-Hervé Gryta, sn, ornamental from Asia
	Fontanesieae	Fontanesia fortunei Carrière
		-T. Jossberger 1829 [BONN], China
	Forsythieae	Forsythia giraldiana Lingelsh.
		-Fliegner et al. SICH1137; coll QBGII-1992.238 [QBG], China
		Forsythia × intermedia Zabel
		-G. Besnard [MPU], ornamental from E. Asia
		Forsythia × mandschurica Uyeki
		-18-40-1968 (Montreal BG, JFRU21), China
	Myxopyreae	Nyctanthes arbor-tristis L.
		-C. Parma 9713 [M], India
	Voucher samples deposited at the herbaria of Kew [K], Munich [M], Bonn [BONN], Montpellier [MPU],
	Missouri [MO], Pretoria [PET] and Antananarivo [TAN].

Table S1 .

 S1 Continued

	Repeat	Start	Match	Start	Identity
	length (bp)	position	direction	position	percentage (%)
	Repeat length (bp)	Start position 114004	Match direction P	Start position 201141	Identity percentage (%) 97.18
		114004	P	135791	98.59
	7975	192316 67020	P D	435791 555343	100.00 97.10
	496	135775 132572	D P	201125 650916	98.39 97.10
	309	172567 513019	D P	610037 552428	99.35 98.55
	176	8692 201778	D D	262772 459579	98.86 98.5
	164	202770 469131	P D	267594 504862	99.39 100.00
	156	261094 1	P P	643905 351561	100.00 100.00
	153	88691 16098	P D	98009 472557	98.69 95.2
	145	130405 308369	D P	513821 638443	100.00 96.77
	129	16246 313708	D P	472714 551989	98.45 98.39
	125	16561 125040	D P	473033 404562	95.20 96.72
	120	183482 19198	D P	272866 513083	93.33 98.31
	118	88726 88785	P P	98009 98009	99.15 100.00
	118	376013 126068	D D	482787 510693	99.15 98.31
	107	204776 238945	P P	500880 552360	100.00 96.61
	105	58304 504868	D P	85353 654477	100.00 100.00
	98	125629 393054	P P	459480 609882	100.00 98.25
	98	527237 45609	D P	654484 537758	100.00 98.21
	95	469137 251733	P P	654440 513082	100.00 98.21
	94	142048 6625	P D	508300 180011	100.00 98.18
	94	341217 19202	P D	615268 251733	98.94 100.00
	90	201688 113944	D P	459479 201435	98.89 96.36
	90	469149 113944	P P	571588 136085	97.78 98.18
	89	125638 19200	P D	201689 341527	98.88 100.00
	87	39758 288438	D D	633208 344694	98.85 96.30
	86	142156 341527	P P	508198 513086	100.00 98.15
	85	15892 14479	D D	472347 199336	95.46 96.23
	83	571595 14479	D P	654440 436693	97.59 96.23
	82	102791 41011	D D	391865 251732	98.78 100.00
	81	215553 52272	D D	428181 433928	100.00 96.23
	80	191683 282272	P D	521201 344623	100.00 98.11
	77	7434 342033	P D	28907 469057	100.00 96.23
	76	494294 19202	D D	540035 41012	97.37 100.00
	75	314298 36584	D P	606097 405527	98.67 96.15
	74	288508 41012	D D	344748 341529	97.30 100.00
	73	502372 41012	D P	513490 513086	98.63 98.08
		251733	D	341529	100.00
		385227	D	654545	96.15
		129290	P	458242	98.04
		314927	D	385227	98.04

Table S1 .

 S1 Continued

	Repeat	Start	Match	Start	Identity
	length (bp)	position	direction	position	percentage (%)
		349634	D	508263	98.04
		469137	P	527237	100.00
		504868	P	527237	100.00
		130529	P	314519	96.00
		168060	D	168096	98.00
		365567	D	497112	98.00
		365613	D	497172	100.00
		494209	D	539960	96.00
		31246	D	128836	100.00
		124229	P	613841	97.96
		313998	P	620051	100.00
		314925	D	527296	95.92
		347295	D	612152	100.00
		16170	D	472638	95.8
		29011	D	83560	97.92
		64383	P	142124	100.00
		320223	D	403438	95.74
		385227	D	527298	97.87
		454881	P	583085	95.74
		494323	D	540064	97.87
		494882	D	540610	95.74
		8124	P	32702	100.00
		45841	P	537696	95.65
		504880	P	571632	97.83
		23408	P	382354	100.00
		41163	D	506842	100.00
		135776	D	576939	95.56
		308386	P	638443	100.00
		342061	D	504832	97.78
		382960	D	641035	95.56
		494160	D	539915	95.56
		8413	D	395045	97.73
		114100	P	137242	95.45
		129292	P	513004	95.45
		321762	D	382414	97.73
		458247	D	513004	97.73
		16055	D	472503	95.3
		41228	P	433845	95.35
		125058	P	404562	100.00

Table S1 .

 S1 End

	Repeat	Start	Match	Start	Identity
	length (bp)	position	direction	position	percentage (%)
		129273	D	433033	100.00
		136153	D	155433	95.35
		136538	D	381742	100.00
		313727	P	551989	100.00
		314925	D	654543	97.67
		45623	P	537758	100.00
		54763	P	248929	95.24
		116025	P	577381	95.24
		450071	D	537324	95.24
		505127	D	516942	95.24
		5935	P	382085	95.12
		6625	P	219574	100.00
		29119	D	83644	95.12
		68397	D	463470	97.56
		83556	D	113089	97.56
		84454	P	381746	95.12
		137257	D	357334	95.12
		148300	D	540879	95.12
		180011	P	219574	100.00
		216731	D	521486	100.00
		382096	D	640153	95.12
		425682	P	507295	95.12
		527235	D	571637	95.12
		3160	P	85352	95.00
		16473	D	472942	95.00
		28975	D	516746	95.00
		29010	D	113092	97.50
		52289	D	433068	95.00
		65224	D	442676	95.00
		65224	P	193366	95.00
		98745	P	163695	97.50
		99893	D	314877	95.00
		111174	P	314296	95.00
		113906	P	136133	95.00
		138673	P	433879	95.00
		331236	P	331238	95.00
		494765	D	540490	95.00

Table S3 .

 S3 Comparison of gene content and number of repeats > 1 kb among the mitogenomes of Lamiales and Nicotiana tabacum (Solanales). The annotations of the mitogenomes were extracted from GenBank. (+: gene presence; -: gene absence; ᴪ: pseudogene)

	Gene	Nicotiana tabacum	Hesperelaea palmeri	Boea hygrometrica	Mimulus guttatus	Salvia miltiorrhiza	Ajuga reptans
	atp1	+	+ (ATG)*	+	+	+	+
	atp4	+	+ (ATG)	+	+	+	+
	atp6	+	+ (ACG)	+	+	+	+
	atp8	-	+ (ATG)	+	+	+	+
	atp9	+	+ (ATG)	+	+	+	+
	ccmB	+	+ (ATG)	+	+	+	+
	ccmC	-	+ (ATG)	+	+	+	+
	ccmFc	+	+ (ATG)	+	+	+	+
	ccmFn	+	+ (ATG)	+	+	+	+
	cox1	+	+ (ATG)	+	+	+	+
	cox2	+	+ (ATG)	+	+	+	+
	cox3	+	+ (ATG)	+	+	+	+
	cytB	+	+ (ATG)	+	+	+	+
	matR	+	+ (ATG)	+	+	+	+
	mttB	+	+ (TTG)	-	+	+	+
	nad1	+	+ (ATG)	+	+	+	+
	nad2	+	+ (ATG)	+	+	+	+
	nad3	+	+ (ATG)	+	+	+	+
	nad4	+	+ (ATG)	+	+	+	+
	nad4L	+	+ (ACG)	+	+	+	+
	nad5	+	+ (ATG)	+	+	+	+
	nad6	+	+ (ATG)	+	+	+	+
	nad7	+	+ (ATG)	+	+	+	+
	nad9	+	+ (ATG)	+	+	+	+
	orf3	+	+ (ATG)	+	+	+	+
	orf101	-	+ (ATG)	-	-	+	-
	orf111a	-	+ (ATG)	-	-	+	-
	orf117a	-	+ (ATG)	-	-	+	-
	orf121	-	+ (ATG)	-	-	+	-
	orf122c	-	+ (ATG)	-	-	+	-
	orf182a	-	+ (ATG)	-	-	+	-
	petG	-	-	-	-	+	-
	petL	-	+ (ATG)	-	-	+	-
	rpl2	-	+ (ATG)	-	ᴪ	+	+
	rpl5	+	+ (ATG)	-	+	+	-
	rpl10	-	+ (ATG)	-	+	+	-
	rpl16	+	+ (GTG)	ᴪ	+	+	-
	rpl23	-	+ (ATG)	-	-	+	+
	* Start codon is given in brackets for each protein-coding gene of Hesperelaea

Table S3 .

 S3 End

	Gene	Nicotiana tabacum	Hesperelaea palmeri	Boea hygrometrica	Mimulus guttatus	Salvia miltiorrhiza	Ajuga reptans
	rps3	+	+ (ATG)	+	+	+	+
	rps4	+	+ (ACG)	+	+	+	ᴪ
	rps7	-	+ (ATG)	+	-	+	-
	rps10	+	+ (ACG)	-	+	+	-
	rps12	+	+ (ATG)	+	+	+	+
	rps13	+	+ (ATG)	+	+	+	+
	rps14	ᴪ	+ (ATG)	-	+	+	-
	rps19	+	-	-	-	-	-
	sdh3	+	+ (ATG)	+	+	-	-
	sdh4	-	+ (GTG)	-	+	+	-
	rrn5	+	+	+	+	+	+
	rrn18	+	+	+	+	+	+
	rrn26	+	+	+	+	+	+
	trnC-GCA	+	+	+	+	+	+
	trnD-GTC	+	+	+	+	+	+
	trnE-TTC	+	+	+	+	+	+
	trnF-GAA	+	+	+	+	+	+
	trnfM-CAT	+	-	-	+	+	+
	trnG-GCC	+	+	+	+	+	-
	trnH-GTG	+	+	+	+	+	-
	trnI-CAT	-	+	-	+	+	+
	trnK-TTT	+	+	+	+	+	-
	trnL-CAA	-	-	+	+	+	-
	trnL-GAG	-	-	-	+	-	-
	trnL-TAA	-	-	+	+	-	-
	trnM-CAT	+	+	+	+	+	+
	trnN-GTT	+	+	+	+	+	+
	trnP-TGG	+	+	+	+	+	+
	trnQ-TTG	+	+	+	+	+	+
	trnR-ACG	-	+	+	-	+	-
	trnR-TCT	-	+	-	-	+	-
	trnS-GCT	-	+	+	+	+	+
	trnS-GGA	+	+	+	+	+	-
	trnS-TGA	-	+	+	+	+	+
	trnT-TGT	-	-	+	+	-	-
	trnV-GAC	-	-	+	-	+	-
	trnW-CCA	+	+	+	+	+	+
	trnY-GTA	+	+	+	+	+	+
	Total protein-coding genes	32	46	30	36	46	30
	Total rRNA genes	3	3	3	3	3	3
	Total tRNA genes	15	19	21	22	22	14
	Number of repeats > 1kb	5	1	1	3	3	0

Table S4 .

 S4 Continued

	ORFs	Length	Start position	Final position	Orientation
	orf40	318	435564	435881	reverse
	orf41	318	193401	193718	reverse
	orf42	321	98825	99145	forward
	orf43	321	266670	266990	reverse
	orf44	321	185364	185684	reverse
	orf45	324	177748	178071	forward
	orf46	324	252473	252796	reverse
	orf47	327	567655	567981	reverse
	orf48	330	252594	252923	forward
	orf49	330	266195	266524	forward
	orf50	330	301985	302314	forward
	orf51	330	596928	597257	reverse
	orf52	333	5083	5415	forward
	orf53	333	21954	22286	forward
	orf54	333	376316	376648	forward
	orf55	333	520389	520721	forward
	orf56	333	475430	475762	reverse
	orf57	336	90115	90450	reverse
	orf58	336	21306	21641	forward
	orf59	336	339734	340069	forward
	orf60	336	454078	454413	forward
	orf61	336	239417	239752	reverse
	orf62	339	621652	621990	forward
	orf63	339	433425	433763	forward
	orf64	339	500932	501270	reverse
	orf65	339	526287	526625	forward
	orf66	342	356961	357302	reverse
	orf67	345	549356	549700	forward
	orf68	348	76426	76773	reverse
	orf69	348	11067	11414	reverse
	orf70	348	100811	101158	forward
	orf71	348	111218	111565	forward
	orf72	348	459806	460153	reverse
	orf73	348	524846	525193	forward
	orf74	348	528254	528601	forward
	orf75	351	139488	139838	reverse
	orf76	351	101442	101792	reverse
	orf77	351	10523	10873	reverse
	orf78	351	321670	322020	forward
	orf79	351	630834	631184	reverse
	orf80	354	74481	74834	reverse
	orf81	354	456675	457028	forward

Table S4 .

 S4 Continued

	ORFs	Length	Start position	Final position	Orientation
	orf82	354	508357	508710	forward
	orf83	354	559239	559592	reverse
	orf84	354	488007	488360	reverse
	orf85	354	121219	121572	forward
	orf86	357	344776	345132	forward
	orf87	357	569464	569820	reverse
	orf88	363	189762	190124	forward
	orf89	366	86180	86545	reverse
	orf90	366	119896	120261	forward
	orf91	366	260060	260425	reverse
	orf92	369	132637	133005	forward
	orf93	372	271528	271899	reverse
	orf94	375	397237	397611	forward
	orf95	375	59584	59958	forward
	orf96	375	386546	386920	reverse
	orf97	378	30320	30697	reverse
	orf98	378	21932	22309	reverse
	orf99	378	43384	43761	forward
	orf100	381	647801	648181	forward
	orf102	381	83575	83955	forward
	orf103	390	380080	380469	forward
	orf104	393	184746	185138	forward
	orf105	393	101488	101880	forward
	orf106	396	33053	33448	forward
	orf107	396	639715	640110	forward
	orf108	399	174047	174445	forward
	orf109	399	233604	234002	forward
	orf110	399	36679	37077	forward
	orf111	399	402022	402420	reverse
	orf112	402	2251	2652	reverse
	orf113	405	300822	301226	reverse
	orf114	408	99588	99995	forward
	orf115	411	176825	177235	reverse
	orf116	414	89235	89648	reverse
	orf117	417	134807	135223	forward
	orf118	417	195194	195610	forward
	orf119	417	440471	440887	reverse
	orf120	420	165428	165847	forward
	orf121	420	406602	407021	forward
	orf122	423	253615	254037	forward
	orf123	423	513960	514382	forward
	orf124	426	342277	342702	forward

Table S4 .

 S4 Continued

	ORFs	Length	Start position	Final position	Orientation
	orf125	426	466575	467	Forward
	orf126	435	161245	161679	reverse
	orf127	435	272203	272637	reverse
	orf128	447	208680	209126	forward
	orf129	450	380719	381168	forward
	orf130	456	78332	78787	forward
	orf131	459	559640	560098	forward
	orf132	462	574819	575280	reverse
	orf133	465	555656	556120	reverse
	orf134	465	417367	417831	reverse
	orf135	471	226011	226481	forward
	orf136	471	631262	631732	reverse
	orf137	486	378625	379110	reverse
	orf138	489	379	867	reverse
	orf139	495	322409	322903	reverse
	orf140	501	309193	309693	forward
	orf141	519	121158	121676	reverse
	orf142	528	508562	509089	forward
	orf143	540	151856	152395	reverse
	orf144	540	104629	105168	reverse
	orf145	546	301463	302008	reverse
	orf146	561	529819	530379	forward
	orf147	564	555795	556358	reverse
	orf148	567	415133	415699	reverse
	orf149	570	149347	149916	reverse
	orf150	570	441530	442099	forward
	orf151	570	193982	194551	reverse
	orf152	576	605529	606104	reverse
	orf153	582	9493	10074	forward
	orf154	594	136632	137225	reverse
	orf155	594	269735	270328	reverse
	orf156	600	379439	380038	forward
	orf157	627	64465	65091	forward
	orf158	633	141386	142018	forward
	orf159	633	516801	517433	forward
	orf160	642	476839	477480	reverse
	orf161	663	207978	208640	forward
	orf162	672	146002	146673	reverse
	orf163	732	180372	181103	reverse
	orf164	744	407244	407987	forward
	orf165	765	445409	446173	reverse
	orf166	774	387306	388079	forward

Table S4 .

 S4 End

	ORFs	Length	Start position	Final position	Orientation
	orf167	822	262017	262838	Reverse
	orf168	834	46796	47629	reverse
	orf169	870	113054	113923	forward
	orf170	885	608880	609764	reverse
	orf171	921	150179	151099	reverse
	orf172	945	206273	207217	forward
	orf173	963	474096	475058	reverse
	orf174	1026	448145	449170	reverse
	orf175	1062	154322	155383	reverse
	orf176	1062	34661	35722	forward
	orf177	1173	388076	389248	forward
	orf178	1176	200288	201463	reverse
	orf179	1224	410964	412187	forward
	orf180	1242	545617	546858	forward
	orf181	1386	418088	419473	reverse
	orf182	1485	478610	480094	reverse
	orf183	1605	585577	587181	reverse
	orf184	1653	415689	417341	reverse
	orf185	1953	510750	512702	forward
	orf186	2283	389149	391431	forward
	orf187	2757	177238	179994	reverse

Table S1 .

 S1 Summary of plastome and mitogenome sequencing and assembly for each individual. (N: number; SD: Standard Deviation)

Table S3 .

 S3 End

	Product group	Gene	E1-e.4 O. e. europaea	E1-l1.1 O. e. laperrinei	E2.1 O. e. europaea	E3.1 O. e. europaea	M-g1.3 O. e. guanchica	C2.13 O. e. cuspidata	Chionanthus rupicolus
	Other proteins	matR	+	+	2+	+	+	+	+
		mttB	+	+	+	2+	+	+	+
		petL*	+	+	ᴪ?	+	+	+	-
		petN*	+	+	+	+	+	+	+
		psbD*	+	+	+	+	+	+	-
	rRNA	rrn5	+	+	+	+	+	+	+
		rrn18	+	+	+	+	+	+	+
		rrn26	+	+	+	+	+	+	+
	tRNA	trnC-GCA	+	+	+	+	+	+	+
		trnC-GCA*	-	-	-	-	-	-	+
		trnD-GTC*	2+	2+	2+	2+	2+	2+	2+
		trnE-TTC	+	+	+	+	+	+	+
		trnE-TTC*	+	+	+	+	+	+	+
		trnF-GAA	+	+	+	+	+	+	+
		trnG-GCC	+	+	+	+	+	+	+
		trnfM-CAT*	+	+	+	+	+	+	-
		trnH-GTG	+	+	+	+	+	+	+
		trnK-TTT	+	+	+	+	+	+	+
		trnM-CAT	3+	3+	3+	3+	3+	3+	3+
		trnM-CAT*	+	+	+	+	+	+	+
		trnN-GTT*	2+	2+	2+	2+	2+	2+	2+
		trnP-TGG	+	+	+	+	+	+	+
		trnQ-TTG	+	+	+	+	+	+	+
		trnS-GCT	+	+	+	+	+	+	+
		trnS-TGA	+	+	+	+	+	+	+
		trnS-GGA*	+	+	+	+	+	+	+
		trnT-GGT*	+	+	+	+	+	+	-
		trnV-TAC*	+	+	+	+	+	+	-
		trnW-CCA*	+	+	+	+	+	+	+
		trnY-GTA	+	+	+	+	+	+	+
		trnY-GTA*	+

Table S4 .

 S4 Repeat content among the mitogenomes of Olea europaea and Chionanthus rupicolus

	Repeat length	E1-e.4	E1-l1.1	E2.1	E3.1	M-g1.3	C2.13	Chionanthus
	(bp)	O. e. europaea	O. e. laperrinei	O. e. europaea	O. e. europaea	O. e. guanchica	O. e. cuspidata	rupicolus
	40-60	65	65	60	65	61	63	78
	61-80	20	20	22	20	21	22	27
	81-100	14	14	15	11	15	10	21
	101-120	1	1	2	3	3	3
	121-140

Table S6 .

 S6 GenBank numbers for each assembled mitochondrial gene region in ten Oleaceae species

		Gene	Chionanthus	Fontanesia	Forestiera	Forsythia×	Fraxinus	Nestegis	Noronhia	Olea	Schrebera	Syringa
			parkinsonii	fortunei	isabelae	intermedia	ornus	apetala	lowryi	exasperata	arborea	vulgaris
	Genomic	Length	Parsimony-		mtDNA		ptDNA
	region	(bp)	informative sites
	mtpt1 mtpt2 mtpt3 mtpt4 mtpt5 mtpt6 mtpt7 mtpt8 mtpt9	497 3110 3154 1898 512 1013 646 712 2183		12 14 52 8 7 98 9 69 23	0.0 2.8×10 -4 ± 2.1×10 -4 6.5×10 -4 ± 2.6×10 -4 4.6×10 -4 ± 3.6×10 -4 1.0×10 -3 ± 1.0×10 -3 7.0×10 -3 ± 5.6×10 -4 3.8×10 -3 ± 1.8×10 -3 1.4×10 -3 ± 8.6×10 -4 7.4×10 -4 ± 4.4×10 -4	0.0 2.1×10 -4 ± 1.6×10 -4 1.1×10 -4 ± 1.1×10 -4 1.8×10 -4 ± 1.6×10 -4 0.0 3.4×10 -4 ± 3.3×10 -4 1.4×10 -3 ± 9.4×10 -4 9.9×10 -4 ± 6.4×10 -4 0.0

Table S7 .

 S7 Nucleotide polymorphisms at the 21 parsimony informative sites among the plastomes of the olive complex. Sites are numbered according to their position in the plastome of O. e. laperrinei E1-l1.1. All sites were located either in the LSC or SSC (Long or Short Single Copies, respectively). When the site is located in a coding sequence, the gene name, the position of the substitution in the codon and the aminoacid change for non-synonymous substitutions are indicated.

												Sites
	Accession	2	9082	13362	18642	45532	48090	51579	52165	56950	64704	67654	83304	85525	85589	112754	114452	115364	118316	122532	127059	129178
		LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	LSC	SSC	SSC	SSC	SSC	SSC	SSC	SSC
	O. e. cuspidata C2.13	A

Table S8 .

 S8 Continued

																Sites
	Accession	259497	265110	276117	279456	281992	282455	282932	283164	295854	300051	301105	301753	302527	302609	303919	308204	311059	311775	326439	334081	334581	338510	339310	349116	351907	354564	356733	380955	383338	392471
						mtpt
	O. e. cuspidata
	C2.13
	Gene location
	Position in codon
	Non syn. changes

Table S8 .

 S8 Continued

																Sites
	Accession	399823	413679	416549	416818	416913	417505	418532	418532	421440	421444	430394	433100	433102	435700	462965	465836	469087	473913	476879	482112	486679	496068	498087	504968	512223	522023	523270	530752	531126	535430
																															mtpt
	O. e. cuspidata	A
	C2.13

Table S8 .

 S8 Continued

																Sites
	Accession	537598	537609	544032	546114	551289	558335	563412	563767	587413	587593	588311	595120	602501	605913	608724	609518	609658	609738	609878	611806	616230	619053	619060	622580	622941	625645	627248	637501	638174
																													mtpt
	O. e. cuspidata	A
	C2.13

Table S8 .

 S8 End

						Sites
		Accession	648972	649951	652195	675733	692527	692716	698722
		O. e. cuspidata	T	C	T	G	-	-	C
		C2.13
		O. e. europaea	T	A	T	G	T	T	-
		E3.1
		O. e. guanchica	T	A	T	T	T	T	C
		M-g1.3
		O. e. europaea	C	C	T	G	C	T	-
		E2.1
		O. e. europaea	C	A	A	T	C	G	A
		E1-e.4
		O. e. laperrinei	C	A	A	T	C	G	A
	Gene location Position in codon	E1.l1.1
	Non syn. changes	Gene location
		Position in codon
		Non syn. changes

  . First, four species belong to the Vachellia lineage: Vachellia tortilis (Forssk.) Galasso & Banfi subsp. raddiana (Savi) Kyal. & Boatwr. (hereafter referred to V. tortilis), Vachellia flava (Forssk.) Kyal. & Boatwr., Vachellia nilotica (L.) P. J. H. Hurter & Mabb. subsp. tomentosa (Benth.) Kyal. & Boatwr. (hereafter referred to V. nilotica), and Vachellia seyal (Delile) P. J. H. Hurter; second, Faidherbia albida (Delile) A. Chev. belongs to tribe Ingeae; and third, Senegalia laeta (R. Br. ex Benth.) Seigler & Ebinger belongs to the Senegalia lineage. These species show a large distribution range toward the Sahelian and southern Africa, as well as on the Asian continent (Table

Table 1

 1 Description of acacia populations analyzed in the present study, including the taxon, the location (with GPS coordinates; Lat., latitude; long., longitude), the location altitude (Alt.; in m), and the number of individuals analyzed per location (number)

	Taxon	Location	GPS coordinates		Alt.	Number	Individuals used for GS
			Lat.	Long.
	Vachellia flava	Adriane	22.47°N	05.35°E	1483	14	BAdriane 1Â
		glala	22.38°N	05.36°E	1485	13	-
	Vachellia nilotica	Amezgin	22.33°N	05.23°E	1255	18	BAmezgin 20V
	achellia tortilis	Nezmet	22.44°N	05.49°E	1338	13	BNezmet 19 and 23Â
		driane	22.47°N	05.35°E	1296	10	-
		Tassena	22.48°N	05.36°E	1451	8	-
		Amezgin	22.33°N	05.23°E	1255	3	-
		Mazoliet	22.58°N	05.40°E	1828	1	-
	Vachellia seyal	Mazoliet	22.58°N	05.40°E	1828	23	BMazoliet 36Â
		karakar	23.02°N	05.42°E	1866	19	-
	Faidherbia albida	In Zbib	22.47°N	05.37°E	1423	10	BIn Zbib 5Ô
		uarsedakfis	22.49°N	05.58°E	1426	4	-
		Ihghi	23.03°N	05.12°E	1084	1	-
		Tassenna	22.48°N	05.36°E	1451	1	-
		Inemrus	23.08°N	05.59°E	1713	1	-
		Inarjiwen	23.11°N	06.02°E	1975	1	-
	Senegalia laeta	In Zbib	22.47°N	05.37°E	1423	12	BIn Zbib 8T
		assena	22.48°N	05.36°E	1451	2	-

Table 2

 2 Primers used to amplify by PCR the 21 cpSSR loci with their allele size range (in bp) on the six studied acacia species, and M13-primer fluorochrome (Flu) used for each locus (Y = YAK, F = 6-FAM, or A = AT550)

	No. of	Primers
	locus

Table 3

 3 Summary of p values estimated by comparing model A versus model A′ and model A versus model A′ using a likelihood ratio test (LRT)We performed these tests under two hypotheses assuming a potential positive selection either on the internal branch ancestral to Faidherbia albida, Inga leiocalycina, and Acacia ligulata (hypothesis H1) or on all branches of the clade including F. albida, I. leiocalycina, and A. ligulata (hypothesis H2).

	Hypothesis	Models	df	Gene
				accD	clpP	rps2	rps3	ycf1	ycf2	ycf4
	H1	A versus M1a	2	0.6393	0.0022***	1	0.9337	0.0584*	0.0595*	0.6311
		A versus A′	1	0.3442	0.0011***	1	0.7111	0.0172**	0.0126**	0.3388
	H2	A versus M1a	2	#	#	#	#	#	#	0.0384**
		A versus A′	1	#	#	0.0563*	#	#	#	0.0444**

Results on other genes are given in

Table S4 df degree(s) of freedom Significance of tests: *p ≤ 0.1; **p ≤ 0.05; ***p ≤ 0.01; # p ≤ 0.001 accD

Table 4

 4 Summary of the Akaike Information Criterion (AIC) used to determine the best-fit hypothesis Hypothesis

Table 6

 6 Plastid haplotype profiles based on length variation at the 21 cpSSR loci Hapl. no. Locus no. a 148 170 232 248 94 118 136 146 166 173 204 262 106 134 153 161 218 241 F. albidaFor each locus, the length of the allele is given in base pair. Based on our sampling in the Hoggar mountains, each haplotype is unique to a taxon Hapl. no. Haplotype number a Alleles sequenced with the Sanger method to check the cause of length polymorphism

	Taxon

C.Van de Paer et al. / Gene 594 (2016) 197-202

Table S2.B

TableS3.B

Table S4.A Continued

Table S4.A End

Table S4.B

Table S4.B Continued

Table S4.B End

Fig. S2.A Continued

Fig. S2.B Continued

Tree Genetics & Genomes (2017) 13:48

b Reverse-primer tailing: gtgtctt

Acknowledgments

This work was funded by the Regional Council Midi-Pyrenees (AAP 13053637, 2014-EDB-UT3-DOCT) and the LABEX entitled TULIP managed by Agence Nationale de la Recherche (ANR-10-LABX-0041). We also thank Jan Hackel for helpful comments, and the Missouri Botanical Garden (MO) for permission to extract DNA from a leaf of Hesperelaea.

Acknowledgments This work has been conducted in the Laboratoire Evolution & Diversité Biologique (EDB) part of the LABEX entitled TULIP managed by Agence Nationale de la Recherche (ANR-10-LABX-0041). CVDP and GB were funded by the Regional Council Midi-Pyrenees (AAP 13053637, 2014-EDB-UT3-DOCT). This work was performed in collaboration with the GeT core facility, Toulouse, France (http://get.genotoul.fr), and was supported by France Génomique National infrastructure, funded as part of BInvestissement d'avenir^program managed by Agence Nationale pour la Recherche (ANR-10-INBS-09). We are grateful to Pascal-Antoine Christin for helpful advices on the use of positive selection tests and Salah Abdelaoui who helped for the plant sampling. Author's contribution statement MM, DBC, and GB conceived the initial project; MM and DBC conducted field expeditions and sampling; MM, SM, OB, and GB performed laboratory works; CVDP and GB analyzed the data; MM, CVDP, and GB wrote the paper; and all authors commented and approved the final version of the manuscript.

Compliance with ethical standards

* One intron of nad1, nad2, nad5 (all species) and nad4 (F. fortunei and S. arborea) was not assembled (and indicated by 100 N in the submitted gene accession)

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2016.09.007.

CHAPITRE III

Genomic consequences of occasional biparental transmission of organelles in two lineages of the olive family (Oleaceae)

VAN DE PAER C., BESNARD G.

In preparation

Abstract

In most of flowering plants, the inheritance of DNA-containing organelles (i.e. chloroplasts and mitochondria) is uniparental, and usually maternal. However, occasional transmission of organelles via pollen (i.e. paternal leakage) has been reported in numerous angiosperm lineages. The genomic consequences of paternal leakage on the evolutionary trajectory of chloroplastic and mitochondrial genomes (hereafter plastomes and mitogenomes, respectively) remain unclear. A link between accelerations of the evolutionary rates with genomic rearrangements of organellar genomes and paternal leakage seems, however, to occur. In this study, we investigated the genomic consequences of paternal leakage in the Oleaceae family, in which both strict maternal inheritance and potential paternal leakage of organelles occur. Two independent lineages, the Jasmineae and a clade of Ligustrinae (LVP), exhibit potential transmission of organellar genomes via pollen. We highlighted large extensions/contractions of the Inverted Repeats (IR) in plastomes of both lineages. In particular, the plastomes of Jasmineae have multiple indels and have lost the gene accD. In addition, we detected an acceleration of the evolutionary rate in plastomes of these two lineages. While genes coding for subunits of photosynthetic complexes appear to be unaffected by change events of the evolutionary rate, an acceleration of the non-synonymous substitution rate (d N ) was detected for 42 and 19% of chloroplastic genes in Jasmineae and the LVP clade, respectively. We found that positive selection occurred on ten genes in Jasmineae, including ycf1, ycf2, and clpP. Although mitochondria were observed in pollen of Jasmineae, we do not detect clear evidences for changes in the evolutionary rates of mitochondrial genes in this lineage.

Key words: accD, clpP, mitochondria, paternal leakage, phylogeny, plastid, positive selection Table 3. List of genes for which the model A (assuming a positive selection on foreground branches) was significantly better than other models under the hypothesis H1, which assumes a potential positive selection on the internal branch of the clade, or the hypothesis H2, which assumes a potential positive selection on all branches of the clade. For ycf2, ccmFc and rps3, both models A were not significantly different under the hypotheses H1 and H2, which assumes a positive selection on at least the internal branch of the clade. S2. List of accessions from GenBank used to reconstruct the phylogenetic tree in Fig.

S5

Table S3. Comparison of gene content among the mitogenomes of Olea europaea and Chionanthus rupicolus Table S4. Repeat content among the mitogenomes of Olea europaea and Chionanthus rupicolus Table S5. Estimates of the nucleotide diversity among the olive complex for the nine mtpt regions and their ptDNA homologous regions Table S6. GenBank numbers for each assembled mitochondrial gene region in ten Oleaceae species Table S7. Nucleotide polymorphisms at the 21 parsimony informative sites among the plastomes of the olive complex Table S8. Nucleotide polymorphisms at the 156 parsimony informative sites among the mitogenomes of the olive complex In preparation

SI Appendix includes:

Table S1. List of species for which we excluded the mtDNA regions.

Table S2. Results of the strict molecular clock violation tests for cpDNA (Table S2.A) and mtDNA (Table S2.B) genes.

Table S3. Results of local molecular clock tests for cpDNA (Table S3.A) and mtDNA (Table S3.B) genes.

Table S4. Results of positive selection tests for cpDNA (Table S4.A) and mtDNA (Table S4.B) genes. S4.A) and mtDNA (Table S4.B) genes, using PAML v.4 (Yang 2007). For each clade independently, we compared AIC values of the model M1a with both models A and A' under the hypothesis H1 (assuming a potential positive selection on the internal branch of the clade) and the hypothesis H2 (assuming a potential positive selection on all branches of the clade). For each gene, models with the lowest AIC value is indicated in bold. We considered that models were non-significantly different if the difference between their AIC was inferior to six (ΔAIC<6). We highlighted in blue for Jasmineae and in gray for LVP the models which were not significantly different from the model with the lowest AIC value. Phylogenies reconstructed from plastomes and nrDNA units show fully congruent relationships between the four Vachellia species. A close relationship is inferred for V. nilotica and V. flava based on both cpDNA and nrDNA, and their non-distinction based on the nrDNA cluster suggests that these taxa may have diverged in a very recent past or belong to a species complex (e.g., Ali andQaiser 1980, 1992). In addition, we assembled two different nrDNA types in S. laeta, and our phylogenetic analysis shows that these two sequences are not resolved in a mono-phyletic lineage within Senegalia but are intermingled with those of S. senegal (Supplementary Methods), supporting that S. laeta belongs to the S. senegal complex [START_REF] Chevalier | La diversité génétique dans le complexe Acacia senegal[END_REF]. Our observation also likely reflects the hybrid status of S. laeta as has been suggested by some authors [START_REF] Giffard | Les gommiers: Acacia senegal Willd., Acacia laeta R. Br[END_REF][START_REF] Amin | Acacia laeta (R. Br.) ex. Benth., considered as a species of hybrid origin[END_REF][START_REF] Ross | A conspectus of the African Acacia species[END_REF]. The reported triploidy of this species [START_REF] Ross | A conspectus of the African Acacia species[END_REF] suggests that S. laeta may be allopolyploid (or segmental autopolyploid) as frequently observed in the S. senegal complex [START_REF] Chevalier | Diversité génétique des acacias[END_REF][START_REF] Assoumane | Highlighting the occurrence of tetraploidy in Acacia senegal (L.) Willd. and genetic variation patterns in its natural range revealed by DNA microsatellite markers[END_REF][START_REF] Odee | Plio-Pleistocene history and phylogeography of Acacia senegal in dry woodlands and savannahs of sub-Saharan tropical Africa: evidence of early colonisation and recent range expansion[END_REF][START_REF] Odee | Rangewide ploidy variation and evolution in Acacia senegal: a north-south divide?[END_REF][START_REF] Diallo | Study of quantitative genetics of gum arabic production complicated by variability in ploidy level of Acacia senegal (L.) Willd[END_REF].

On contrasted evolutionary rates in plastomes of mimosoids

Our phylogenetic analyses based on plastomes confirm that the evolutionary rates are heterogeneous among mimosoids [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF][START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF]. Compared to previous studies, adding members of Faidherbia (Ingeae), Vachellia, and Senegalia in the mimosoid plastome phylogeny allows delimiting a clade (tribe Ingeae + Acacia s.s.) with longer branches resulting from accelerated global substitution rates (Fig. S5). Interestingly, the three analyzed species of this clade also share an extension of the IR (Fig. S3) as already noticed by [START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF] and [START_REF] Williams | The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene[END_REF], respectively, on Acacia and Inga. Our analyses confirm that several genes [(encoding proteins involved in different functions; caseinolytic protease (clpP), ribosomal protein (rps2, rps3), acetyl-coA carboxylase (accD), probable ATPase (ycf2), photosystem-I assembly protein (ycf4)] show accelerated synonymous and non-synonymous substitution rates in tribe Ingeae + Acacia s.s., with evidence for positive selection (Fig. 2; Table 3). These analyses also suggest that positive selection was not episodic (in relation to the IR extension) but seems widespread on all branches of the clade following the IR extension (i.e., hypothesis H2; Table 4), especially on clpP, accD, ycf1, and ycf2 as similarly observed in several lineages of Sileneae (Erixon and Oxelman 2008;Sloan et al. 2014;[START_REF] Rockenbach | Positive selection in rapidly evolving plastid-nuclear enzyme complexes[END_REF]). An evolutionary transition related to the IR extension could be associated to changes of some selective pressures offering new evolutionary trajectories to some genes. IR extension or reduction coupled to an accelerated evolutionary rate have been also observed in plastomes of Papilionoideae legumes (e.g., [START_REF] Guo | Rapid evolutionary change of common bean (Phaseolus vulgaris L.) plastome, and the genomic diversification of legume chloroplasts[END_REF]Magee et al. 2010;[START_REF] Dugas | Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP[END_REF] and other plant families [e.g., Geraniaceae (Chumley et al. 2006;Guisinger et al. 2011;[START_REF] Blazier | Variable presence of the inverted repeat and plastome stability in Erodium[END_REF], Oleaceae (Lee et al. 2007; C. Van de Paer et al., in prep.), Apocynaceae [START_REF] Ku | The complete plastid genome sequence of Madagascar periwinkle Catharanthus roseus (L.) G. Don: plastid genome evolution, molecular marker identification, and phylogenetic implications in asterids[END_REF], Caryophyllaceae (Sloan et al. 2014), Apiaceae (Downie and Jansen 2015), Eucommiaceae (Wang et al. 2016)]. The reasons for such recurrent switches need to be unraveled. In tribe Ingeae and Acacia s.s., this could be an indirect consequence of a non-strict maternal inheritance of chloroplasts (Reboud and Zeyl 1994) potentially leading to heteroplasmy than in turn could modify selective pressures. We suggest that the plastid inheritance mode should be better investigated in mimosoids to test for this hypothesis. Finally, contrasted Vachellia flava

Conflict of interest The authors declare that they have no conflict of interest.

Author : Céline VAN DE PAER Title : Structural diversity and contrasted evolution of cytoplasmic genomes in flowering plants: a phylogenomic approach in Oleaceae Supervisor : Guillaume BESNARD

Abstract :

In plants, the structural dynamics and concerted evolution of nuclear and cytoplasmic genomes are poorly understood. The objective of this thesis was to study the structural diversity and evolution of mitogenomes and plastomes in the family Oleaceae with a phylogenomic approach. First, we assembled mitogenomes from low-coverage sequencing data obtained from live and herbarium material. Considerable structural variation of mitogenomes was observed in the olive, and a chimeric gene potentially associated to a type of male sterility was detected. Finally, we studied the evolution of plastomes and mitochondrial genes in the Oleaceae. Accelerated evolution of plastomes was observed in two independent lineages. This change of evolutionary rate could be the consequence of an occasional transmission of plastids with pollen, modifying selective pressures on some genes.

Key