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Congruence Entre Différenciation Morphologique Et Moléculaire (Barcode Moléculaire) Pour Sept Espèces De La Famille Des Phytoseiidae (Acari : Mesostigmata)

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Congruence Entre Différenciation Morphologique Et Moléculaire (Barcode Moléculaire) Pour Sept Espèces De La Famille Des Phytoseiidae (Acari : Mesostigmata) MINISTÈRE DE L’ALIMENTATION, DE L’AGRICULTURE ET DE LA PÊCHE MONTPELLIER SUPAGRO THÈSE présentée à Montpellier SupAgro pour obtenir le diplôme de Doctorat Discipline : Évolution, Écologie, Ressources génétiques, Paléontologie École doctorale : Systèmes Intégrés en Biologie, Agronomie, Géosciences, Hydrosciences, Environnement Laboratoire d’accueil : Unité Mixte de Recherche Centre de Biologie et de Gestion des Populations 1062, 2 Place Pierre Viala, 34060 Montpellier cedex Congruence entre différenciation morphologique et moléculaire (Barcode moléculaire) pour sept espèces de la famille des Phytoseiidae (Acari : Mesostigmata). Présentée et soutenue publiquement par Mireille Huguette Okassa Oyieyi le 10 décembre 2010 Membres du JURY Marie-Claude Bon Chercheur USDA, Montpellier Examinatrice Emmanuel Douzery Professeur, Université Montpellier II Représentant de l'ED Francisco Ferragut Professeur, Université de Valencia, Espagne Rapporteur Serge Kreiter Professeur, Montpellier SupAgro Co-directeur de thèse Sarah Samadi Maître de conférences, Université Paris 6 Rapporteur Marie-Stéphane Tixier Maître de conférences, Montpellier SupAgro Co-directrice de thèse 1 RESUME Congruence entre différenciation morphologique et moléculaire (Barcode moléculaire) pour sept espèces de la famille des Phytoseiidae (Acari : Mesostigmata) La famille des Phytoseiidae comprend des espèces d’intérêt agronomique dont certaines sont utilisées pour lutter contre des acariens ravageurs appartenant principalement aux familles des Tetranychidae et Eriophyidae. L’identification et la description des espèces de Phytoseiidae sont essentiellement basées sur des caractères morphologiques. Cependant, plusieurs problèmes d’identification sont rencontrés du fait : (1) de leur petite taille (faible nombre de caractères observables), (2) du manque de recul sur la valeur diagnostique de certains caractères dits discriminants, (3) et enfin du diagnostic fiable uniquement au stade femelle. Dans ce contexte, ce travail de thèse a pour objectif de déterminer dans quelle mesure l’approche moléculaire peut assister le diagnostic traditionnel. Pour cela, nous avons étudié l’étendue des variations intra- et interspécifiques (moléculaires et morphologiques) de sept espèces de Phytoseiidae d’intérêt agronomique, ceci pour comparer la capacité discriminante de quatre marqueurs moléculaires (12S ARNr, Cytb, COI et ITS). Dans ce travail, nous mettons en évidence l’utilité de ces marqueurs pour le diagnostic de tous les stades de développement et montrons l’importance de la forme du calyx de la spermathèque pour différencier deux espèces du genre Euseius , ce qui nous a permis de décrire une nouvelle espèce. Nous montrons également que les marqueurs mitochondriaux et nucléaires peuvent différencier des espèces morphologiquement proches. Cependant, il faut rester prudent quand aux conclusions tirées (espèces différentes ou populations différentes au sein d’une même espèce) sur la base des résultats obtenus avec les marqueurs mitochondriaux. Enfin, nous montrons que les règles de décision (valeurs seuils) sont difficiles à (1) établir avec ces mêmes marqueurs et (2) surtout à généraliser pour la différenciation des espèces appartenant aux deux sous-familles étudiées. Pour conclure, ce travail met en évidence de façon claire la nécessité d’une approche taxonomique intégrative, incluant un va-et-vient entre données moléculaires, morphologiques et écologiques, pour la garantie d’un diagnostic fiable des espèces de Phytoseiidae. Mots clés. Phytoseiidae, lutte biologique, diagnostic spécifique, barcode moléculaire, morphologie. 2 ABSTRACT Congruence between morphological and molecular differentiation (barcoding) for seven species of the family Phytoseiidae (Acari : Mesostigmata). The family Phytoseiidae includes numerous species of huge interest for biological control of mite pests, essentially belonging to the families Tetranychidae and Eriophyidae. Identification and description of Phytoseiidae species are mainly based on morphological characters. However, taxonomists have to face several problems for species diagnostic because of : (1) their small size (low number of visible characters), (2) little information on the diagnostic value of some characters considered as discriminant, and at last (3) diagnostic only reliable at female stage. In this context, the present works aims to determine how molecular approaches can assist traditionnal diagnostic. For this, we studied the intra- and interspecific variations (morphological and molecular) of seven agronomic important species, in order to compare the ability of four molecular markers (12S rRNA, Cytb mtDNA, COI mtDNA et ITS) to discriminate between species. We first show that molecular markers could be of great help for differentiating species at all life stages. Moreover, we show the importance of the spermatheca calyx shape for separating two species of the genus Euseius , and a new specie was then described. We also emphasize that both mitochondrial and nuclear markers differentiate morphological close species. However, some cautions have to be paid on the conclusions developped (different species or different populations within a same species) when only mitochondrial markers are used. At last, we underline that decision rules (treshold cutt-off values) are difficult (1) to establish with these latter markers and also (2) to generalise for the accurate differentiation of the Phytoseiidae species belonging to the two sub-families considered. To conclude, this work clearly emphasizes the importance of integrative taxonomy, including an « up-and-down » analysis of molecular, morphological and ecological data, to secure the specific diagnostic among the family Phytoseiidae. Key-words. Phytoseiidae, biological control, specific diagnostic, barcoding, morphology. 3 REMERCIEMENTS Cette thèse n’a pu se réaliser que grâce au soutien sans faille de mes deux co- encadrants, Marie-Stéphane Tixier et Serge Kreiter, envers qui j’exprime ma profonde gratitude pour l’encadrement scientifique dont j’ai bénéficié pendant ces trois années. Je suis également reconnaissante à Brigitte, Jean-François, Marie-claude, Maxime Galan, Maxime Ferrero, Roula, Gwen, Fabio, Martial, Sandrine, Christian et Mohamad pour leurs aides scientifique et technique. Merci particulièrement à Sabine pour son soutien quotidien lors de la rédaction de mon manuscrit. Merci à Angham, Fabien, Guillaume, Jean-marie et Sylvie pour leur bonne humeur et leur disponibilité. Merci de tout cœur à ma famille, notamment à mon père Eugène, à ma mère Léonie, à mes frères et à ma sœur d’avoir toujours cru en moi et de m’avoir soutenue moralement et matériellement tout au long de ma formation. Merci aussi aux familles Marie-jeanne, Makosso et Okamba pour leur assistance et leur encouragement. Merci à Sarah Tchicou ma sœur de cœur depuis toujours et mes amis Stéphane, Virginie, Yvette et Fabien Yeloukoudila pour leur soutien. Merci à Sabria et Nass de s’être gentiment occupés de mon fils pendant les derniers jours de la rédaction de mon manuscrit. Merci également à Jean- Casimir, Gervilis, Elie et Goberte pour leur aide. Mes plus grands et affectueux remerciements vont à mes deux amours mon fiancé Aymar et mon fils Isaac pour leur soutien. Pour terminer, je tiens sincèrement à remercier les membres du jury Sarah Samadi, Marie-Claude Bon, Emmanuel Douzery et francisco Ferragut d’avoir accepté d’évaluer ce travail de thèse. 4 LISTE DES FIGURES Figure 1. Chaetotaxie du bouclier dorsal des trois sous- familles : Amlyseiinae Muma (a) Phytoseiinae Berlese (b) et Typhlodrominae Scheuten (c) (Kreiter, 1991) 26 Figure 2. Morphologie des chélicères d’ Euseius gallicus Kreiter et Tixier de la femelle (x 50) (photo réalisée au microscope optique à contraste de phase, Okassa 2010) 27 Figure 3. Face ventrale d’une femelle adulte d’ Euseius gallicus Kreiter et Tixier présentant les boucliers : sternal (a) et ventrianal (b) (x 40) et les soies qu’ils portent. (photos réalisées au microscope optique à contraste de phase, Okassa 2010) 29 Figure 4. Face ventrale d’un mâle adulte d’Euseius gallicus Kreiter et Tixier présentant les deux boucliers sterno-génital et ventrianal (x20) (photo réalisée au microscope optique à contraste de phase, Okassa 2010). 29 Figure 5. Spermathèque d’une femelle adulte d’ Euseius stipulatus Kreiter et Tixier (x 40) montrant les différentes parties la constituant (photo réalisée au microscope optique à contraste de phase, Okassa, 2010). 30 Figure 6. Cycle de développement des Phytoseiidae (Kreiter, 1991) 32 Figure 7. Importance de l’expertise taxonomique des Phytoseiidae dans le succès des programmes de lutte contre les espèces nuisibles des cultures. 37 Figure 8. Liste des différents concepts d’espèces d’après Mayden (1997) 42 Figure 9. Représentation schématique de la notion d’espèce (Samadi et Barberousse, 2005, 2006) 43 Figure 10. Schéma représentant les fréquences des distances génétiques à l’intérieur d’une espèce (intraspécifique) et entre des espèces (interspécifiques). Ces variations peuvent être chevauchantes (absence de « gap ») ou non chevauchantes (présence de « gap »). 52 Figure 11. Localisation des gènes de l’ADN mitochondrial au sein de la plupart des arthropodes (Simon et al., 1994) 66 Figure 12. Ensemble des étapes réalisées pour les analyses moléculaires et morphologiques sur chaque spécimen collecté 75 Figure 13. Corrélation entre la moyenne intraspécifique et le nombre d’individus séquencés pour les marqueurs (a) 12S ARNr, (b) Cytb et (c) ITS. 226 5 Figure 14.
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