FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN CENTRE FOR SOCIAL EVOLUTION DEPARTMENT OF BIOLOGY PhD thesis Luigi Pontieri Discrimination behavior in the supercolonial pharaoh ant Academic advisor Jes Søe Pedersen June 2014 faculty of science university of copenhagen centre for social evolution department of biology Danish National Research Foundation Centre for Social Evolution Discrimination behavior in the supercolonial pharaoh ant This thesis dissertation has been submitted in accordance with the requirements for the degree of PhD, at the PhD School of The Faculty of Science, University of Copenhagen, Denmark to be defended publicly before a panel of examiners. by Luigi Pontieri June 2014 Academic advisor Assoc. Prof. Jes Søe Pedersen Cover: artwork of queens, workers and brood of Monomorium pharaonis © Luigi Pontieri PREFACE This thesis is the result of a three year PhD project carried out at the Centre for Social Evolution (CSE), Department of Biology, University of Copenhagen in Denmark under the supervision of Assoc. Prof. Jes Søe Pedersen. During my PhD period I spent three months in the laboratory of Assoc. Prof. Timothy Linksvayer, Department of Biology, University of Pennsylvania. I additionally spent a total of three weeks at the Laboratoire d’Ethologie Expérimentale et Comparée (LEEC), Université Paris 13, Sorbonne Paris Cité, hosted by Prof. Patrizia d’Ettorre. The projects in this thesis were funded by CSE via a grant from the Danish National Research Foundation (grant DNRF 57). The thesis is comprised of a general introduction on the problem of biological invasion with a special focus on invasive ant species, the traits that makes them successful invaders and how genetic diversity can affect the recognition system and the intra- and interspecific interactions that these species present. I then present my model organisms, a summary of the main findings of my projects and suggested future directions. This is followed by four chapters of original empirical work, three of which are in preparation for submission in peer reviewed journals and one is currently submitted. Luigi Pontieri Copenhagen, June 2014 CONTENTS SUMMARIES . 7 SUMMARY . 8 RESUMÉ . 10 GENERAL INTRODUCTION . 13 INTRODUCED SPECIES AND THE PROBLEM OF BIOLOGICAL INVASIONS . 14 INVASIVE ANTS . 14 THE “INVASIVE ANT SYNDROME”. 15 THE STRUCTURE OF UNICOLONIAL POPULATIONS AND THE PROBLEM OF RECOGNITION . 16 ANT RECOGNITION SYSTEM . 18 THE EFFECT OF GENETIC DIVERSITY ON NESTMATE RECOGNITION AND SUPERCOLONY INTERACTIONS . 18 THE EFFECT OF UNICOLONIALITY ON DISEASE SPREAD . 20 MODEL ORGANISMS. 22 The Pharaoh ant 22 Metarhizium brunneum 26 SHORT SUMMARY OF THE THESIS CHAPTERS . 27 FUTURE PERSPECTIVES . 28 REFERENCES. 30 CHAPTER 1 . 39 GENETIC DIVERSITY IS MORE IMPORTANT THAN RELATEDNESS FOR NESTMATE DISCRIMINATION IN THE PHARAOH ANT CHAPTER 2 . 63 UNEXPECTED FUSION OF GENETICALLY AND CHEMICALLY DIVERGENT COLONIES OF THE INVASIVE PHARAOH ANT CHAPTER 3 . 91 THE STATISTICAL APPROACH TO IDENTIFY NESTMATE RECOGNITION CUES CHAPTER 4 . 115 ANT COLONIES PREFER INFECTED OVER UNINFECTED NEST SITES ACKNOWLEDGEMENTS . 137 ENGLISH, DANSK Summaries 7 SUMMARY The majority of eusocial insect species live in small, kin structured colonies that are mutually aggressive and rarely interact. By contrast, a restricted group of ant species show a peculiar social organization called unicoloniality, where colonies can grow to vast networks of geographically separated but mutually tolerant nests, also referred to as “supercolonies”. Many unicolonial ants are invasive, as their introduced supercolonies attain huge size and cause severe economic and ecological damage, affecting in particular species composition and functioning of ecosystems. There is therefore an increasing need to understand which factors promote the ecological dominance of these species, and particularly how the discrimination of both conspecifics and heterospecifics (including parasites) might influence structure and ecological success of invasive populations. In this PhD thesis I investigated the discrimination behavior of the invasive pharaoh ant (Monomorium pharaonis) as a model for other invasive and supercolonial ant species. The pharaoh ant is one of the few ant species that can be reared in the laboratory for many generations. Furthermore, the possibility to do controlled crosses of colonies provides the unique opportunity to establish colonies of different genetic composition. These traits make this species a suitable study subject to set up behavioral experiments that aim to investigate which factors, and to which extent, might influence the inter- and intra- specific discrimination abilities of invasive ants. In the first chapter I focused on the nestmate recognition system of pharaoh ants, investigating whether the cues used for discrimination had a genetic origin and how different level of within-colony genetic diversity and relatedness influenced the discrimination abilities of the colonies. I show that, despite a general ability of discriminating nestmates from non-nestmates, the degree of relatedness between colonies did not influence the overall level of discrimination. Furthermore, I found that genetically low-diverse colonies displayed better discrimination abilities than high diverse ones and that low genetically diverse colonies discriminated high diverse non-nestmates better than vice versa. In the second chapter I investigated whether the high genetic differentiation characterizing natural colonies of pharaoh ants is sufficient to prevent unrelated colonies to fuse and how different levels of genetic similarity shape the outcome. By pairing laboratory colonies in a fusion assay, I show that the majority of unrelated colonies fused despite high initial levels of aggression. Moreover, I also found that the initial aggression was positively correlated with the chemical and genetic distance between colony pairs, further confirming the important role of endogenous cues in the nestmate recognition of this species. The third chapter presents a methodological study on the best procedures for identifying chemical compounds used for nestmate recognition in social insects. We first compiled datasets of cuticular hydrocarbons (CHCs) and aggression between colonies of three species of ants (Formica exsecta, Camponotus aethiops and Monomorium pharaonis) and a simulated dataset. Then, using the available 8 information about the exact cues used for nestmate recognition in F. exsecta, we evaluated the power of different combinations of data transformation and chemical distance calculation in differentiating between true nestmate recognition (NMR) cues and other compounds. We found that particular combinations of statistical procedures are more effective in differentiating NMR cues from other compounds. We also developed a new method for centroid calculation that increased the power of the analysis and can therefore be used in future studies that aim to identify nestmate recognition cues in other species. In the fourth chapter I investigated the nest site preference of pharaoh ant colonies and, specifically, their ability to avoid nests containing infectious pathogens as invasive, supercolonial ants are hypothesized to be particularly prone to disease. Using binary choice tests between three types of nests, I found that migrating colonies surprisingly preferred nest sites containing nestmate corpses overgrown with sporulating mycelium of the generalist fungus Metarhizium brunneum. This unexpected finding can provide new insight into the important co-evolution of social insects and their pathogens. 9 RESUMÉ Størstedelen af eusociale insektarter lever i små kolonier bestående af beslægtede individer. Kolonierne er typisk aggressive over for hinanden og interagere sjældent. Der findes imidlertid en begrænset gruppe af myrearter som har en speciel social organisation kaldet unikolonialitet, hvor kolonier kan vokse til et enormt koloninetværk, som er geografisk adskilte men ikke aggressive over for hinanden. Dette fænomen kaldes også “superkolonier”. Mange unikoloniale myrer er invasive, fordi indførte superkolonier kan opnå enorme størrelser og påvirker især artssammensætningen og økosystems funktioner, og derved forårsage alvorlig økonomisk samt økologisk skade. Der er derfor, et stigende behov for at forstå, hvilke faktorer der forårsager disse arters økologiske dominans, og især hvordan diskrimination af både artsfæller og ikke artsfæller (herunder parasitter) kan have indflydelse på økologisk succes og strukturen af invasive populationer. I denne ph.d.-afhandling, undersøgte jeg diskriminationsadfærden hos den invasive faraomyre (Monomorium pharaonis) som en model for andre invasive og superkoloniale myrearter. Faraomyren er en af de få myrearter, der kan opdrættes i laboratoriet i mange generationer. Det er tilmed muligt at lave kontrollerede krydsninger mellem kolonier, hvilket giver en unik mulighed for at etablere kolonier med forskellig genetisk sammensætning. Disse egenskaber gør denne art perfekt egnet til at udføre adfærdseksperimenter, der har til formål at undersøge hvilke faktorer der påvirker diskriminationsadfærden mellem både artsfæller og ikke- artsfæller hos invasive myrer. I det første kapitel, fokuserede jeg på bofællegenkendelsessystemet hos faraomyrer. Jeg undersøgte om de kemiske forbindelser der anvendes til diskrimination var påvirket af den genetiske sammensætning, og hvordan forskellige niveauer af genetisk diversitet samt slægtskab påvirket diskriminationsevnen hos kolonierne. Jeg viser,