Evolution of the Leafcutter Bees and Chemical Ecology of Bee-Flower Relationships
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Phylogenetics as a tool for evolutionary studies: evolution of the leafcutter bees and chemical ecology of bee-flower relationships Dissertation submitted to the University of Neuchatel by Vincent Trunz Thesis committee Dr. Christophe Praz (co-director) Prof. Sergio Rasmann (co-director) Dr. Betty Benrey Prof. Denis Michez Dr. Mathieu Perret Thesis defended on th 26 June 2017 IMPRIMATUR POUR THESE DE DOCTORAT La Faculté des sciences de l'Université de Neuchâtel autorise l'impression de la présente thèse soutenue par Monsieur Vincent TRUNZ Titre: “Phylogenetics as a tool for evolutionary studies: evolution of leafcutter bees and chemical ecology of bee-flower relationships” sur le rapport des membres du jury composé comme suit: Dr Christophe Praz, co-directeur de thèse, UniNE Prof. ass. Sergio Rasmann, co-directeur de thèse, UniNE MER Betty Benrey, UniNE Prof. ass. Denis Michez, Université de Mons, Belgique Dr Mathieu Perret, Conservatoire et Jardin Botaniques, Genève Neuchâtel, le 21 août 2017 Le Doyen, Prof. R. Bshary Remerciements Une thèse de doctorat ressemble probablement pour tous ceux qui s’y frottent à l’ascension d’une montagne. Pour moi cette montagne était l’équivalent de l’Everest, et j’avais besoin d’un guide. Mon superviseur de thèse, Christophe Praz, a joué ce rôle de tout son cœur, et je ne saurais le remercier assez de m’avoir mené là où moi-même je ne pensais pas pouvoir arriver. Il y a de nombreuses personnes qui m’ont aussi aidé tout au long du chemin, parfois en travaillant avec moi sur certains points de ma thèse. Un immense merci à vous en particulier, Dimitri Bénon, Anne-Marie Labouche et Romain Piault. Merci à Gaétan Glauser et Matteo Lucchetti pour toute l’aide pour les analyses chimiques. Un grand merci à l’équipe du Jardin Botanique de Neuchâtel qui m’a accueilli et aidé pour la réalisation de mes élevages. Je tiens à remercier chaleureusement les membres de mon comité de thèse pour leur bienveillance et leur intérêt pour cette étude, mais aussi pour leurs nombreuses questions et commentaires qui ont aidé à améliorer ce document. Je ne suis pas arrivé sur cette voie par un complet hasard. Un petit caillou m’a fait sortir des ornières du chemin tranquille que je m’étais donné de suivre il y a une quinzaine d’années. Cette voie alternative s’est révélée extrêmement plus difficile, mais incommensurablement plus riche. Ce petit caillou, ce battement d’ailes d’un papillon, c’est toi, Flo Chardo, et je t’en suis infiniment reconnaissant. Merci à ma famille et à mes amis pour leur soutien, et plus que tout, merci à toi, Marie, pour ton appui indéfectible durant cette période parfois mouvementée. Mon humeur n’a pas toujours été au beau fixe, loin s’en faut, mais tu as bravé la tempête et tu m’as aidé à tenir le cap. Merci. 5 Table of content Abstract of the thesis 9 Résumé de la these 13 General introduction 17 What are bees? 19 The evolution of bees 20 Bees and flowers 21 Objectives of this thesis 24 Chapter 1 25 Foreword 25 Manuscript : 26 Comprehensive phylogeny, biogeography and new classification of the diverse bee tribe Megachilini: Can we use DNA barcodes in phylogenies of large genera? Chapters 2 63 Foreword 63 Manuscript 65 To bee or not to bee? Chemical ecology and the complex nature of bee-flower relationships in the family Boraginaceae 7 Chapter 3 101 Foreword 101 Manuscript : 103 How pollen chemistry complements pollination syndromes: Sinningia pollen secondary compounds in bee- and bird-pollinated flowers of the genus (Gesneriaceae) General discussion 123 Phylogeny of the leafcutting bees: the recent rise of a cosmopolitan genus 123 Pollen: Resource or reward? 125 When specialist bees and specialist plants meet 129 Conclusion and perspectives 130 References for: introduction, forewords and general discussion 131 Appendix 1: 135 Manuscript: Hofferia 137 Stenoheriades Palaearctic osmiine bees of the genera and (Megachilidae, Osmiini): biology, taxonomy and key to species Appendix 2: 149 Manuscript: 151 Ochreriades fasciatus Nesting biology, flower preferences, and larval morphology of the little-known Old World bee (Apoidea: Megachilidae: Megachilinae) 8 Abstract of the thesis Bees are present in almost all terrestrial ecosystems and are of vital importance for the pollination of wild flowers and cultivated crops. Despite being well investigated compared to many other insects, numerous open questions remain on their taxonomy, phylogeny and ecology. The present thesis aims at filling some of the gaps in knowledge concerning the evolutionary and natural history of bees. In addition, it aims at obtaining a better understanding of their complex relationships with flowers. Throughout the thesis, phylogenetics is used as a tool for examining an observed pattern in an evolutionary context. In the first chapter, the evolutionary history and the biogeography of the second largest bee genus worldwide is examined; in the second and third, pollen chemical ecology is investigated in two plant lineages each exhibiting multiple transitions in their pollination biology. In the first chapter of this thesis, we inferred a phylogeny based on five nuclear genes for the bee tribe Megachilini, one of the most diverse andMegachile widely distributed tribe of bees. This tribe includes the large and cosmopolitan genus , or the leafcutting bees, as well as two cleptoparasitic lineages - "cuckoo" bees that do not build their own nests but hide their eggs into the nest cells of other bees. We use our phylogenetic framework to examine the evolutionary history of these bees, reconstruct their biogeography and propose a new classification. In addition, we test a new methodological approach for the reconstruction of nearly fully-resolved phylogenetic trees using DNA barcodes, approximately 600 bp long sequences of the mitochondrial gene cytochrome oxidase. Our results indicate that the two cleptoparasiMegachiletic lineages form a monophyletic group nested within the nest-building genus , thus suggesting a single origin of cleptoparasitism in this bee clade. Biogeography reconstruction analyses suggest a center of origin in the Paleotropical area some 30 million years ago and numerous colonization events of the other geographic regions. Contrary to expectations, there were very few long-distance dispersal events and the current distribution of this large bee tribe can largely be explained by geodispersal between adjacent continents. When used alone, DNA barcodes had little phylogenetic signal: only nodes < 5 million years old could be recovered in such analyses; however, the addition of a single nuclear gene was enough to recover a majority of nodes up to 20 million years old. Lastly, we propose a 9 revised classification at the generic and subgeneric levels for this important group of bees. In the second part of this thesis (chapters 2 and 3), we focus on bee-pollinated plant clades and examine a little studied floral trait: the presence of secondary defense compounds in the pollen. While numerous studies have examined the ecological significance of nectar secondary compounds, pollen secondary compounds represent a nearly unexplored field of research. Because bee-flower relationships are likely centered on pollen, not nectar, they may strongly be impacted by pollen chemistry. In chapter two, we focus on the plant family Boraginaceae and on its western Palearctic members. This plant family represents an important resource for solitary bees, bumblebees as well as honeybees. We survey the alkaloids in the pollen of 23 species and use comparative phylogenetic analyses to test hypotheses on their evolutionary origin. Second, we examine the possible impact of these alkaloids on solitary bees in bioassays where natural pollen and nectar provisions of three solitary bees (one specialist, two generalists) were supplemented with realistic levels of pollen alkaloids. Eggs of these three species of bees were transplanted onto these provisions and their development was examined. These bioassays indicated that two of the three species, one generalist and one specialist, failed to develop on provisions supplemented with realistic levels of alkaloids. This result demonstrates for the first time that pollen secondary compounds can impact bee development and thus ultimately bee-flower relationships. Based on our comparative phylogenetic analyses, we postulate that the presence of alkaloids in the pollen of some species of Boraginaceae is not directly linked to bees, but rather that alkaloids are part of the chemical defense of the reproductive tissues of the flower. However, six independent lineages exhibiting a sophisticated, bee-adapted morphology had significantly lower levels of alkaloids in the pollen than other species of Boraginaceae. We postulate that in these species, pollen may be considered a reward to pollinating bees, while in other species pollinated by nectar-seeking visitors, pollen does not act as a reward. This important difference allows us to re-examine the complex nature of bee-flower relationships in this plant family. The focus of chapter three is anotherSinningia plant clade where bees are important pollinators, the Neotropical plant genus in Gessneriaceae. This genus is a remarkable 10 model group as it exhibits strongly defined pollination syndromes (mostly hummingbirds and bees), and as there has been numerous, independent switches between these syndromes. As in chapter two, we survey all the secondary metabolites of the pollen and ask the question whether pollen secondary chemistry differs among the different