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A PhD is a unique adventure, and I got the chance to cross the path of amazing people that I would like to acknowledge here. Mine brought me to many places overseas and I wish to reach the people I met by using a language they speak. Consequently, this section will be a mix of French, English and Japanese.

I would like to start with the jury. I cannot thank you enough for making me the honour to read this manuscript and get interested in my research. Thank you, Alexander Blanke and Pierre Moretto, for accepting to be reporters of my thesis. I hope you will find this manuscript of interest and I cannot wait to read your comments. Many thanks also go to Adria LeBoeuf and Martine Maïbèche for consenting to examine my thesis. I am looking forward to our discussions on the defence day. Special thanks also go to Julien Gasparini for taking me under his wing after the loss of my supervisor. I appreciate that you spontaneously offered me your help and even though our research topics diverge, you made significant contributions to my introduction and guided me for all administrative procedures.

Then, I would like to thank the great myrmecologists who advised me as members of my PhD committee meetings: Roberto Keller, Mathieu Molet, Vincent Fourcassié and Corrie Moreau. These meetings helped me to plan and adapt my PhD, and you gave me light to proceed even through the darkest paths.

My first year of PhD brought me to Japan, first at the Okinawa Institute of Science and Technology. Many thanks go to Evan Economo for welcoming me there for six months and collaborating ever since. I am glad you got a full professor position, and to count you as a collaborator. I really admire the dynamic lab you put up and manage in a kind and clever way.

I would also like to thank the entire AriLab, with special thanks to Paco and Georg for teaching me how to work on microCT, from specimen preparation to 3D modelling. Thanks again Paco for being so welcoming and driving me around a lot, I appreciated the time we spent together. Big hugs to Sam and Julian, you guys were the best lab buddies I could ever ask for and I am looking forward to hearing more from you both in the academic sphere. And I could count on the help of two batches of fantastic interns to have a merry atmosphere outside work hours. You are so numerous I cannot thank you all, but touring Okinawa with you was a blast. Two special thanks though: to Ines for being so smart, curious and happy-golucky, and Merey for how unique and sweet you are and the trust you put in me.

Second part of my stay in Japan brought me to Kagawa prefecture, in the small town of Miki. No more English signs or menus there, so 日本語でやらなきゃいけないです!まず 、伊藤先生、三ヶ月お世話になりました。伊東研の動物園はいい勉強になりました。もう 一度一緒にうどんトリップでお話しましょうね。あとタンクトップ研の皆さん、僕の下手 な日本語でも一緒に時間を過ごしてありがとうございました。特に水野くんに感謝してい ます。必ず立派なミルメコロジストになるから全力でがんばってください。香川大の優しい 豊田先生とカラオケの女王赤城さん、ありがとうございました。Je remercie également Mathilde et Baghdad pour les bons moments passés ensemble, vous apportiez la French Touch dont j'avais besoin pendant ces trois mois à Kagawa. そしてもちろん、井戸川くんに も感謝しています。ブラジル、フレンチギアナやSWARMはいい思い出になりました。博士 課程で頑張ってください、どこかでまた会おうぜ。最後に、蟻への興味を掻き立ててくれた 辻先生に感謝しています。This is also the paragraph I choose to warmly thank Johan Billen, the king of histology, who helped me understand the importance of glands in ants. Johan, you are a great storyteller and I thoroughly enjoyed our long email communications and our meetings in Japan. I am looking forward to hearing more anecdotes from you.

Most of my PhD took place in France, aussi je repasse en français pour remercier les personnes qui ont contribué à rendre mon quotidien parisien plus agréable. Je vais commencer par les collègues à iEES Paris qui m'ont accompagné sur le lieu de cette aventure. Merci Mathieu pour tes conseils lors de mes comités de thèse et pour le temps que tu consacres à faire vivre notre équipe multi-sites. Je regrette qu'on ait si peu parlé morphologie ensemble mais j'espère avoir à nouveau l'occasion d'échanger avec toi à l'avenir. Je remercie également Thibaud d'être un pilier de cette équipe et du laboratoire en général Merci à Romain P. pour avoir veillé sur nos fourmis et ces moments partagés au labo, particulièrement au début de ma thèse et souvent en musique. Et que serait l'équipe fourmis sans ses autres doctorants ! Romain H., merci pour ta gentillesse et ton sens des responsabilités que tu caches allègrement derrière de bonnes vannes et une barbe bien fournie, tu ne sais pas à quel point je suis chanceux d'avoir commencé ma thèse en même temps que toi. J'admire ta capacité à t'investir sur beaucoup de choses. Donne des nouvelles depuis la Suissi-la-famille ! Lauren, on s'est rencontré bien avant la thèse, mais quel plaisir d'avoir pu apprendre à mieux te connaître ces deux dernières années ! Merci pour ta compagnie, au labo, au bureau et en vocal sur Discord, au plaisir de parler fourmis et de geeker (et rager) avec toi pendant encore quelques temps. Et le petit dernier, Basile, tu t'es parfaitement intégré à notre groupe de joyeux lurons et on ne pourrait plus se passer de toi. Bon courage pour la suite de ta thèse, et continue à illuminer le labo et le bureau de ton sourire et de ta bonne humeur si communicative.

J'ai aussi eu l'opportunité de passer du temps avec plein d'autres jeunes chercheurs au cours de ma thèse, même si je suis sûr que je vais en oublier, je me dois les remercier. Tout d'abord, les personnes avec qui j'ai pu partager le bureau 508. Alexis, tu auras eu le temps de me raconter un paquet d'histoires, le bureau n'a jamais été aussi animé qu'en ta présence.

Merci pour ta bonne humeur, et n'oublie pas que tu as toujours le choix. Je te souhaite de réussir tout ce que tu choisiras d'entreprendre. Merci encore pour ta disponibilité sur la fin de ma thèse, j'ai apprécié que tu me proposes de l'aide Charlotte, tu es la première interaction pied-pied que j'ai eue dans ce bureau, et aussi une interlocutrice posée et bienveillante, aussi je garde un bon souvenir de ces moments passés ensemble. Clarence, l'aura protectrice de pigeon puissant ajoutait la petite dose de sérieux qu'il fallait dans ce bureau, dommage que tu sois partie si vite ! Chloé D., tellement discrète qu'Alexis t'aura enfermée plusieurs fois dans le bureau, tu as toujours "surveillé mes arrières" et t'entendre travailler m'a aidé à me concentrer moi aussi. Chloé C., si vite montée du 2ème quand elle en a eu l'occasion pour amener dans notre bureau les lézards vivipares, ton superbe casque de vélo et des horaires de déjeuner bien trop décents, merci pour ton enthousiasme au quotidien.

Deux bureaux à côté, en 512, il y avait aussi de joyeux lurons ! Iry, la diva au grand coeur, merci pour ta gentillesse et tes éclats de rire (sans oublier ta tarte au potimarron !), tu es de bon conseil et j'ai toujours apprécié le temps passé avec toi au cours de ma thèse. David, ton humour presque aussi douteux que le mien m'a bien diverti, et ton engagement dans la recherche m'impressionne (mais pas autant que ton amour pour Paris). Continue à tout faire pour atteindre tes objectifs, et on sort la Switch ensemble quand tu veux. Vincent, camarade à bouclettes, merci d'avoir partagé cette affection pour les Hyménoptères. Charles, le "vieux sage" du bureau, nos séquences d'imitations vont me manquer. Un peu plus loin dans le couloir, je n'oublie pas Corentin, le breton dopé à la proline, merci d'avoir partagé de plus en plus de déjeuners et de pauses sur la fin de ma thèse.

En descendant d'un étage et en changeant d'aile, le bureau 413 a lui aussi abrité son lot de jeunes chercheurs. D'abord, merci au club des cinq comme on les appelle, qui ont entretenu une forte dynamique dans le laboratoire au tout début de ma thèse, qui n'était plus si bonne après leur départ. Avril, j'ai eu de moins en moins d'occasions de te voir, mais je te connais depuis assez longtemps pour savoir que tu as toujours beaucoup de choses à gérer.

Merci d'être venu au Japon avec Clément, j'ai passé un bon moment avec vous, et j'espère qu'on aura bientôt l'occasion de se refaire une soirée à revoir les photos de ces moments.

Youssef, je garde un bon souvenir de sessions d'ultimate en début de thèse et de ton enthousiasme lors des événements entre jeunes chercheurs. Jérôme, alias Jeschen, tu as préféré rester près de l'Orge depuis le premier confinement, mais je n'oublie pas ton investissement dans les JRJC de Roscoff et ton enthousiasme au moment d'aller manger un morceau. Paul, j'ai rarement vu un naturaliste aussi bien sapé, continue à mener ta recherche avec passion et prends confiance en toi. Une bise à la relève : Thomas, Samuel, Alice, et d'autres que je n'ai pas eu l'occasion de rencontrer, ce n'est pas le meilleur contexte sanitaire pour commencer une thèse.

En dehors du laboratoire, j'ai interagi avec deux passionnés Rennais que je dois remercier. Merci à toi Cédric, depuis notre rencontre à Ant Course en Guyane j'ai eu l'occasion d'être impressionné par ton parcours, ta rigueur et tes connaissances. Je me plongerai dans ta thèse (brillante, j'en suis sûr) avec grand plaisir, et j'ai hâte de publier ces études sur Melissotarsus fossile avec toi. Et merci à toi Corentin pour tes visites régulières et ton intérêt pour nos recherches, à Christian et moi. Lorsque tu n'étais qu'en L3, l'étendue de tes connaissances sur les fourmis m'a clairement impressionné. Je te souhaite de voyager, d'apprendre encore plus de choses, et j'ai hâte de te voir devenir un expert en myrmécologie.

Je me dois aussi de remercier un certain nombre de personnes en dehors de la sphère professionnelle, qui m'ont accompagné à leur manière au cours de cette aventure. Et que serait la vie parisienne sans quelques poireaux ? Merci aux Negitachi, la chorale geek parisienne à ne pas rater, qui m'aura diverti sur une très large proportion de mon temps libre. Gabrielle. Tu as été à mes côtés pendant une grande partie de ma thèse, à laquelle tu es définitivement associée. Merci pour tous ces moments ensemble, je sais que je ne suis pas quelqu'un de facile à supporter, ce que tu fais étrangement bien. Merci pour toute l'aide que tu as pu me proposer en dépit de ton manque de confiance en toi. Même si je ne l'ai pas toujours acceptée parce que je suis du genre à m'occuper seul de certaines choses, te savoir à mes côtés et disponible était d'un grand soutien.

Finally, I would like to dedicate this last paragraph to someone very special, who taught me so much. Like the thorax is the power core of ants, Christian Peeters was the power core of my PhD. Christian, I planned to thank you in this section and during my defence. You were a fantastic mentor, not only to me but to other young myrmecologists. You trusted me from the start and took me under your wing as your last PhD student. It took me some time to grow up and meet your expectations, but I hope you would be proud of this manuscript and my defence. Obviously, you would likely kick my butt after reading Chapter 3 and the Discussion because I am just a clumsy scientist when you are not around. I missed our discussions these past two months, and I will miss them along my research career. I will do my best to pursue and publish our projects. Your last paper as first author was a huge success, and I hope our Khalife and Peeters will be published soon. For now on, I will proudly say I was your PhD student and give my best to honour your memory. Rest in peace Christian, I will take it over from here.

As a transition between acknowledgements and the main course, here is one of Christian's favourite Tweets to introduce the topic of my PhD.

Table of Contents

Introduction 1-The importance of body size

The size of things is a fascinating topic. For far too long, this question has been investigated through an anthropocentric prism. Anthropocentrism comes from the Ancient Greek: anthropos means 'human being', and kentron means 'centre'. Anthropocentrism sets man as the centre of everything and grants major importance to religious texts, blindfolding even the brightest minds on topics such as the origin of the Earth and its place in the Solar System or the evolution of life. A major change of mentality occurred in the 16 th century during the Copernican Revolution, named after Nicolaus Copernicus. He presented a heliocentric model where the Earth would rotate around the Sun in contrast to Ptolemy's geocentric model -widely accepted at that time -that stated the opposite. The novelty of his model startled the mind of other astronomists such as Brahe, Kepler and Galilei who completed and validated it before it was used by Newton to establish his laws of universal gravitation and motion. Beyond astronomy, the Copernican Revolution had profound consequences on mentalities: as Kant wrote in 1787, proceeding assuming that "knowledge must conform to objects" instead of "objects must conform to our a priori knowledge" is an important step to take for the scientific community.

Freed from anthropocentrism and fuelled by technical progress, physicists put more efforts into the search for the minimal and the maximal size of matter. The past few centuries have been marked by major findings, from the tiniest building blocks to the largest supermassive objects. In 2020, the tiniest known building blocks of matter are quarks, and have an approximate size of 10 -19 m. The largest objects that have been found across the universe are galaxies and supermassive black holes, larger than 10 21 m. The size of multiple systems is shown in figure 1. Human beings stand in the middle of this scale not because of anthropocentrism, but because of how challenging it is to observe matter at scales that are far away from us. There probably are smaller entities than quarks and larger entities than galaxies waiting to be discovered. knowledge we got on body size during the 19th century is what we call biological rules.

These rules are numerous and result from global trends observed on living organisms. They know exceptions and how they interact with each other is poorly understood. For example, Bergmann's rule states that populations and species in a cold environment are larger than in warmer environments while Cope's rule states that animal body size tends to increase over time. Rules can overlap but also be in contradiction. Fortunately, during that same century, Darwin and Wallace introduced the concept of natural selection to the scientific community.

Natural selection theory states that individuals have differential survival and reproduction success (called fitness) depending on their phenotype. If a given phenotype is favoured and heritable, it will spread in a population. This theory has since been accepted and verified with strong examples such as the development of a resistance against insecticides in mosquitoes [START_REF] Curtis | Selection for and against insecticide resistance and possible methods of inhibiting the evolution of resistance in mosquitoes[END_REF].

How does natural selection drive the evolution of body size? This question naturally came up after Darwin and Wallace's work and is still entertaining the scientific community. For a trait such as body size, natural selection can act in four different ways. First, it can be directional: for example, larger individuals can forage more efficiently and produce more offspring than smaller individuals, resulting in an increase of body size over generation known as Cope's rule [START_REF] Brown | Body size, ecological dominance and Cope's rule[END_REF]. Second, natural selection can be stabilizing: individuals with intermediate body size can have a higher fitness than the extremes, for example as the result of two opposite selective pressures [START_REF] Holand | Stabilizing selection and adaptive evolution in a combination of two traits in an arctic ungulate[END_REF]. Third, it can be disruptive, and favour both extremes over the intermediates [START_REF] Gross | Disruptive selection for alternative life histories in salmon[END_REF]. Fourth, body size and fitness could be unrelated, and evolution of body size could be the consequence of genetic drift (neutral evolution, [START_REF] Kimura | The Neutral Theory and Molecular Evolution[END_REF]. Selective pressures on body size can be numerous, in different directions, and operate at any life stage from egg to adult. The observed body size of a given organism results from compromises (trade-offs) to cope with all pressures.

Directional and disruptive selection for body size should favour extreme phenotypes, and animals should grow or shrink more at every generation. However, the size range we observe for animals remains limited compared to what we could expect following natural selection.

An important fact to consider is that body size is correlated with the morphology, physiology and lifestyle of an organism [START_REF] Rensch | Histological Changes Correlated with Evolutionary Changes of Body Size[END_REF], likely acting as stabilizing selective pressures creating a boundary. Organisms cannot grow or shrink boundlessly, even though evolution found interesting ways to push these boundaries in some organisms. That is what miniaturisation is all about.

2-Miniaturisation drives the evolution of new species and phenotypes

Size distribution after an adaptive radiation favours diversity to occupy as many ecological niches as possible but following rules imposed by the body plan (bauplan) of the ancestor. Therefore, during a radiation, two phenomena take place to further increase the body size range: gigantism and miniaturisation. Both involve pushing the limits towards extremely large or small body size. Miniaturisation is the evolution of extremely small body size within a lineage [START_REF] Hanken | Miniaturization of Body Size: Organismal Consequences and Evolutionary Significance[END_REF] and is the consequence of drastic morphological adaptations. Defining an empirical threshold for "extremely small" valid for all animals is impossible because size variations are limited by a given bauplan. For example, plethodontid salamanders are a fantastic example of miniaturisation even though the smallest are 20mmlong [START_REF] Roth | Miniaturization in plethodontid salamanders (Caudata: Plethodontidae) and its consequences for the brain and visual system[END_REF]. They are still larger than most insects but reaching such a small size is a considerate accomplishment regarding the bauplan of vertebrates, and tetrapods in particular. Other examples of miniaturisation in vertebrates include frogs [START_REF] Yeh | The Effect of Miniaturized Body Size on Skeletal Morphology in Frogs[END_REF], fish [START_REF] Rüber | Evolution of miniaturization and the phylogenetic position of Paedocypris, comprising the world's smallest vertebrate[END_REF] and lizards [START_REF] Pianka | Evolution of Body Size: Varanid Lizards as a Model System[END_REF] (see [START_REF] Hanken | Miniaturization of Body Size: Organismal Consequences and Evolutionary Significance[END_REF] for a review).

Miniaturisation is a dominant phenomenon in the evolution of insects, a much more diverse group than vertebrates. Gigantism in insects is limited by the respiratory system, which takes relatively more space in larger insects and limits the maximum possible size around 17cm [START_REF] Kaiser | Increase in tracheal investment with beetle size supports hypothesis of oxygen limitation on insect gigantism[END_REF]. Thus, the largest insects can hardly compete with the largest species of other lineages to become top predators, which contributes to explain why natural selection favoured individuals with smaller body size instead. On a side note, insects used to be larger during the Paleozoic because greater oxygen concentrations in the atmosphere made the respiratory system more efficient, and a similar volume of trachea could oxygenate a larger volume of organs. Miniaturisation in insects is spectacular. Some minute insects are equivalent in body size to unicellular organisms (reviewed by Polilov, 2015, figure 2): the world record is held by some genera of the family Mymaridae, 139µm for Dicopomorpha males (wingless) and 158µm/170µm for Kikiki/Megaphragma. This extreme example of miniaturisation is made possible by a parasitic lifestyle: these organisms use the resources of a host for development and are not self-sufficient. The smallest non-parasitic insects are Nanosella and Scydosella beetles, 300µm, less spectacular but still matching the of ant species had a head width below 1mm whereas no social bee or wasp was this small. Wasps and bees fly, and their body size determines the size of their flight muscles. Larger flight muscles allow foragers to carry heavier prey or pollen/nectar loads to feed the colony, hence extremely low size cannot be selected. In contrast, ant workers do not fly, which removes the constraint of wing muscles on miniaturisation (see part 4 of this introduction).

Earthbound life enables the evolution of swarm behaviours such as collective transportation that allow multiple minute workers to retrieve large prey. Furthermore, ants have a morphologically pronounced division of labour: there is one reproductive caste, the queen(s), and at least one helper caste, the workers. Miniaturisation is most striking in the worker caste, which I will focus on throughout this manuscript, but can also concern the queen caste (e.g. in subterranean ants). Size divergence between queens and workers promoted the evolution of intermediate phenotypes called soldiers, following different allometric growth rules [START_REF] Molet | Evolution of Novel Mosaic Castes in Ants: Modularity, Phenotypic Plasticity, and Colonial Buffering[END_REF]. This soldier caste takes care of specific tasks (detailed in Chapter 1) in a colony and gets division of labour to a next level.

In rough agreement with Polilov's definition of microinsects [START_REF] Polilov | Small Is Beautiful: Features of the Smallest Insects and Limits to Miniaturization[END_REF] I will use the term "minute" for ant workers with a total length below 2mm. Some notorious genera with minute workers are Pheidole (1100 species, most diverse genus of ants), Monomorium (400 species), Carebara (>240 species), Solenopsis (>200 species, including the red imported fire ant), Tapinoma (>90 species), Plagiolepis (>70 species), Brachymyrmex (>40 species), and Leptanilla (>40 species). Now that we know that selection towards smaller size shapes the evolution of ants, the question that naturally comes to our mind is: why getting smaller, and why not even smaller?

These are questions I will address in the two following parts of this introduction and throughout my thesis.

4-The adaptive role of miniaturisation

Habitat perception and interstitial niches

How one organism perceives a given habitat is linked to its body size: the smaller you are, the more rugose the habitat is. This concept was formulated as the size-grain hypothesis [START_REF] Kaspari | The size-grain hypothesis and interspecific scaling in ants[END_REF]) and illustrated with the example ofa savannah (Figure 4). Animals of various sizes inhabit this ecosystem, including elephants, mice and ants. From the the colony fitness. However, exploiting interstitial niches can be beneficial for at least two reasons. First, it provides a shelter against harsh environmental conditions. Indeed, small interstices (e.g. in tree bark and leaf litter) offer microclimates that only minute foragers can access and help to handle inclement foraging conditions (discussed in [START_REF] Kaspari | Thermal adaptation generates a diversity of thermal limits in a rainforest ant community[END_REF].

Higher survival of foragers and longer foraging trips increase the resource pool of an ant colony. Second, it provides an efficient way to avoid predation. Indeed, all members of an ant colony live in a perennial nest, where the queen and young workers stay. This nest requires as much protection as possible, in particular because the colony dies if the queen dies. Ant workers create nest entrances and tunnels that match their own size. Smaller body size means narrow entrances to the nest, which makes it possible only for other minute animals to get inside. Only foragers leave the nest, meaning they are the most vulnerable ants in a colony. However, they are the oldest workers that already served the colony well and they can make use of interstices during their foraging trips not only for microclimates but also to hide from predators.

A whole new world of physics

Minute insects live in a world where physics acts differently. From a human perspective, gravity is the main force ruling the world. However, gravity is linked to the mass of a system, with mass being proportional to a volume (cube length). Ants live on the ground where they are confronted with other forces that are proportional to a surface (square length).

When ants are minute (i.e. at low length values), cube to length ratio decreases, meaning other forces that can outpower gravity. Such forces include surface tension forces, hydrophobic effects and van der Waals adhesion. Surface tension is the tendency of a liquid to minimize its surface area. This phenomenon relies on the attraction between molecules inside this liquid. If we take the example of a pond, the water surface is flat. Water molecules are made of two hydrogen atoms and one oxygen atom. Even though a water molecule has a neutral charge, electrons from covalent bonds are pulled toward the oxygen atom, creating a slightly positive charge on hydrogen atoms and a negative charge on the oxygen atoms. Positive hydrogen atoms from one water molecule are attracted to negative oxygen atoms from other water molecules, creating a strong attraction force between water molecules. Under the surface, this force pulls molecules in all directions. In contrast, at the surface, the attraction between water molecules and the medium above (air in our case) is negligible, so the pull is lateral and downward, Hydrophobic effects are the property of molecules to be repelled from a mass of water.

Ant cuticle is hydrophobic, and hydrophobic effects can help minute ants carrying heavy moist objects or water itself. For example, if an ant carries a water droplet, the weight of the droplet will pull it down whereas hydrophobic interaction with the ant mandibles (below the droplet) will pull it up.

Van der Waals forces are intermolecular interactions that are highly distancedependent and are involved in dry adhesion mechanisms in biological systems. The most famous example of van der Waals adhesion is the gecko. Gecko's feet are exceptionally large and their sole is covered with millions of tiny hairs called setae. These setae engage van der Waals interactions with the surface they stand on and allow them to walk even on smooth walls and ceilings [START_REF] Autumn | Mechanisms of Adhesion in Geckos[END_REF]. Ants use wet adhesion mechanisms as mentioned earlier, but they also use dry adhesion mechanisms, relying on van der Waals forces [START_REF] Peng | The microstructure morphology on ant footpads and its effect on ant adhesion[END_REF]. These forces vary according to the surface area and are more efficient on minute ants.

Consequently, all these forces exceeding the effects of gravity in minute ants contribute to explain how ants can carry several times their own mass, a scientific trivia that has fascinated us for thousands of years.

Colonial economy

In ants, cuticle thickness is correlated with body size and phylogeny (Peeters et al., 2017a). Insect cuticle is made of chitin and proteins that are built using ingested nitrogen. This means that smaller workers have a thinner cuticle and are cheaper to produce. With a given nitrogen budget for a colony, there is a trade-off between colony size and worker size: workers can be few but big, or numerous but minute. Large colonies can be advantageous for central place foragers. For example, in scavenger species, one worker needs to be at the right place at the right time to find a carcass before competitors: this probability increases with the number of foragers. In addition, swarm hunting and aphid-tending are more efficient with as many individuals as possible. On the flipside, this thin cuticle makes minute individuals more vulnerable to bites and stings.

Colony founding

Colony founding is a critical phase for an ant colony. There are two main strategies for colony founding in ants (reviewed by [START_REF] Peeters | Nonflying Reproductives in Ants[END_REF]. On one hand, the new founding queen leaves the mother colony with nestmate workers: this strategy is called dependent colony founding (DCF) as the queen relies on nestmates. On the other hand, the new foundress is alone to raise her first daughters: this strategy is called independent colony founding (ICF).

Queens during ICF can either forage (non-claustral) or stay safe and rely entirely on the metabolic reserves they accumulated before aerial dispersal (claustral). Claustral queens must raise their first daughters as soon as possible in order to get new resources from foraging.

Therefore, in some species, claustral queens raise a first generation of workers that are smaller than average. These so-called nanitic workers are less expensive, faster to produce than standard workers, and increase the success probability of the incipient colony. Nanitic workers constitute an interesting case of phenotypic plasticity that highlights the advantages of miniaturisation during colony founding.

Nanitics forage: even though their small size jeopardizes their success in hunting species, they perform well as scavengers or honeydew eaters, as clearly shown in Ectatomma tuberculatum [START_REF] Dejean | Growth-related changes in predation behavior in incipient colonies of the ponerine ant Ectatomma tuberculatum (Olivier)[END_REF]. This species has a mixed diet with carbohydrates from nectaries and honeydew and proteins from live prey and dead insects. Foundation is non-claustral and the queen forages to breed a first batch of approximately 12 nanitic workers.

These nanitics forage actively and mostly bring back dead insects and honey. In contrast, larger "post-nanitic" workers born after them are efficient hunters and use a sophisticated ambush strategy to capture live prey. This observation shows that nanitics do not have the same behavioural repertoire as workers but perform well the tasks they specialise for.

Nanitic workers occur in species with medium-and large-sized workers: Ectatomma tuberculatum (10mm, [START_REF] Dejean | Growth-related changes in predation behavior in incipient colonies of the ponerine ant Ectatomma tuberculatum (Olivier)[END_REF], Myrmica rubra (4-5mm, [START_REF] Brian | Food distribution and larval size in cultures of the ant Myrmica rubra L[END_REF], Camponotus japonicus (6-10mm, [START_REF] Hasegawa | A trade-off between number and size within the first workers of the ant Camponotus japonicus[END_REF], Oecophylla longinoda (6mm, [START_REF] Ouagoussounon | Founding weaver ant queens (Oecophylla longinoda) increase production and nanitic worker size when adopting non-nestmate pupae[END_REF] and Solenopsis invicta (smallest species with nanitics described, 3-6mm, [START_REF] Porter | Adaptive Value of Nanitic Workers in Newly Founded Red Imported Fire Ant Colonies (Hymenoptera: Formicidae)[END_REF]. Species with minute workers cannot have nanitics if standard workers are already reduced to a minimal size. Furthermore, worker miniaturisation is usually linked to a higher worker/queen dimorphism, meaning that a queen can produce many minute workers just with her own metabolic reserves. Miniaturisation and nanitics are two distinct phenomena, the first one being an evolutionary process across generations and the second one linked to phenotypic plasticity in one generation, even though both provide similar benefits for colony foundation. Fast development of workers at all colony stages means that minute ant colonies grow fast, which is essential in an environment with competitors and limited resources.

Symbioses and parabioses

Ants get involved in various interactions with their biotic environment (see Chomicki and Renner, 2017 for a review). In particular, trophobiosis (food-based mutualism) between ants and aphids is iconic. Aphids suck the sap of plants and excrete a sugar droplet called honeydew. Formicine ants collect this honeydew and protect the plant and the aphids from predators and invaders [START_REF] Way | Mutualism Between Ants and Honeydew-Producing Homoptera[END_REF] and clean the colony from aphid exuviae [START_REF] Buckley | Ant-Plant-Homopteran Interactions[END_REF]. Aphids are just one example from the order Hemiptera that also includes stink bugs and scale insects. Ant-Hemiptera mutualism exists in species of various sizes, but because the size of the ant usually matches the size of the Hemiptera, miniaturisation gives access to new potential interaction partners. In addition, miniaturisation is associated with larger colonies (see Colonial economy), meaning that more workers are available to tend more Hemiptera. A non-extensive overview of ant-Hemiptera mutualism is provided in Table 1. All Hemiptera reported in this table produce honeydew but belong to different families. Ants involved in these mutualisms belong to three of the four most diverse subfamilies (Dolichoderinae, Formicinae and Myrmicinae), which suggests that ant-Hemiptera interactions play a role in species diversification.

Other remarkable cases of mutualism exist in Myrmicinae, with the example of Melissotarsus. These 2.5mm ants live below the bark of multiple families of trees of various trunk width and breed armoured scale insects that do not produce honeydew [START_REF] Schneider | Mutualism between armoured scale insects and ants: new species and observations on a unique trophobiosis (Hemiptera: Diaspididae; Hymenoptera: Formicidae: Melissotarsus Emery): Ants and diaspidids: new species and observations[END_REF]. The scale insects can live without the ants, on the bark of trees, and produce a shield that protects them from predators. But with the ants, they live below the bark, which provides them with more direct access to the parenchyma tissues they feed on, but also a shelter and hygiene. Scale insects bred by Melissotarsus do not produce their shield anymore and the ants feed on their secretions as well as exuviae and adult individuals (Peeters et al., 2017b, Cyrille et al. in prep). Ants show total commitment to life inside tunnels as workers show a range of adaptations to tunnelling through live wood, such as modified midlegs that prevent them from walking on flat surfaces (Delage-Darchen, 1972, Appendix 2). Ants and scale insects benefit from one another but can only survive if they are not deleterious to the tree. Melissotarsus chew extensive networks of galleries below the bark and multiple colonies can merge into a single super-colony, which could make them a remarkable pest for tree plantations and forests [START_REF] Mony | Behavioural Ecology of Bark-digging Ants of the Genus Melissotarsus[END_REF]. However, they do not seem to harm trees that can survive for decades in virtue of the small size of the ants. Larger ants would chew wider tunnels that would kill most trees, meaning they could only live in large (old) trees and would be forced to move their nest frequently anyway. In contrast, Melissotarsus can colonize young trees, and the colony will grow with the tree. This system shows a combination of advantages provided by miniaturisation in a mutualistic context. Besides, miniaturisation can open the door to new types of interactions. For example, two species of ants can share a common nest but keep their brood separated. This phenomenon, called parabiosis (literally "living beside"), often involves ants of different sizes. In South America, small ants from the genus Crematogaster (around 3mm-long) cohabit with larger species from genera Camponotus, Odontomachus, Pachycondyla and Dolichoderus. They bring epiphyte seeds to their carton nests to form "ant gardens" [START_REF] Davidson | Ecological Studies of Neotropical Ant Gardens[END_REF][START_REF] Menzel | Selective interspecific tolerance in tropical Crematogaster-Camponotus associations[END_REF][START_REF] Orivel | Ant gardens: interspecific recognition in parabiotic ant species[END_REF][START_REF] Swain | Trophic competition among parabiotic ants[END_REF][START_REF] Vantaux | Parasitism versus mutualism in the ant-garden parabiosis between Camponotus femoratus and Crematogaster levior[END_REF] Benefits of this cohabitation are shared: the larger species builds and protects the nest, but is able to follow foraging trails of the smaller species and steal their prey. A great size difference between both species seems necessary to prevent competition for resources and congestion inside the nest. 5-Why not even smaller? Constraints of miniaturisation.

Sensory organs, nervous system and metabolic cost

Smaller animals have proportionally larger brains than large relatives. This brain allometry rule, known as Haller's rule [START_REF] Rensch | Histological Changes Correlated with Evolutionary Changes of Body Size[END_REF] has been studied thoroughly in vertebrates [START_REF] Harvey | Comparing brains[END_REF]) and more recently in invertebrates. Information processing in miniaturised nervous systems is challenged by noise and energy consumption, which should cause behavioural deficits (reviewed by [START_REF] Niven | Miniaturization of Nervous Systems and Neurons[END_REF]. Consequently, workers of Pheidole were shown to have a higher mass-specific metabolic rate than soldiers [START_REF] Shik | The metabolic costs of building ant colonies from variably sized subunits[END_REF], which could explain a shift towards carbohydrate-based diets as frequently observed in minute workers [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF]. Miniaturised nervous systems have smaller neurons packed densely and have an increased density of connections and energy consumption, but smaller neurons have less space for mitochondria that produce the energy required for neural signalling. In ants, one early study showed Haller's rule holds true and suggested a correlation between brain size and behavioural complexity [START_REF] Cole | Size and behavior in ants: Constraints on complexity[END_REF].

However, a more recent study showed that the scaling coefficient changes at small body sizes within and between species, supporting Haller's rule, but predicted that behaviour of minute ants should be comparable to larger ants [START_REF] Seid | The Allometry of Brain Miniaturization in Ants[END_REF]). Cole's results could be biased because he looked at the behavioural repertoire of the smallest workers in species with different modes of division of labour: workers and soldiers, polymorphic workers, and monomorphic workers. Worker size may have simply been correlated to division of labour, which influences the tasks a worker has to take care of. Similarly, in orb-weaving spiders ranging from 0.6 to 7mm in size, web construction behaviour was not impacted by miniaturisation, which contradicts the behavioural simplification hypothesis intra-and interspecifically [START_REF] Eberhard | Are smaller animals behaviourally limited? Lack of clear constraints in miniature spiders[END_REF].

One real impact of miniaturisation on behaviour in ants involves sensory organs and associated brain regions. Indeed, smaller species have smaller eyes and lower contrast sensitivity, spatial resolving and obstacle avoidance ability (Palavalli-Nettimi and Narendra, 2018;[START_REF] Palavalli-Nettimi | Miniaturisation reduces contrast sensitivity and spatial resolving power in ants[END_REF]. However, in social insects, chemical cues play an important role, in particular for nestmate recognition [START_REF] Nielsen | Colony-level and season-specific variation in cuticular hydrocarbon profiles of individual workers in the ant Formica truncorum[END_REF] but also for foraging trails in ants [START_REF] Jackson | Communication in ants[END_REF]. Depending on the species, ants respond more to visual cues, chemical cues, or a combination of both. Visual cues are only relevant for aboveground navigation [START_REF] Heyman | Ants Use Multiple Spatial Memories and Chemical Pointers to Navigate Their Nest[END_REF] and used most extensively by desert or nocturnal ants (large) with big eyes such as Cataglyphis, Melophorus and Mystrium [START_REF] Freas | The View from the Trees: Nocturnal Bull Ants, Myrmecia midas, Use the Surrounding Panorama While Descending from Trees[END_REF][START_REF] Narendra | Learning, retention and coding of nest-associated visual cues by the Australian desert ant, Melophorus bagoti[END_REF][START_REF] Steck | Desert ants benefit from combining visual and olfactory landmarks[END_REF]. In contrast, many species of minute ants live underground (discussed in Chapter I) and rely more on chemical cues. Large eyes and optic lobes are conserved exclusively in alates that need to locate a mate and a suitable nest.

Egg size, not a problem in ant workers

One limit hypothesized by [START_REF] Rensch | Histological Changes Correlated with Evolutionary Changes of Body Size[END_REF] is the size of the reproductive system and egg. Increasing egg size with decreasing body size has been observed in Coleoptera and Lepidoptera (reviewed by [START_REF] Polilov | Small Is Beautiful: Features of the Smallest Insects and Limits to Miniaturization[END_REF]. However, some organisms developed a way to push this limit. Indeed, an egg has two main parts: the zygote, and the yolk that provides nutrients required for the zygote development. In minute parasitoid wasps (Myrmaridae and Trichogrammatidae), yolk size is considerably reduced because the zygote grows using the host's resources [START_REF] Polilov | Small Is Beautiful: Features of the Smallest Insects and Limits to Miniaturization[END_REF]. In ants, the size of the reproductive system and egg unlikely limits miniaturisation for two reasons. First, concerning worker miniaturisation, worker ants are sterile in most genera: they lack a spermatheca [START_REF] Gotoh | Degeneration patterns of the worker spermatheca during morphogenesis in ants (Hymenoptera: Formicidae)[END_REF], have few undeveloped ovarioles, and do not lay eggs. Second, concerning queen miniaturisation, an ant zygote mostly develops at the larval phase when fed by other workers. Hence, similarly to parasitoid, we can expect little yolk is necessary for the zygote development, which reduces the size of eggs.

Flipside of physics: surface tension and Reynolds number

As we wrote earlier, minute ants move through a world where gravity can be exceeded by other forces. For example, surface tension acts at liquid-air interfaces and explains the cohesion of water drops. Interestingly, water drops have an average volume of 50mm 3 . While most insects are not worried of getting caught in such a small volume in virtue of their size and their hydrophobic cuticle [START_REF] Watson | Anti-Wetting on Insect Cuticle -Structuring to Minimise Adhesion and Weight[END_REF], the smallest ants have a volume close to 1mm 3 and can get trapped inside a raindrop. Their low mass means they have a low kinetic energy and they can hardly break through tension surface forces, making minute workers vulnerable to rainfall events. More importantly, size also shapes the way an organism perceives the viscosity of a fluid, including air. One mathematical number that summarises it is Reynolds number, introduced to biologists by [START_REF] Purcell | Life at low Reynolds number[END_REF], given by the expression Re = lv/𝛎 where l is the length of the organism, v its velocity, and 𝛎 the kinematic viscosity of the

Wing muscles, not a problem anymore

Wing muscles fill most of the thorax of alate insects [START_REF] Snodgrass | The thorax of the Hymenoptera[END_REF] and certainly limit miniaturisation in Hymenoptera. In an ant queen of Cataglyphis savignyi, wing muscles account for 52% of the thorax volume (personal data). The smallest flying insects underwent drastic modifications of their flight apparatus: wings are ciliate and wing muscles are extremely reduced, making these insects "passive flyers" using air flow [START_REF] Cheer | Paddles and rakes: Fluid flow through bristled appendages of small organisms[END_REF]. However, such modifications do not concern ant workers: they do not fly and have lost wing muscles, which frees a considerable space inside the thorax (figure 7). This space permitted an enlargement of neck, leg and petiole muscles useful for ground labour [START_REF] Keller | Evolution of thorax architecture in ant castes highlights trade-off between flight and ground behaviors[END_REF], Appendix 1) and the miniaturisation of the thorax in workers.

Thorax endoskeleton and muscles?

However, new constraints may arise from this worker thorax. Large muscles are what make ant workers fit for ground labours that include carrying, walking/running, stinging and spraying acid, more than any other minute insect. The thorax is the power core of an ant worker because it hosts muscles that move the head, the legs, and the abdomen. A common explanation given to explain the strength of ants is called the square-cube law: the volume of an organism is a volume -a cube length, l 3 -whereas muscle strength is proportional to crosssectional area -a square length, l 2 . The ratio between muscle strength and body volume (1/l) increases when size decreases, explaining why small organisms can lift relatively heavier objects. However, despite the square-cube law, many muscles with different functions have to fit with vital organs of the nervous, digestive and endocrine systems. Miniaturisation likely involves trade-offs inside the thorax that remain to be understood, between muscles and other organs but also between different muscles. Characterizing these trade-offs would help to explain how minute ants can perform as well as larger ants and reveal new limits to miniaturisation. This is the question I chose to focus on during my PhD. As a conclusion, positive and negative effects of miniaturisation on ants are summarized in table 2.

6-The thorax, power core of workers

Before diving into the core of my thesis, I would like to make you familiar with the ant thorax (Figure 8, Appendix 1). First of all, what we call "thorax" is usually referred to as "mesosoma" because in ants, like in all Apocrita, it includes the three thoracic segments (T1, T2, T3) as well as the first abdominal segment (A1). Moreover, abdominal segments 2 and sometimes 3 (A2 and A3) become distinct body parts called petiole and post-petiole (postpetiole is specific to subfamily Myrmicinae). Petiole and post-petiole are additional articulations that amplify the movement range of what we call the abdomen (usually referred to as the "gaster"). Even though petiole and post-petiole include muscles that move the next segment, muscles included inside the thorax are much larger and are responsible for the overall power that moves the abdomen.

What can be found inside the thorax? First, the digestive system. Inside the thorax, it is the oesophagus. There is no digestion ongoing yet inside this section of the digestive tube, so the primary function of the oesophagus is to shuttle food from the infrabuccal pocket to the crop (located inside the abdomen) where nutrients are stored, or the other way for regurgitation to nestmates through mouth-to-mouth (stomodeal) trophallaxis. Trophallaxis is a crucial behaviour in most ant societies: it involves all colony members from larvae to queen and permits to finely tune trophic flow inside the colony and circulate a wide range of molecules involved in growth and development [START_REF] Leboeuf | Oral transfer of chemical cues, growth proteins and hormones in social insects[END_REF]. Third, the respiratory system. In the thorax, respiration relies on small openings through the exoskeleton connected to air sacs by particular structures called spiracles. The oxygen then moves through a network of tracheoles. Studying the respiratory system specifically is a tedious task, and this would deserve a PhD for itself, which is why I will rarely mention it.

Advantages

Fourth, the nervous system. All nerves inside the thorax are connected to a ventral nerve chord that passes through from the brain to the abdomen. The chord becomes a ganglion in all three thorax segments, anteriorly to each leg pair, referred to as the three neural ganglia (Figure 7). I will focus on this nerve chord and these ganglia and ignore other nerves.

Fifth, linked to the neural ganglia is the endoskeleton. Indeed, the exoskeleton shows several infoldings inside the thoracic cavity, including three forked pillars posterior to each ganglion. These pillars are called furcae and act as attachment surfaces for muscles. They are named profurca (in T1), mesofurca (in T2) and metafurca (in T3).

Last but far from least, the ant thorax includes many muscles. In ant queens, the largest muscles are wing muscles, longitudinal and dorsoventral, that fill up 52% of the thorax in Cataglyphis savignyi. Muscles I chose to focus on to study miniaturisation move the head, trochanters (second segment of the legs) and abdomen, turning the thorax into a power core.

However, many other muscles are inside the thorax: some connect exoskeleton and endoskeleton or two furcae and make the ant thorax more robust, others articulate the coxae (leg first segment) to provide a wide range of motions used for grooming, and others ensure the cohesion between sclerites.

As mentioned earlier, the thorax is the power core of ant workers and what makes them legendarily strong. Even though my focus on muscles influenced the design of my workflow, I tried to give to other components of the thorax sufficient attention.

7-Thesis goals, tools and outline

During my PhD, I investigated the effects of miniaturisation on ant workers, with a particular focus on allometric relationships inside the thorax. This manuscript is written in three chapters, all in English and formatted as scientific articles. The first one is currently in review in Journal of Natural History. The second one will soon be submitted to Arthropod Structure and Development. The third one will be refined before submission to Myrmecological News. Following these three chapters is a general discussion to intertwine all results and draw conclusions on a global scale. Then you will find two appendices, corresponding to two articles published with collaborators: the first one compares worker and queen thorax and is required to understand how unique the worker

Introduction

Food storage is an essential trait that distinguishes perennial from annual colonies in social Hymenoptera [START_REF] Judd | Social insects structure, function, and behavior[END_REF]. In addition to withstanding unfavourable periods of the year, the ability to store food buffers the resources retrieved by foragers for the nutrition of larvae, especially the expensive sexuals that are reared during a few months only. The alimentary tract of Hymenoptera is divided into crop, midgut and hindgut. In social species, ingested nutrients can either be held in the crop and regurgitated to nestmates through stomodeal (mouth to mouth) trophallaxis [START_REF] Leboeuf | [END_REF] or move to the midgut where digestion and absorption proceed. Because social wasps and bees need to fly for quotidian tasks, their crop can only store a limited amount of food, mostly nectar from single foraging trips. However, wasps and bees use carton or wax to build receptacles inside their nest, to store pollen as well as nectar. In sharp contrast, ant workers can store food internally, one of the many benefits of losing flight ability [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF]. A key adaptation for augmented internal storage in many ant lineages is the evolution of a more complex proventriculus, a cuticular valve that finely controls nutrient flow between crop and midgut [START_REF] Eisner | A comparative morphological study of the proventriculus of ants (Hymenoptera: Formicidae)[END_REF][START_REF] Eisner | The evolution and social significance of the ant proventriculus[END_REF]. Another adaptation unique to about fifteen ant genera is the convergent evolution of a soldier caste that is intermediate in gaster size between queens and workers and allometrically different (Peeters and Ito, unpublished data). Soldiers are defined as a discrete caste of large individuals that follow allometric growth rules distinct from workers, contrary to major workers that occur in species with polymorphic workers that follow the same growth rules [START_REF] Molet | Evolution of Novel Mosaic Castes in Ants: Modularity, Phenotypic Plasticity, and Colonial Buffering[END_REF]. Soldiers seem restricted to some of the lineages that have workers of extremely small size (head width below 1mm, [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF]. Soldiers are few in a colony and perform specific functions that are enhanced by the larger heads -nest defence, phragmosis (nest entrance blocking), seed-grinding -as well as food storage. Soldiers are more effective than workers to store food without any further adaptation in virtue of their larger gasters. However, it is worth mentioning extreme examples of replete workers such as Myrmecocystus and Prenolepis, which have gasters distended to the point that intersegmental membranes become stretched and visible. Though these replete workers have an unequalled storage capacity, they are restricted to a storage function:

Myrmecocystus replete workers hang from the nest roof and can only exchange food [START_REF] Burgett | Lipid Storage by Honey Ant Repletes[END_REF].

The ant genus Carebara (Myrmicinae: Crematogastrini) currently includes 253 described species occurring in tropical and subtropical regions on all continents [START_REF] Azorsa | Taxonomy of the ant genus Carebara Westwood (Formicidae, Myrmicinae) in the Malagasy Region[END_REF]. Following synonymy of Oligomyrmex [START_REF] Fernández | The American species of the myrmicine ant genus Carebara Westwood (Hymenoptera: Formicidae)[END_REF]) and especially

Pheidologeton [START_REF] Fischer | The ant genus Carebara Westwood (Hymenoptera, Formicidae): synonymisation of Pheidologeton Mayr under Carebara, establishment and revision of the C. polita species group[END_REF], Carebara species show a large variation in the degree of morphological differences within colonies. Workers are minute (body length around 1.5 mm) but can be monomorphic with huge queens (C. vidua = Carebara sensu stricto) or coexist with a distinct soldier caste (formerly Oligomyrmex); other species (formerly Pheidologeton)

show extreme polymorphism in body size among workers and soldiers. In parallel, life habits range from hypogaeic to large-scale aboveground raiding [START_REF] Lepage | Observations on the ant Carebara vidua F. Smith preying on termites in Kenya[END_REF][START_REF] Moffett | Foraging dynamics in the group-hunting myrmicine ant, Pheidologeton diversus[END_REF], but field behaviour has been studied in only few species. Log-nesting C.

urichi (C. polita group) is reported to be a scavenger and predator on minute arthropods [START_REF] Wilson | Caste and division of labor in Erebomyrma, a Genus of dimorphic ants (Hymenoptera: Formicidae: Myrmicinae)[END_REF], while subterranean C. vidua is a suspected predator of Macrotermes termites [START_REF] Lepage | Observations on the ant Carebara vidua F. Smith preying on termites in Kenya[END_REF]. Morphological studies identified two Southeast Asian and three Afrotropical Carebara species with phragmotic ("door-head") soldiers [START_REF] Fischer | Two new phragmotic ant species from Africa: morphology and next-generation sequencing solve a caste association problem in the genus Carebara Westwood[END_REF]. Though many Carebara species are subterranean, only C. vidua has been studied. In general, the biology of subterranean ants remains poorly understood despite their diversity [START_REF] Wong | Subterranean ants: summary and perspectives on field sampling methods, with notes on diversity and ecology (Hymenoptera: Formicidae)[END_REF] due to difficulties in both observation and sampling of whole colonies. Here we provide information on the biology of C. perpusilla (Emery, 1895), an underground species formerly classified as Oligomyrmex [START_REF] Ettershank | A generic revision of the world Myrmicinae related to Solenopsis and Pheidologeton (Hymenoptera: Formicidae)[END_REF] that has blind 1.5mm-long workers as well as much larger, morphologically disjunct soldiers. We describe differences in functional morphology between workers and soldiers. We use food dyes, dissections and micro-CT to compare crop size variation for food storage. Furthermore, our laboratory observations provide novel behavioural data and document the natural history of a species of Carebara restricted to a subterranean lifestyle.

Materials and Methods

Live ants

An incomplete colony of C. perpusilla was collected on 3 August 2016 (dry season) in a riverine forest in Gorongosa National Park, Sofala province, Mozambique (S 19.00133 E 34.37730, +/-100m elevation). The colony was first kept in laboratory in its original soil chamber transferred to a 120 x 90 x 45 mm box with plaster and frequently sprayed with water to maintain a moist environment. Then the colony was moved to a bigger artificial nest (285 x 285 x 90 mm) with glass-covered galleries and chambers dug inside plaster. Many chambers were available, and the colony occasionally migrated from one to another. We sterilized a beaker of gardening soil (heated at 80°C for 4 hours) and added it to the nest and foraging areas in addition to the original soil where the colony was found and brought with.

Ants were provided with big dead insects: crickets, mealworms and occasionally cockroaches.

Many springtails (Folsomia candida) were given occasionally and could hide in the soil, consistent with the abundance of tiny invertebrates in the soil. Ants did not show any interest towards sugar water. This study summarizes more than 300 hours of daytime observations over 2 years.

Between September and November 2018, a Plagiolepis pygmaea (Latreille, 1798) colony of unknown origin invaded uninhabited chambers of the C. perpusilla nest.

Plagiolepis are minute formicine ants with a worker caste that matches the size of C. perpusilla workers, which allowed us to observe C. perpusilla in a context of competition.

After a while, we culled half of this quickly growing P. pygmaea colony because it threatened to overwhelm C. perpusilla, then the rest of the colony left the nest 4 days later.

Food colouring

We prepared two solutions with dyes. The first one, 'blue', was obtained by mixing methylene blue powder with water: this solution is hydrophilic and highlights carbohydrates.

The second one, 'red', was prepared with Sudan Red III powder and alimentary oil (colza and sunflower): this solution is hydrophobic and highlights fat. Both solutions had a final concentration of 10 g/L. Mealworms were injected 0.1 mL and given to the ants after at least thirty minutes. Correct diffusion of the dye was easily assessed by eye and checked by cutting the mealworms.

CT-scanning (3 specimens)

Three specimens were micro-CT scanned to exemplify the castes and complement dissection data. Indeed, workers are exceedingly difficult to dissect, and micro-CT permits non-destructive visualization. Micro-CT scans were performed at the Okinawa Institute of Science and Technology Graduate University (OIST, Japan) using a Zeiss Xradia 510 Versa 3D X-ray microscope operated with the Zeiss Scout-and-Scan Control System software (version 11.1.6411.17883). Specimens were chosen randomly in order to compare castes, not to distinguish repletes and non-repletes. One worker, one replete soldier and one dealate reproductive queen of C. perpusilla were preserved in 90% ethanol, stained in a 2 M iodine solution for 24 h, and then transferred to microtubes filled with 99% ethanol for scanning.

Scan settings were selected accordingly to yield optimum scan quality. Full 360 degree rotations were based on 1601 projections. The resulting scan resolutions and parameter settings are provided (see Table S1). Post-imaging 3D reconstruction was done with the Zeiss Scout-and-Scan Control System Reconstructor software (version 11.1.6411.17883), and the output files saved in DICOM format. 3D reconstructions of image stacks were visualized with Drishti 2.6.5 [START_REF] Limaye | Drishti: a volume exploration and presentation tool[END_REF], a software that builds 3D models from the grey levels of voxels (volumetric pixels). Transfer Function Editor, Clipping Planes and Viewport tools were used to navigate through the gaster at any desired angle.

Measurements

Crop to gaster ratio calculated from micro-CT data in the three specimens we scanned.

We used Drishti to create three virtual perpendicular sections (longitudinal, frontal and transversal) through the middle of the gaster. We then measured the ratio between crop area and whole gaster area on all three views using ImageJ. We calculated the average of these three values, giving one crop proportion value for the worker and one for the soldier. In addition, we segmented the crop of the worker and the soldier using ITK-SNAP to determine the absolute crop volume for these two castes.

We measured head, mesosoma and gaster length from pictures in dorsal view of C. perpusilla specimens in their nest through the glass. We measured 8 individuals per caste, chosen in order to represent the morphological variability observed in the colony.

Measurements were taken as follows: head length = from clypeus to occiput; mesosoma length = from the occiput to the anterior tip of the petiole; gaster length = from the posterior tip of the postpetiole to the end of the gaster. We compared the mean length and variance for head, mesosoma and gaster length between castes. We used the Bartlett test to compare variances. In cases of homoscedasticity, mean lengths were compared using the one-way ANOVA followed by the post-hoc Tukey test. In cases of heteroscedasticity, mean lengths were compared using the Kruskal-Wallis rank test followed by pairwise comparisons using the Dunn test. All p-values above 0.05 were considered statistically non-significant.

Colony life

Brood care. Carebara perpusilla workers performed daily tasks related to feeding (see Intracolonial trophic flow), grooming, and transporting brood. Ordinarily, only early and late larval instars were moved around the nest. We observed several emigration events that lasted less than a day. A few workers first visited the future nest, sometimes a few days in advance. Later, we counted 50 to 100 workers and 2 to 5 soldiers visiting the nest before queens and brood get relocated in a few hours (three out of seven queens left the old nest within half an hour). We did not observe adult transport. Over the whole emigration process, only workers carried the brood, including large sexual larvae and pupae that were dragged backward over short distances by individual workers. Interestingly, a group of five workers tried to carry a male pupa, but they moved slower than a single worker. Only once, a soldier was seen carrying a big male larva.

Meconium.

As in all Apocrita, the midgut of ant larvae does not connect to the rectum, thus feces aggregate at the end of the digestive tract. Older C. perpusilla larvae have two kinds of aggregates at the end of the digestive tract: one viscous and white, the other solid and black (Figure S2A). When both occurred inside a larva, the white one was the most posterior. In late larval instars, the white aggregate pushed through the soft larval cuticle. One worker came to extract the big droplet, held this "white meconium" between the mandibles and allowed other workers and larvae to lick it before carrying it to a midden chamber inside the nest or outside to drop it. Also in late larval stages, the black aggregate grew bigger and when a part of it was extruded, any worker that walked by started licking and pulling it. In the following minutes other workers (up to seven) joined to lick and pull the black meconium (Figure S2B). Removal lasted from two minutes to half an hour. Then the meconium was licked for several additional minutes, and one worker discarded it in the midden or outside.

Carcass burial and retrieval

Individual workers were observed walking randomly all over the foraging area. Any big dead insect was quickly found by one or more workers that antennated it multiple times, then walked to another part of the carcass and repeated the antennation behaviour. This sequence went on for one to five minutes, often followed by a few extra minutes of nondirectional walking, before the worker walked back to the nest. Workers were observed leaving a trail by touching the ground with the distal tip of their gaster. This trail was not

In parallel, individual workers chased springtails. Stinging was not observed, instead workers gripped and crushed their prey. When they tried to catch springtails above ground, these often tried to escape by jumping. Carebara perpusilla workers showed remarkable speed and strength, usually managing to bite and grip the springtails before they jump. When they escaped, springtails often landed next to another worker and got captured. In addition, workers were particularly efficient to capture springtails hiding below the ground (i.e. where jumping was impossible). A few cases of collaboration between two workers were observed:

if one worker could not secure a good grip on a springtail, another worker came and bit it also. The first worker could adjust its grip and carry it alone. Springtails were chased even by workers returning from a big prey to the nest with a full crop (visualized in the case of coloured mealworms). Once in the nest, several workers fed on springtails, and solid pieces or even entire preys were given to medium larvae.

Interactions with Plagiolepis pygmaea

Carebara perpusilla workers moved soil pellets and corpses to build thick barricades inside their tunnels that separated them from the Plagiolepis pygmaea colony (Figure S3). Some non-replete soldiers stayed close to these barricades. Therefore, interactions between P. pygmaea and C. perpusilla workers happened exclusively outside the nest and were most aggressive next to common food resources (Figure 6). Three P. pygmaea foragers tried to bite the legs of a C. perpusilla worker. One P. pygmaea was spotted carrying an immobile C.

perpusilla worker back to its nest, but dropped it when picked up with forceps, and the immobile worker started moving again and walked back to its nest. Multiple dead C. perpusilla workers and soldiers were found inside P. pygmaea chambers. Surprisingly, several P. pygmaea scouts walked a few millimetres away from a freshly buried mealworm without stopping.

One day, two C. perpusilla workers chased away a P. pygmaea scout that was drinking hemolymph from a dead mealworm (five C. perpusilla were already on it). But that scout came back two minutes later with five other workers, one seen with the gaster raised.

After another two minutes, the number of P. pygmaea workers became ten and one bent the gaster down (possibly to spray formic acid, not seen). Two minutes later, twenty-two P.

pygmaea were facing the five C. perpusilla workers. One C. perpusilla opened its mandibles and made one P. pygmaea walk away, but the Carebara were outnumbered. After six more minutes, forty-five P. pygmaea were on the mealworm. Three C. perpusilla remained: two pulling and licking it for a few minutes before discarding it outside the nest. We suppose that something at its surface attracts workers so that it is pulled out before the larva pupates. This attractive substance is ingested by workers and would thus participate in nutrient recycling. [START_REF] Wilson | A social ethogram of the neotropical arboreal ant </i>Zacryptocerus varians</i> (Fr. Smith)[END_REF] observed one similar meconium removal in Cephalotes varians: it was pulled by a single worker, licked by multiple workers who then passed it along the nest from one ant to another and discarded it outside. The meconium shrank in size though no solid piece was apparently taken away. The potential nutritive role of meconium disposal in ants has been neglected in past studies.

Burial of insect carcasses

The foraging strategy of C. perpusilla does not rely on soldiers, which rarely come out of the nest and only for a few minutes. In contrast, Pheidole pallidula soldiers are active in the foraging process: they are recruited to transport carcasses and protect foraging trails [START_REF] Detrain | Field study on foraging by the polymorphic ant species, Pheidole pallidula[END_REF]. Instead of bringing dead mealworms or crickets to the nest, C. perpusilla buries them in the ground. Such food exploitation strategy has already been reported in Solenopsis invicta that switches from transporting to burying when fed to satiation [START_REF] Qin | Food-burying behavior in red imported fire ants (Hymenoptera: Formicidae)[END_REF] and ant breeders have also observed food burial in the pavement ant Tetramorium caespitum. Interestingly, in C. perpusilla, collective transportation by workers was never observed, not even to relocate the heavy sexual brood. On one hand, burying a carcass bypasses the retrieving phase that can be fastidious for subterranean ants. Indeed, C. perpusilla workers move through narrow channels in soil that match their size. Dragging a big prey to the nest would either destroy underground tunnels or dangerously expose the ants to aboveground predators. On the other hand, burying a dead insect hides it from other potential competitors -P. pygmaea in our observations -and parasites. Inside the moist foraging area, mealworm carcasses were prone to rot in a few days; however, mould did not develop on buried carcasses. Some stayed buried for a long time, unnoticed and unspoiled, while workers took time to fully exploit it below the soil cover. Fierce competition with P. pygmaea was observed before carcasses were fully buried. P. pygmaea workers are similar in body size, walk fast, spray acid, and recruit efficiently. However, the C. perpusilla colony had few foragers and only 14 soldiers at the time of confrontation with P. pygmaea, and this may have affected the outcome of encounters. Carcass burial seems particularly adapted for subterranean ants, and we suppose this behaviour occurs more widely since most species are understudied (reviewed by [START_REF] Wong | Subterranean ants: summary and perspectives on field sampling methods, with notes on diversity and ecology (Hymenoptera: Formicidae)[END_REF]. Interestingly, workers of a laboratory colony of Pheidole flaveria were observed burying a dead cricket with soil pellets (C. Peeters, personal data), although nothing is known about the natural ecology of this species.

In addition to scavenging big dead arthropods, C. perpusilla workers actively chased springtails. Such microinsects are abundant in the soil and are a constant and reliable food resource that workers can deal with individually. Similarly, [START_REF] Wilson | Caste and division of labor in Erebomyrma, a Genus of dimorphic ants (Hymenoptera: Formicidae: Myrmicinae)[END_REF] observed workers of Carebara urichi bringing to the nest tiny prey: a mite, a springtail and arthropod eggs. In contrast to Strumigenys trap-jaw ants that have morphologically specialized mandibles to hunt springtails [START_REF] Gronenberg | The trap-jaw mechanism in the dacetine ants Daceton armigerum and Strumigenys sp[END_REF], C. perpusilla workers have a more generalist morphology allowing them to exploit a wider range of resources.

Repletes for food storage

All castes were found to have an expandable crop (Figure 8), associated with prolonged gaster distension (repletion) in soldiers and queens only. Food-colouring and observations of trophallaxis showed that newly acquired food first goes to workers and larvae before soldiers and queens, underlining the replete function of the latter.

Food storage is an emergent property of insect colonies. Only a small proportion of colony members forage at any given time, and food brought back to the nest is shared with younger nestmates and the queen(s). Excess nutrients (i.e. not immediately used for larval growth or adult maintenance) can be stored in various ways. Ponerine and other predatory ants can keep their prey alive for a few days by immobilizing them with venom, although excess food is also stored as lipids in the hemolymph of young workers [START_REF] Smith | Nutritional Asymmetries Are Related to Division of Labor in a Queenless Ant[END_REF].

Harvester ants (e.g. Messor and Pogonomyrmex) forage on seeds that are hoarded inside underground chambers called granaries [START_REF] Johnson | Seed-harvester ants (Hymenoptera: Formicidae) of North America: an overview of ecology and biogeography[END_REF][START_REF] Reyes-Lopez | Food storage in the nest and seed selectivity in the harvester ant Messor barbarus (Hymenoptera: Formicidae)[END_REF]. A major shift to internal food storage evolved in species foraging on honeydew (subfamily Formicinae), that repletes store in their crop inside the colony. An extreme example is the honeypot ant Myrmecocystus (Formicinae) where replete workers hang from the nest roofs with a ball-like distended gaster full of lipids and carbohydrates [START_REF] Burgett | Lipid Storage by Honey Ant Repletes[END_REF]. When repletes are removed from a colony, other large workers become repletes [START_REF] Rissing | Replete Caste Production and Allometry of Workers in the Honey Ant, Myrmecocystus mexicanus Wesmael (Hymenoptera: Formicidae)[END_REF]. In honeydew-eating ants, a distensible crop does not only improve storage but also foraging efficiency as a way to transport more food. In contrast, examples of replete workers in Myrmicinae are scarce. A first reason is that these ants generally do not feed on honeydew. A second reason is that myrmicine workers are usually minute, and thus not very efficient to transport or store food internally, even with a distensible crop. The only example of replete workers in Myrmicinae is in Monomorium pharaonis. In this species, only larvae can swallow solid food and they convert it into liquid secretions for workers and queens [START_REF] Børgesen | A new aspect of the role of larvae in the pharaoh's ant society (Monomorium pharaonis (L.) -Formicidae, Myrmicinae): Producer of fecundity-increasing substances to the queen[END_REF]. Some young workers become repletes and store the excess of liquid secretions to feed queens in a period of starvation and maintain egg-laying rate [START_REF] Børgesen | Nutritional function of replete workers in the pharaoh's ant, Monomorium pharaonis (L.)[END_REF].

In genera where a soldier caste has evolved, morphological specialisation for nutrient storage exists, though often less obvious than head adaptations (mandible shape and volume of head capsule largely reflect the mandibular closer muscles). In Colobopsis (Formicinae), soldiers are phragmotic, i.e. they use their modified head to block nest entrances. In C.

fraxinicola and C. nipponica, these soldiers have gasters distended with highly developed fat bodies [START_REF] Hasegawa | Caste specialization in food storage in the dimorphic ant Colobopsis nipponicus (Wheeler)[END_REF][START_REF] Wilson | The soldier of the ant Camponotus (Colobopsis) fraxinicola as a trophic caste[END_REF]. In Crematogaster (subgenus Orthocrema, Myrmicinae: Crematogastrini), the soldier caste has ovaries with intermediate numbers of ovarioles between workers and queens, and soldiers lay trophic eggs [START_REF] Peeters | Evolution of a soldier caste specialized to lay unfertilized eggs in the ant genus Crematogaster (subgenus Orthocrema)[END_REF]. Fat body storage occurs in Cataglyphis bombycina desert ants (Formicinae): soldiers have long sabre-shaped mandibles suited to fight vertebrate predators, as well as distended abdomens [START_REF] Molet | Bigger Helpers in the Ant Cataglyphis bombycina: Increased Worker Polymorphism or Novel Soldier Caste?[END_REF]. Lizards are the main predators and are probably able to dig to reach underground chambers: the soldiers can arguably both defend and store food by staying inside. In C. perpusilla, soldiers were rarely observed outside the nest and when they were, they always had a small gaster; replete soldiers were exclusively inside. We expect soldiers to defend and store food inside the nest while a minority of non-replete soldiers walk out to help dismember dead insects. Interestingly, even though replete workers exist in very few Myrmicinae, the most widespread occurrence of replete soldiers may be in Pheidole (1100 species), even though Wilson (2003, p.17) reported scant evidence for this role. Soldiers are larger than workers and therefore better candidates for food storage adaptations in general. [START_REF] Eguchi | A revision of Northern Vietnamese species of the ant genus Pheidole (Insecta: Hymenoptera: Formicidae: Myrmicinae)[END_REF] recorded replete soldiers in nine Pheidole species from Vietnam, some of which are seed-gatherers. In P. ryukyuensis, young soldiers can become repletes, and workers starved together with a replete soldier survived four times longer than with a non-replete soldier [START_REF] Tsuji | Nutrient Storage in the Major Workers of Pheidole ryukyuensis (Hymenoptera: Formicidae)[END_REF]. In P. morrisi, replete soldiers have twice as much fat as non-repletes, they are more numerous before winter and avoid confrontation with fire ants more than nonrepletes [START_REF] Yang | Seasonality, division of labor, and dynamics of colony-level nutrient storage in the ant Pheidole morrisi[END_REF]. Pheidole pallidula replete soldiers store lipids exclusively and are heavier as well as behaviourally distinct in their avoidance of defensive tasks, compared to non-repletes [START_REF] Lachaud | Lipid storage by major workers and starvation resistance in the ant Pheidole pallidula (Hymenoptera, Formicidae)[END_REF]. Similarly, our food dye data indicate that C. perpusilla repletes accumulate lipids. Fat storage inside the crop was also shown in repletes of Solenopsis invicta [START_REF] Glancey | Evidence of a Replete Caste in the Fire Ant Solenopsis invicta[END_REF]. In Carebara, replete soldiers were already observed in C. urichi but with no evidence of fat storage as workers ate insects but also sugar water [START_REF] Wilson | Caste and division of labor in Erebomyrma, a Genus of dimorphic ants (Hymenoptera: Formicidae: Myrmicinae)[END_REF]. These multiple examples show that soldiers in Myrmicinae are preadapted for food storage and repletes are not restricted to honeydew-eating ants.

Swollen gasters are commonly observed in ant queens due to enlarged ovaries and a high egg-laying rate (physogastry), but we found virgin dealate queens of C. perpusilla with distended crops. Carebara perpusilla is polygynous: multiple egg-laying queens and replete queens co-existed in our laboratory colony. Twenty-nine gynes stayed inside the nest and severed their wings. Observations and dissections suggest that all these virgin gynes became repletes. Micro-CT examination of oesophagus and abdominal crop showed membranes to be highly wrinkled, denoting elasticity (data not shown). Though C. perpusilla queens can store more food than soldiers due to body size differences, this slight advantage is unlikely to compensate the huge costs of gyne production. Gynes are expensive because of wing muscles, so replete gynes are probably a laboratory artefact, unlikely to occur in wild colonies.

Extreme size difference between workers and soldiers

The evolution of minute workers along with larger soldiers has various advantages.

Such workers are cheap to produce and permit an increase in colony size, together with novel diet specialisation and nesting sites [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF]. In contrast, soldiers are more expensive and few in a colony. In C. perpusilla one soldier can store as much food as 25 workers, and even in species lacking conspicuous repletes, one soldier can store more food than several workers in virtue of the size difference. Furthermore, contrary to replete workers that are restricted to food storage, replete soldiers can have multiple roles. Examples in the literature show that soldiers of a given species can combine two functions: food storage with defence [START_REF] Molet | Bigger Helpers in the Ant Cataglyphis bombycina: Increased Worker Polymorphism or Novel Soldier Caste?[END_REF], phragmosis [START_REF] Hasegawa | Caste specialization in food storage in the dimorphic ant Colobopsis nipponicus (Wheeler)[END_REF][START_REF] Wilson | The soldier of the ant Camponotus (Colobopsis) fraxinicola as a trophic caste[END_REF] or seed-milling [START_REF] Gobin | Queens and major workers of Acanthomyrmex ferox redistribute nutrients with trophic eggs[END_REF]. We argue that the balance between a replete soldier's big head and big gaster keeps the centre of mass around the mesosoma (discussed in [START_REF] Anderson | Simple" Biomechanical Model for Ants Reveals How Correlated Evolution among Body Segments Minimizes Variation in Center of Mass as Heads Get Larger[END_REF], contrary to small-headed replete workers (e.g. Myrmecocystus) where it shifts posteriorly and throws off balance. Having multi-tasking soldiers in a colony counterbalances their high production costs, moreover only few soldiers are needed in a colony. In species with minute workers but lacking soldiers, these tasks are either absent or performed by queens. However, queens have wing muscles and are hence more costly to produce than soldiers. The adaptive value of soldiers explains their parallel evolution in distant genera with high worker-queen difference in body size. The most striking example is the hyperdiverse genus Pheidole [START_REF] Moreau | Unraveling the evolutionary history of the hyperdiverse ant genus Pheidole (Hymenoptera: Formicidae)[END_REF], in which all species have soldiers that promoted an effective division of labour [START_REF] Wilson | Pheidole in the New World: A Dominant, Hyperdiverse Ant Genus[END_REF].

Chapter 2: Minute workers and large soldiers in the subterranean ant Carebara perpusilla: functional consequences of muscle allometry in the thorax

Abstract

Ants can carry multiple times their own weight over long distances. Loss of flight in worker ants, accompanied by the loss of wing muscles, freed space inside the thorax. Muscles that articulate the head, legs and petiole fill a larger portion of the thorax, which maximizes strength. However, most genera have minute workers (body length below 2mm). Regardless of body size, workers are capable of carrying multiple times their own weight. This is commonly explained by physics and the square-cube law: an animal size is determined by its volume (cube length) whereas muscle strength is linked to cross-sectional area (square length), making small animals stronger. However, how can muscles and organs fit inside the thorax at the lower size limit has not been investigated. Using X-ray computed microtomography (microCT), we studied the exoskeleton and internal anatomy of Carebara perpusilla, an African ant species that has minute workers (1.5mm-long) and larger soldiers (3.0mm-long), allowing strong intraspecific comparisons. We compared the relative volume of the neural ganglia and muscles that move the head, legs and abdomen inside the thorax and the cuticle thickness of the head and thorax. We highlight strong allometric differences inside the thorax of two wingless castes reflecting trade-offs linked to miniaturisation and division of labour, two phenomena that play major roles in the evolutionary success of ants.

Differences in cuticle thickness show that the exoskeleton does not miniaturise, which decreases the difference in production cost between workers and soldiers. We discuss how not only physics but also thorax design underlies the strength of minute ants.

Introduction

Ant workers are well-known for their ability to carry heavy objects or food items multiple times their own weight. This ability is essential because ants are central place foragers: workers bring food to a perennial nest to share it with nestmates instead of eating on the spot. The carrying power of ants lies in their thorax -usually referred to as 'mesosoma' in ants because in Apocrita the three thoracic segments are fused with the first abdominal segment, called 'propodeum' -which houses muscles that control the head, legs and abdomen, necessary for foraging and locomotion. Ant workers, in contrast to queens and other winged Hymenoptera, lack wing muscles, that normally fill 40-50% of the thorax volume, and instead other muscles expanded to the vacant space (Keller et al., 2014, Appendix 1).

Miniaturisation is the evolution of extremely small body size within a lineage [START_REF] Hanken | Miniaturization of Body Size: Organismal Consequences and Evolutionary Significance[END_REF]. This phenomenon is widespread in insects and has driven the evolution of the smallest metazoans (parasitoid wasps, see [START_REF] Polilov | Small Is Beautiful: Features of the Smallest Insects and Limits to Miniaturization[END_REF] for examples) as well as the evolution of ants. Indeed, flight loss alleviated constraints on body size in worker ants, thus permitting miniaturisation in most genera, contrary to social bees and wasps [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF]. During 140 Mya of evolution, relatively few species of ants became large (e.g. in Dinoponera, 30mm, 8 spp, Paraponera, 25mm, 1 sp, and Dinomyrmex, 28mm, 2 spp) while workers became minute -body size below 2mm -in most extant species (e.g. in Monomorium, 400 spp, Carebara, 250 spp, and Pheidole, 1100 spp, all Myrmicinae).

Miniaturisation brings many benefits to ants, such as opening up new interstitial niches and reducing the cost and development time of individuals [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF]. However, miniaturisation is more than a simple downscaling of individuals as the size of some organs changes allometrically with body size. A famous example is Haller's rule brain-body allometry, defined as the brain getting relatively bigger with decreasing body size [START_REF] Rensch | Histological Changes Correlated with Evolutionary Changes of Body Size[END_REF]. This rule has been validated empirically in ants [START_REF] Cole | Size and behavior in ants: Constraints on complexity[END_REF][START_REF] Seid | The Allometry of Brain Miniaturization in Ants[END_REF] as well as many vertebrate and invertebrate taxa (reviewed by [START_REF] Eberhard | Grade Changes in Brain-Body Allometry: Morphological and Behavioural Correlates of Brain Size in Miniature Spiders, Insects and Other Invertebrates[END_REF].

Moreover, miniaturisation involves a reduction of sensory organs like the eyes, and smaller ants -with less ommatidia -have a lower spatial resolution and contrast sensitivity (Palavalli-Nettimi and Narendra, 2018;[START_REF] Palavalli-Nettimi | Miniaturisation reduces contrast sensitivity and spatial resolving power in ants[END_REF]. Nonetheless, how vital organs and muscles scale with miniaturisation inside the thorax is unknown. The internal morphology of the thorax has only been studied in large ants that are not representative of the majority of ants (Liu et al., 2019, Appendix 1). The strength of small ants is commonly explained with physics and the square-cube law: the volume of the thorax is a cube measurement (x 3 ) whereas muscle strength is proportional to its cross-sectional area, a square measurement (x 2 ). The ratio between muscle strength and thorax volume (x 2 /x 3 = 1/x) increases when size (x) decreases, explaining why smaller animals can carry relatively heavier weights in general. However, ants stand out in terms of load carrying, and a minute ant thorax must integrate functional organs along with numerous muscles in a tiny volume in order to complete worker tasks.

Using X-ray computed microtomography, we compared the cuticle and internal thorax morphology of Carebara perpusilla workers and soldiers, focusing on the neural ganglia and muscles that move the head, legs and abdomen. Carebara perpusilla is a subterranean African species with two wingless infertile castes -1.5mm-long workers and 3mm-long soldiersallowing a powerful intraspecific comparison. Workers perform brood care and foraging.

They hunt soil microinsects such as springtails, as well as scavenge on large insect carcasses that they bury with soil, retrieving only small meat pieces and hemolymph (see Chapter 1). In contrast, soldiers rarely leave the nest: some guard the entrances while others store food in their distended abdomen (repletes). Soldiers can be recruited to insect carcasses where they use their powerful mandibles to cut through the cuticle, so that workers can access the meat.

Material and Methods

Specimens and scanning

Specimens of C. perpusilla were collected in a riverine forest on August 2016 in Gorongosa National Park, Sofala province, Mozambique (S 19.00133 E 34.37730, ± 100m elevation) and stored in 90% ethanol. Micro-CT scans were performed at the Okinawa Institute of Science and Technology Graduate University (OIST, Japan) using a Zeiss Xradia 510 Versa 3D X-ray microscope operated with the Zeiss Scout-and-Scan Control System software (version 11.1.6411.17883). One worker and one soldier, selected randomly, were stained in a 2 M iodine solution for 24 h, and then transferred into microtubes filled with 99% ethanol for scanning. Scan settings were selected to yield optimum scan quality. Full 360degree rotations were based on 1601 projections. The resulting scan resolutions and parameter settings are provided in Table S1. Post-imaging 3D reconstruction was done with the Zeiss Scout-and-Scan Control System Reconstructor software (version 11.1.6411.17883), and the output files saved in DICOM format.

Segmentation

Active labelling of voxels (volumetric pixels), i.e. segmentation, was performed from reconstructed stacks using ITK-SNAP 3.6.0 [START_REF] Yushkevich | User-guided 3D active contour segmentation of anatomical structures: Significantly improved efficiency and reliability[END_REF]. For most structures, segmentation was achieved semi-automatically with the 'region competition' algorithm followed by manual segmentation to correct errors. Other structures such as the furcae were segmented manually every 5 slices and filled in using the 'Interpolate' tool. Identification of muscles and skeletal structures was done by homology relative to Apis mellifera following [START_REF] Snodgrass | Anatomy of the honey bee[END_REF]. The whole thorax cuticle, neural ganglia and oesophagus were segmented, but muscles were only segmented in one half of the thorax (left or right) with the exception of muscle 46 (segmented for both sides). The thorax hosts many muscles, and we selected muscles with a function that can easily be determined and linked to particular tasks. First, muscles that insert on the occiput and cervical apodemes are respectively direct and indirect head muscles that move the head. Second, most leg muscles move the coxae but only one muscle per leg hosted inside the thorax inserts on the second leg segment and supports the body during locomotion. Third, muscles that insert on the petiole are responsible for abdomen movement and support.

Volume measurements

We used ITK-SNAP to determine the volume of the nervous system, directly computed using the count of labelled voxels and voxel size. However, this method would underestimate the actual volume one muscle occupies in the thorax: the space observed between the fibres of a muscle is due to shrinkage and needs to be included to determine physiological muscle volume. Therefore, we exported mesh files (.stl) from ITK-SNAP to Blender 2.80. We fitted a 3D object -usually an Icosphere -to each muscle mesh. Good fitting was achieved using 1) the 'Subsurface' modifier on the 3D object to create more faces and 2) the 'Shrinkwrap' modifier to fit this 3D object on the muscle mesh. The volume of this 3D object was measured and used as the physiological muscle volume. In case of poor fitting, we split the muscle mesh into two meshes of simpler shapes and fitted a 3D object to both separately. Volume values were multiplied by two for muscles segmented only on one side, and then normalized using the volume of the inner thorax -segmented with ITK-SNAPleading to proportions presented in figure 3 and table S2. Soldiers and workers are two distinct morphological castes following different allometric rules, which is why we used the whole thorax volume to scale our volume comparisons.

Cuticle thickness measurement

We measured cuticle thickness on 3D models using Drishti [START_REF] Limaye | Drishti: a volume exploration and presentation tool[END_REF]. We created 3D models directly from greyscale images (without segmentation) and inserted clipping planes through the head (behind the eyes), pronotum (first dorsal thoracic plate) and the propodeum (first abdominal plate but fused to the rest of the thorax in ants). Twelve measurements were taken for each individual along the left-right axis using the "Path" function. Statistical analyses were performed using R 3.3.2 to compare worker and soldier cuticle thickness normalized by head width values provided in Chapter 1. We tested data normal distribution using Shapiro-Wilk's test and homoscedasticity using Bartlett's test. We applied Student's two sample t-test when previous hypotheses were verified or Mann-Whitney's U test when not.

Results

Nervous system

Hymenoptera have a ventral nervous system inside the thorax, consisting of three ganglia connected by a nerve cord. In C. perpusilla, these ganglia are located near the insertion of the three leg pairs (Figure 1). Ganglia and nerve cord occupy 19.08% of the thorax of the worker compared to 11.61% for the soldier (Table S2). In addition, ganglia are tightly associated to endoskeletal structures called furcae. The three furcae are infoldings of the cuticle that rise like pillars inside the thorax anteriorly to each leg pair and terminate with a platform where muscles attach. Their position is slightly posterior to the neural ganglia. The first furca (or profurca) is particularly well-developed to attach muscles that move the head (Figure 2). The profurca partly encloses the first ganglion, hence a relatively bigger first ganglion in the worker implies a relatively higher profurca. Only a tiny space is left between the profurca platform and the dorsal cuticle (pronotum), filled exclusively by the oesophagus (Figure 1). In comparison, soldiers have more space between profurca and dorsal cuticle, used by the oesophagus but also muscle 46 (see Head muscles).

contrast, head and thorax had similar cuticle thickness values for the worker (5.4 ± 0.6 µm for the pronotum, 6.2 ± 1.0 µm for the propodeum). After normalizing by head width, the thorax cuticle of the worker was thicker than of the soldier (pronotum: Mann-Whitney U test, U = 139.5, p<0.001; propodeum: two sample t-test, t = 5.7894, p<0.001, Figure 4). Carebara perpusilla workers and soldiers have moderate cuticle sculptures on the propodeum, which explains the higher standard deviation we observed for this sclerite.

Discussion

No miniaturisation of the nervous system

The three thoracic ganglia are relatively larger in minute workers. In particular, the first ganglion occupies most of the prothorax and limits the reduction of the profurca, leaving only a tiny passage dorsally for the oesophagus. A similar study used microCT to look at the head internal anatomy of workers and soldiers of Pheidole, another genus with minute workers [START_REF] Lillico-Ouachour | Internal head morphology of minor workers and soldiers in the hyperdiverse ant genus Pheidole[END_REF]. The authors showed that workers have a relatively and absolutely larger brain than soldiers, which illustrates Haller's rule in terms of volumes.

Our results inside the thorax can be interpreted similarly. Though Haller's rule has not been investigated there, we suspect an identical biological phenomenon: there must be a lower limit to the size of the neural ganglia to ensure sufficient motor control of all six legs. The nervous system of ants, especially brain and thoracic ganglia, appears to be the main constraint on body size miniaturisation. In parallel, Haller's rule has been linked to behavioural repertoire in Eciton army ants [START_REF] O'donnell | Brain investment under colony-level selection: soldier specialization in Eciton army ants (Formicidae: Dorylinae)[END_REF]. In this genus, large soldiers are specialized for colony defence against vertebrates whereas polymorphic workers (smaller) forage, assemble the bivouac, and take care of the queen and brood. Soldiers have a relatively smaller brain with smaller sensory region, as a result of a colony-level selection for minimal investment in brain tissue [START_REF] O'donnell | Brain investment under colony-level selection: soldier specialization in Eciton army ants (Formicidae: Dorylinae)[END_REF]. Haller's rule can thus be linked to both miniaturisation and division of labour.

Head muscles are linked to division of labour

For both soldiers and workers, we focused on three direct muscles (42, 43 and 44) in the prothorax. Larger ants previously studied have an additional direct muscle (41) that is missing in C. perpusilla [START_REF] Liu | The mesosomal anatomy of Myrmecia nigrocincta workers and evolutionary transformations in Formicidae (Hymenoptera)[END_REF]; Appendix 1). To investigate whether the lack of muscle 41 is specific to subfamily Myrmicinae, we examined the thorax of larger myrmicines (Messor barbarus and Tetramorium fhg046) and found muscle 41. In addition, 41 was also found in the minute workers of an Asian Carebara, C. castanea (personal data). The function of muscle 41 is to lift the head up, which is redundant with muscles 42 and 43 that can likely balance the loss of 41 in C. perpusilla and possibly other species with minute workers.

Even though workers and soldiers had the same direct and indirect head muscles, their differed. Direct muscles -especially depressor muscles -occupied a slightly larger proportion of the thorax in workers. In C. perpusilla, only workers carry loads: they can catch and bring prey to the nest, but also move soil pellets to bury insect carcasses they feed on (see Chapter 1). Direct muscles are responsible for the up and down movement of the head and are likely involved in these tasks. Remarkably, the head/inner thorax volume ratio is higher for workers than soldiers despite the apparently large head of soldiers, explaining why workers require relatively larger direct head muscles. Moreover, soldiers do not carry anything but their head, hence direct muscles are only required for unloaded up and down movements.

Conversely, indirect head muscles are much bigger in soldiers. This major difference is explained by muscle 46 that fills most of the soldier prothorax. In C. perpusilla, older soldiers either guard the nest entrance or bite through the cuticle of insect carcasses to create holes so that workers can reach inside (see Chapter 1). Their large head is packed with powerful mandible muscles and needs to be manoeuvred sideways efficiently for fighting and biting.

Division of labour can thus explain differences in head muscle allocation between workers and soldiers.

Leg muscles and body balance

All three ET muscles are relatively larger in workers than in soldiers. Since only workers carry objects, loaded locomotion requires strong ET muscles to support the combined weight of the worker and its load across all legs. However, some leg pairs have specific functions that we can discuss in more details. First, C. perpusilla workers carry their loads with their mandibles, which likely shifts their centre of mass forward in a similar way as harvester ants [START_REF] Merienne | Walking kinematics in the polymorphic seed harvester ant Messor barbarus: influence of body size and load carriage[END_REF]: strong ET 1 muscles are required to support a greater weight with the forelegs. Second, soldiers have a huge head packed with mandible muscles, and young soldiers stay inside the nest and store food inside their abdomen, functioning as repletes (see Chapter 1). Therefore, the weight of their head is constant whereas the weight of their abdomen can vary considerably throughout their life. Consequently, their centre of mass move along the anteroposterior axis and ET 1 and 3 are more important to counterbalance this shift than ET 2 muscles, which are drastically reduced in soldiers. This is a result of stabilizing selection that constraints an ant's centre of mass to the thorax, as modelled by [START_REF] Anderson | Simple" Biomechanical Model for Ants Reveals How Correlated Evolution among Body Segments Minimizes Variation in Center of Mass as Heads Get Larger[END_REF].

Petiole muscles for abdomen support

All four petiole muscles are relatively larger in the soldier than in the worker. Petiole muscles are responsible for moving the abdomen: petiole and post-petiole are small segments compared to the posterior thorax (propodeum) so the muscles they house are extremely small in comparison. C. perpusilla soldiers can store food in their crop up to 92% of the abdomen volume (see Chapter 1). Replete soldiers' abdomen is 2.75 times bigger than the inner thorax, hence large petiole muscles are required to support and lift off the ground a replete abdomen.

In comparison, workers store food in their abdomen more moderately and their abdomen is only 1.52 times bigger than their inner thorax, so they do not require large petiole muscles.

The basis of strength in minute ants

The square-cube law is commonly used to explain the strength of minute ants. A smaller insect has a greater area to volume ratio, so muscles are relatively stronger. Our results show that in addition to physics, the space allocated to muscles and not to other structures is crucial. For example, many solitary wasps are minute but do not show the same physical abilities for two proximate reasons. First, ant workers lack wing muscles, freeing a considerable space inside the thorax for other muscles (Appendix 1). Second, ant workers do not need to disperse and reproduce, functions fulfilled by queens and males, whereas a solitary wasp has to be an autonomous unit. Therefore miniaturisation of ant workers is less constrained than in solitary Hymenoptera.

Cuticle thickness: miniaturisation and colonial economy

After normalisation by head width, workers had a thicker thorax cuticle than soldiers whereas no significant difference was observed for head cuticle. This observation suggests that there is a limit to the miniaturisation of the thorax cuticle in ants. The smallest insects have a poorly developed and weakly sclerotized exoskeleton with a reduction of number of segmented structures [START_REF] Polilov | Small Is Beautiful: Features of the Smallest Insects and Limits to Miniaturization[END_REF]. Here C. perpusilla workers have a sclerotized exoskeleton with no missing sclerite. The exoskeleton of ants is a scaffold where many muscles find their insertion and would likely lose some crucial biomechanical properties if simplified. In addition, the small difference in absolute cuticle thickness of the thorax between workers and soldiers minimizes the extra cost of soldiers. This result contrasts with what Peeters et al., (2017a) found in Carebara diversa where minute workers (head width 0.55-0.61 mm) with a cuticle thickness between 5.4 and 8.4 µm coexist with soldiers (head width 4.27-4.74 mm) with a cuticle thickness between 37.6 and 50.6 µm (Peeters et al., 2017a). In C. perpusilla worker/soldier dimorphism is lower, thorax cuticle thickness is similar, and more soldiers can be produced at the colony scale. However, the head cuticle was thicker than the thorax cuticle in soldiers. Even though it increases the production cost of soldiers, thicker head cuticle confers an adapted robustness to guard the nest against invaders and attach big mandible muscles.

Conclusion

Our results show intraspecific differences in space allocation inside the thorax between minute workers and larger soldiers. The lack of reduction in neural structures appears to be the principal constraint on miniaturisation in ants. Because the first thoracic ganglion remains large, the profurca must be large enough to encircle it, which influences the organisation of all head muscles. Miniaturisation is all about trade-offs: with less space available, some muscles are reduced whereas other stay large. In C. perpusilla, workers have reduced petiole muscles but substantial nervous system and leg muscles. However, these trade-offs are likely linked to the behavioural repertoire of minute ants, that can vary between species. Besides, the thorax includes many other important structures such as the oesophagus and important glands. Thorax design remains to be compared between minute ant species with different lifestyles.

Introduction

In 1721, Jonathan Swift wrote Gulliver's Travels. This science-fiction book tells the adventures of Gulliver, a man who travelled to two remarkable places: Lilliput, where humans are 12 times smaller (15cm-tall), and Brobdingnag, where they are 12 times larger (18m-tall).

Such scaling is called isometry: Lilliputians and Brobdingnagnans are simply scaled-down and scaled-up humans with identical proportions. However, Swift did not consider some physical rules that make these two kinds of humans unrealistic. As a consequence, in the real world, the brain is relatively larger in smaller animals: this is known as Haller's rule [START_REF] Rensch | Histological Changes Correlated with Evolutionary Changes of Body Size[END_REF].

Body size variation across the animal kingdom is limited by these allometric relationships. While some are shared by all animals, others are lineage specific. The evolution of extremely large or small body size within a lineage, known as gigantism and miniaturisation, involves pushing the limits of these relationships through morphological, physiological and behavioural adaptations. In particular, at the lower size level, fitting all organs, muscles and skeletal structures becomes challenging. Miniaturisation is a dominant phenomenon in insects [START_REF] Polilov | Small Is Beautiful: Features of the Smallest Insects and Limits to Miniaturization[END_REF], including ants. Interestingly, ants are the only eusocial Hymenoptera (that also includes bees and wasps) to show this trend [START_REF] Peeters | Wingless and dwarf workers underlie the ecological success of ants (Hymenoptera: Formicidae)[END_REF].

In parallel, they are the most successful in terms of species number, repartition and biomass [START_REF] Fittkau | On Biomass and Trophic Structure of the Central Amazonian Rain Forest Ecosystem[END_REF][START_REF] Schultz | In search of ant ancestors[END_REF], and their strength is legendary. The strength of ants comes from their thorax: contrary to other Hymenoptera, ant workers lost the ability to flight and their wing muscles, that account for half of the volume of the inner thorax, and this space is used for enlargement of head, leg and petiole muscles (Appendix 1).

Despite the square-cube law, which provides a strength advantage to minute insects over larger relatives, numerous muscles with different functions have to be accommodated with other vital organs inside a small exoskeletal capsule. For example, Haller's rule, which holds true for ant brains [START_REF] Seid | The Allometry of Brain Miniaturization in Ants[END_REF], likely applies to the thoracic nervous system (see Carebara (e.g. reduced spines) but is arboreal like Melissotarsus (Francisco Hita Garcia, pers. comm.). These three genera were chosen for their size and close phylogenetic relationship: they belong to the Carebara group (tribe Crematogastrini, subfamily Myrmicinae, [START_REF] Blaimer | Paleotropical Diversification Dominates the Evolution of the Hyperdiverse Ant Tribe Crematogastrini (Hymenoptera: Formicidae)[END_REF]. We expected to observe Haller's rule with the thorax nervous system and extremely reduced petiole muscles in Carebara and Melissotarsus, two ants that do not sting, compared to Tetramorium. Moreover, we hypothesized Melissotarsus had larger head muscles due to their large head and wood-tunnelling behaviour (Appendix 2). Remaining muscles should scale isometrically with body size.

Throughout this chapter, genus names will often be used to refer to Carebara perpusilla, Melissotarsus sp. and Tetramorium fhg046.

Materials and Methods

3D visualisation and linear measurements

Using Drishti [START_REF] Limaye | Drishti: a volume exploration and presentation tool[END_REF], 3D models of the entire thorax were created directly from the grayscale DICOM images. Inserting of clipping planes through the thorax at desired angles allows us to perform "virtual histology" and see all structures inside. In addition, the 'Path' function was used to take measurements on the 3D model and insert correct scale bars on figures. Thorax (mesosoma) length was measured from the anterior tip of the pronotum to the posterior end of the propodeum above the petiole.

Segmentation

Segmentation workflow was identical to Chapter 2. Head muscles, external trochanter muscles, petiole muscles, nervous system, exoskeleton, endoskeleton (furcae) and additional structures were modelled in 3D using ITK-SNAP 3.6.0 [START_REF] Yushkevich | User-guided 3D active contour segmentation of anatomical structures: Significantly improved efficiency and reliability[END_REF]. Voxel labelling (i.e. segmentation) was mostly achieved semi-automatically using the 'region competition' algorithm and complemented by manual segmentation. Some structures such as the furcae were labelled manually every 5 slices and filled with the 'Interpolate' tool. Muscles were segmented in one half of the thorax except muscle 46, segmented for both sides.

Volume calculations

Similarly to Chapter 2, we used ITK-SNAP to get the volume of the nervous system, computed from the count of labelled voxels and voxel size, but used Blender to determine the volume of muscles. Muscles shrink in ethanol and the space we observe between fibres needs to be integrated in the volume calculation. Segmented muscles were exported as mesh files Indirect head muscles were relatively much larger in Melissotarsus (14.20%) than in Carebara (5.95%) and Tetramorium (4.77%). In particular, muscle 46 expands much more posteriorly in Melissotarsus (figure 3).

External trochanter muscles

For each leg, one muscle originates from the thorax roof, crosses the coxa (first leg segment) as a tendon and inserts on the trochanter (second leg segment). These muscles, called external trochanter (ET) muscles, act as traction cables bracing the body up while walking (figure 3). For all legs, Melissotarsus had the relatively smallest ET muscles (figure 2). ET1 and ET3 were relatively larger in Carebara than Tetramorium but the opposite was observed for ET2.

Petiole muscles

The petiole is moved by four different muscle pairs that insert directly on its anterior end to move it up (120), down (118) and sideways (119 and 121). Muscles 118 and 119 find their origin on the third furca and are extremely small in all three species observed (<0.1% of the thorax volume, not shown in figure 3). In contrast, muscles 120 and 121 attach to the propodeum roof and are much larger (figure 3). Both were relatively larger in Tetramorium, then in Melissotarsus and finally in Carebara (figure 2).

Other structures

Observation of the inner thorax of all three specimens highlights particular features besides muscles and the nervous system. In particular, the dorsal space (filled by wing muscles in queens) changes considerably between species (figure 4). In Carebara and Tetramorium, much space is taken by the oesophagus, which accounts for 13.93% of the inner thorax volume in Tetramorium. In Melissotarsus, this space is filled by the labial gland. In ants, the labial gland is usually located in the first segment of the thorax (prothorax) and secretes enzymes that are transported to the infrabuccal pocket through a duct. In Carebara and Tetramorium, the labial gland is at its ordinary location, but in Melissotarsus this gland expands posteriorly and fills an important portion of the thorax (no volume data available due to the structural complexity of this gland).

the tree bark [START_REF] Billen | Glandular innovations for a tunnelling life: Silk and associated leg glands in Melissotarsus and Rhopalomastix queen and worker ants[END_REF]. Large indirect muscles are required to maneuver their large head sideways to chew tunnels efficiently.

ET muscles

External trochanter muscles were extremely reduced in Melissotarsus. ET muscles cause the depression of the trochanter, which lifts the ant body up against gravity. These muscles are enlarged in workers compared to queens as an adaptation to ground locomotion (Appendix 2). Remarkably, Melissotarsus workers display a unique locomotion and cannot walk on flat surfaces [START_REF] Delage-Darchen | Une Fourmi de Côte-d'Ivoire : Melissotarsus titubans Del[END_REF]. Their forelegs are reduced and used exclusively to spin silk while their mid-and hindlegs have hypertrophied coxae and 90 degrees-bent trochanters keeping their legs close to the body (Appendix 2). Midlegs and hindlegs are orienting upward and downward and brace the ant body against the tunnel walls in four directions. ET muscles play a very minor role in this locomotion. Instead, the coxae host large muscles to move the trochanter and provide efficient bracing in any direction, and more space of the thorax is allocated to muscles that rotate these large coxae.

Differences between Carebara and Tetramorium are more difficult to interpret. Both invest a similar space to ET1, but ET2 is relatively larger in Tetramorium and ET3 in Carebara. One hypothesis is that ET3 muscles in Tetramorium have a conflicting position with petiole muscles (Figure 3). Otherwise, there might be differences in use of midlegs and hindlegs between subterranean and arboreal ants that remain to be characterized. These observations suggest that ET muscles can scale isometrically with body size and that other factors affecting single leg pairs are more impactful.

Hyper-reduction of petiole muscles in miniaturisation

Interestingly, relative volume of petiole muscles was associated with the ant body size.

Most ants in the subfamily Myrmicinae have a sting, as is the case in Tetramorium fhg046.

However, Melissotarsus and Carebara do not exhibit stinging behaviour, and microCT observation of their abdomen shows an extremely reduced stinging apparatus (sting and associated muscles). Therefore, both Carebara and Melissotarsus only use their abdomen for tasks that do not require strength, e.g. laying a pheromone trail. This can explain why petiole muscles can be extremely reduced in these minute species to make space for other organs or muscles. How petiole muscles limit miniaturisation in species that sting or spray acid (subfamily Formicinae) remains to be investigated.

Different miniaturisation strategies

Carebara workers are at the lower size limit. Nervous and digestive systems as well as head muscles fill a large proportion of the thorax, petiole muscles are extremely reduced, and little space is left for remaining muscles and glands. They have eyes with only one ommatidium, do not run fast and have no sting (see Chapter 1). However, they are numerous, can bury their prey and store food with replete soldiers. The competitive strategy of Carebara relies on their number, and being at the smallest possible size makes workers cheaper to produce.

In contrast, Melissotarsus is slightly larger but has a unique cube-shaped thorax that maximises the thorax volume space despite its short length. This additional volumecompared to an hourglass-shaped thorax of the same length -is allocated to more fibres of muscle 46 and hypertrophied labial glands. One could argue that 2.5mm-long Melissotarsus are not minute ants. However, the fossil record shows extinct Melissotarsus species that reached 1.5mm in length (Chény, Khalife et al., in prep). Miniaturisation likely was a crucial step for these ants to colonize their new niche, i.e. reaching a suitable size to interact with scale insects and living below the bark without harming the tree. Then, ants strictly committed to life below the tree bark when a new locomotion evolved. Worker enlargement became adaptive to have a larger head with more silk glands and stronger mandible muscles, larger coxae to brace their body efficiently while tunnelling, and a larger thorax to accommodate more head muscle fibres for tunnelling and huge labial glands for digestion. Workers reached an optimal size for their environment, still small enough to minimize their deleterious effects to their host tree.

These examples show that there is not a single way to miniaturise. Habitat, biotic interactions and behavioural repertoire dictate trade-offs inside the thorax of minute ants.

Ecology and natural history are connected to morphology [START_REF] Sosiak | Multidimensional trait morphology predicts ecology across ant lineages[END_REF]. For example, we can expect all minute ants that do not sting or spray acid to have extremely reduced petiole muscles. Our results underline the importance of studying the anatomy of ant workers to understand their evolution and behaviour.

The third chapter focused on interspecific miniaturisation. We compared two minute ants closely related but with different lifestyles, Carebara perpusilla and Melissotarsus sp., and a larger relative, Tetramorium fhg046. Remarkably, we did not find isometric relationships between muscles and thorax volume when comparing all three ants. Moreover, Haller's rule was not strictly respected for the neural ganglia, because like all rules it knows exceptions. Interesting differences in head, leg and petiole muscles were connected to the lifestyle of the ants and not to body size. These findings suggest that all minute ants are scaled differently according to their lifestyle. The inner thorax morphology could be used to predict ecological traits and behaviour. Similarly, a study predicted the ecological niche of 160 species of ants with ~80% accuracy based on their external morphology through geometric morphometrics [START_REF] Sosiak | Multidimensional trait morphology predicts ecology across ant lineages[END_REF]. I believe that muscle proportions in the thorax could be a good predictor of ecology and behaviour, and combining external and internal morphological analysis of rare species (e.g. Martialis heureka, [START_REF] Rabeling | Newly discovered sister lineage sheds light on early ant evolution[END_REF] could teach us a lot about their lifestyle.

Weaknesses

One weakness of this work is, of course, the lack of biological replicates. Compared to microscopy or other imaging techniques, microCT is a new technology, and the data analysis step remains to be optimized. While huge progress has been made in terms of scanning machines and protocols [START_REF] Du Plessis | Laboratory xray micro-computed tomography: a user guideline for biological samples[END_REF], segmentation remains a time-consuming step. However, progress has already been made in the medical field to automate segmentation through machine learning [START_REF] Moeskops | Automatic Segmentation of MR Brain Images With a Convolutional Neural Network[END_REF] that should be applicable to biology soon (Evan Economo, pers. comm.). Variations between individuals and populations were not accounted for during my PhD despite the size variations we observe e.g. in Melissotarsus.

Studying multiple individuals for each species would make our comparisons more robust. To compensate, we performed dissections and histology on several individuals Carebara and Melissotarsus to validate our 3D observations. Tetramorium fhg046 is a rare ant with limited specimens, which did not allow us to perform such additional work, which is why Francisco Hita Garcia, a specialist who has scanned thousands of ants, helped me to set and check the scan of this specimen.

Another weakness of this work, also arising from a lack of replicates, is how muscles volumes are scaled. Indeed, it involves the segmentation of the entire inner thorax, that has relatively clear anterior and posterior limits but is difficult to define on the ventral side, where the coxae are inserted. Even though I segmented it myself on all specimens, small variations might exist in how this inner thorax was segmented that could slightly under-or overestimate all muscle proportion values for each specimen.

Besides, the ant neck is a complex articulation. The articulation between the head and a thorax is wrapped by a soft membrane that plays a big role in load-carrying [START_REF] Nguyen | The exoskeletal structure and tensile loading behavior of an ant neck joint[END_REF]. Furthermore, we consider that the ventral plates of the thorax (propleura) slide along the anteroposterior axis, but observations of grooming behaviour suggest that they can move dorsoventrally like a bellows. Indirect muscles and other small muscles that move the propleura might have more functions than turning the head sideways. Cutting-edge protocols, such as in vivo microtomography [START_REF] Walker | In Vivo Time-Resolved Microtomography Reveals the Mechanics of the Blowfly Flight Motor[END_REF], could allow us to characterize the degrees of freedom of the head and propleura.

Finally, some might consider this work too superficial because of the lack of biomechanical analysis. We described differences in allocation of space but never linked it directly to increased strength for muscles. These differences are teaching us what the tradeoffs are in minute ants, and which muscles or organs are scaling allometrically with thorax volume through miniaturisation. Biomechanics is one supplementary step that would connect these differences to strength and will be discussed in the next part of this discussion.

Perspectives

During my PhD, I studied how miniaturisation affected allometric relationships inside the thorax by comparing two castes or distinct species. In order to complement these studies, investigating how allometric relationships change within a species with polymorphic workers would be of great interest. In particular, Messor barbarus is a granivorous ant that has a continuous range of worker sizes with a positive allometry between head size and body size: larger workers have relatively larger heads [START_REF] Bernadou | Ergonomics of load transport in the seed harvesting ant Messor barbarus: morphology influences transportation method and efficiency[END_REF]. Furthermore, workers carry seeds that match their own size (Figure 1), which helps to explain how a wide range of body sizes can be adaptive. I would expect most muscles to scale isometrically with body size but head muscles would likely fill more space in the largest workers, that have relatively larger heads. Instead, smaller workers would have a relatively larger nervous system, following Haller's rule.

As mentioned earlier, the final step of this work would be biomechanical analysis to link differences in muscles and exoskeleton to output forces. A workflow for insects has been proposed by [START_REF] Blanke | Computational biomechanics changes our view on insect head evolution[END_REF]. First, a multibody dynamics analysis (MDA) on our segmented 3D models would give us values for muscle forces and joint reaction. These values can be used as an input for a finite element analysis (FEA), a computational technique that allows visualization of stress and strain patterns through the body during simulated behaviours. FEA also requires physical parameters of the cuticle such as Young's modulus (linked to elasticity), which are usually taken from the literature. This kind of analysis would shed light on biomechanical limits to miniaturisation and compare the strength of minute ants and larger ants.

Conclusion

To sum up, we showed that despite their small size, minute ants are not behaviourally deficient. Their thorax includes the same muscles and organs as larger ants, but with different proportions following trade-offs that reflect their lifestyle and habitat. The diversity of ants suggests an equivalent diversity of thorax designs. Ants are a fantastic example of how miniaturisation can be adaptive by allowing colonization of new ecological niches and drive the evolution of new species.

La miniaturisation chez les fourmis : adaptations squeletto-musculaires et succès évolutif

Résumé :

Les fourmis existent dans tous les écosystèmes terrestres et constituent une part importante de la biomasse animale, surtout dans les tropiques. Ce sont des insectes eusociaux, constituant des colonies pérennes avec une division du travail : les reines se reproduisent tandis que les ouvrières s'occupent du couvain et cherchent de la nourriture. Les fourmis ouvrières sont les seuls hyménoptères sociaux à avoir perdu la capacité de voler et à montrer une tendance à la miniaturisation -l'évolution d'une très petite taille au sein d'une lignée en outrepassant les limites fixées par le plan d'organisation du corps. Elles sont également renommées pour leur force, étant notamment capable de porter plusieurs fois leur poids sur de longues distances. Force et agilité au sol requièrent l'intégration de nombreux muscles au sein du thorax pour bouger la tête, les pattes, et l'abdomen via le pétiole. La miniaturisation n'a jamais été étudiée chez un insecte social, et comment le volume des muscles et autres organes évolue avec la taille du corps reste un mystère. Dans cette thèse, j'ai étudié Carebara perpusilla, l'une des plus petites fourmis du monde, par une approche de morphologie fonctionnelle. D'une part, j'ai observé la division du travail chez cette espèce qui possède deux castes non-reproductrices, ouvrières et soldats. D'autre part, j'ai utilisé la microtomographie aux rayons X (microCT) pour reconstituer des modèles 3D du thorax et comparer le volume des organes entre castes et entre espèces. Mes résultats suggèrent que la miniaturisation peut prendre différentes formes, en lien avec l'écologie et le répertoire comportemental, ouvrant la voie à des approches prédictives.

Mots-clés : évolution, morphologie fonctionnelle, allométrie, modélisation 3D

Miniaturisation in ants: from skeletomuscular adaptations for ground behaviours to evolutionary success

Abstract:

Ants are present in all terrestrial ecosystems and represent an important part of the animal biomass, particularly in the tropics. They are eusocial insects, constituting perennial colonies with division of labour: queens reproduce while workers take care of the brood and find food. Ant workers are the only social Hymenoptera to have lost the ability to fly and show a tendency to miniaturise, unlike bees and wasps. Miniaturisation is the evolution of extremely small size within a lineage, exceeding the limits set by the body organization plan. At the same time, workers are famous for their strength, being able to lift multiple times their own weight over long distances. The strength and agility of workers on the ground requires the integration of many muscles inside the thorax to articulate the head, legs, and abdomen via the petiole. Miniaturisation in social insects has never been studied, and how the volume of muscles and other organs changes with body size remains a mystery. In this thesis, I studied Carebara perpusilla, one of the smallest ants in the world, with an approach of functional morphology. On the one hand, I observed the division of labour in this species which has two nonreproductive castes, workers and soldiers. On the other hand, I used X-ray microtomography (microCT) to reconstruct 3D models of the thorax and to compare the volume of the organs intra-and interspecifically. My results suggest that miniaturization can take different forms, related to the ecology and behavioural repertoire of minute ants, paving the way for predictive approaches.

Keywords: evolution, functional morphology, allometry, 3D modelling

  Je continuerai à suivre vos activités d'un peu plus loin, et arranger des morceaux selon mon temps et mon inspiration. Spéciale dédicace à Eve, Vincent et Gaelle, notre petit quartet était très chouette et a poussé mes exigences individuelles toujours plus loin. Bon courage pour la suite ! Côté gaming, je remercie la team Win Diesel (parce qu'on gagne, mais que ça prend du temps) pour les parties endiablées de Fortnite et Paladins qui m'ont bien diverties pendant que notre ami COVID-19 sévissait dehors. Une bise spéciale à Pauline avec qui j'ai passé de nombreuses soirées à rager et massacrer des joueurs innocents. Je poursuis mes remerciements avec des pensées pour ma famille : Alice et Gérald, j'ai toujours pu compter sur votre aide. J'espère vous donner de quoi être fiers, sachez que de mon côté je suis fier de vous, et je ne connais pas beaucoup de couples qui ont le courage de commencer une nouvelle vie à votre âge. Je vous souhaite bon courage au Japon et je viendrai vous voir dès que l'occasion se présentera. Abel, si jamais tu envisages une carrière en sciences, je serais ravi que tu survoles ma thèse et que tu y apprennes des choses. En attendant, ne rate pas tes années de lycée, elles passent vite, et dépasse vite mon niveau de japonais. Une pensée pour maître Ulysse, l'autre frère, trop peu d'occasions de te revoir (surtout avec ce virus) mais "je suis fier de ton réussissement !" Des bisous à mes grands-parents dans le Sud et en Normandie, en particulier mon papy qui m'envoie chaque article sur les fourmis qu'il rencontre au cours de ses lectures scientifiques et m'a appris à regarder les plantes et les animaux dès le plus jeune âge. Il me reste aussi à remercier Louis, mon bro', que tu sois à Nancy, Cork ou Perpignan, on trouve toujours le moyen de se voir et c'est toujours avec plaisir. Depuis l'époque où on jouait avec des bâtons, le temps me semble long mais notre amitié n'a pas vraiment changé et j'espère te revoir très vite. Et Clémentine bien sûr, comment t'oublier ? Ces colos ensemble ont créé un sacré lien entre nous, et les soirées, week-ends et coups de fil pendant ma thèse étaient une bonne bouffée d'air frais. Prends soin de toi, et j'ai hâte de revenir faire griller quelques saucisses à Dreux. Pour finir, je tiens à adresser des remerciements spéciaux à

  inside the thorax?

  First, regarding the giant Brobdingnagnans, there is a principle called the square-cube law: the volume of an organism changes by a cube length whereas muscle strength and bone support are proportional to its cross-sectional area, a square length. This concept was first formulated by Galileo Galilei in 1638 and discussed by J.B.S. Haldane in 1928. The legs of a Brobdingnagnan, as an isometrically-upscaled human by a factor 12, would have legs 12² = 144 times as strong as Gulliver. But his weight, proportional to his volume, would be 12 3 = 1728 times as great as Gulliver. His giant legs would collapse under his own body weight. This explains why an elephant has proportionally larger legs than a mouse: there is a positive allometry between body size and leg width in terrestrial mammals. Despite this allometry, elephant legs still barely support the body weight, and changes in behaviour are also necessary: elephants walk slowly and do not jump. This example shows how morphology and behaviour are connected and that changes in body size involve allometric modifications. Besides, concerning the tiny Lilliputians, their brain would have too few or too small neurons and would not function well.

Table 1 : Examples of ant-Hemiptera trophobiotic interactions, from

 1

larger (top) to smaller (bottom) ants. In most cases, worker size is linked to Hemiptera size.

Table 2 : Summary of the positive and negative effects of miniaturisation on fitness.

 2

  First, I studied the division of labour in Carebara perpusilla, one of the world's smallest ants and a model species I used at all steps of my PhD. This species has two helper castes, workers (minute) and soldiers (larger), and I assessed which caste took care of which task. My hypothesis was that workers took care of the brood and foraged whereas soldiers, fewer, helped for nest defence and retrieval of heavy prey. In order to test this hypothesis, I observed a colony fragment of C. perpusilla in the laboratory, used food dyes, took pictures and performed dissections.Second, I investigated how miniaturisation affected allometric relationships inside the thorax at the species level, comparing workers and soldiers of C. perpusilla. I expected muscle proportions to be linked to the tasks I observed workers and soldiers perform in

chapter 1, but also the nervous system to be relatively larger in workers than in soldiers. I used X-ray computed microtomography (microCT) to investigate the internal anatomy of both castes, complemented by dissections. MicroCT is a cutting-edge technology that allows to build 3D models and perform virtual dissections as well as quantitative analyses. The freeware-based workflow I developed to analyse microCT scans is detailed in figure

9

.

Third, I investigated miniaturisation at the interspecific level, comparing two phylogenetically related minute workers, C. perpusilla and Melissotarsus sp. (the genus is currently revised). Both species have vastly different lifestyles: C. perpusilla has two helper castes, nests underground and feeds on small insects and carcasses, whereas Melissotarsus have a single helper caste, exclusively lives below the bark of trees and eats scale insects they breed. I predicted different allometric relationships between muscles following their behaviour (described in Chapter 1 for C. perpusilla and Appendix 2 for Melissotarsus) but a similar proportion of the thorax filled by the nervous system. Once again, I used microCT as my main tool to test these predictions, using Tetramorium fhg046 as a closely related but larger ant species as a reference point for comparison.

  Chapter 2). Furthermore, ants show an important diversity of lifestyles and behavioural repertoires. Whether two minute ants picked up in two different habitats have similar muscle and organ proportions inside the thorax is unknown. Using X-ray computed microtomography, a cutting-edge technology that allows 3D visualisation and quantification, we compared allometric relationships inside the thorax of workers of three species. First, Carebara perpusilla is among the smallest ants (body length of 1.5mm) and is subterranean (see Chapter 1). Second, Melissotarsus sp. is longer (body length of 2-2.5mm) but has a short thorax, and lives below the bark of trees (see Appendix 2). Third, Tetramorium fhg046 is a medium-sized ant (body length of 3.5mm) that shares morphological similarities with

  Tetramorium fhg046 (T. dumezi group) was collected in August 2007 in Kenya (Kakamega Forest, 1650m elevation). Specimens were stored in 90% ethanol. Micro-CT scans were performed using a Zeiss Xradia 510 Versa 3D X-ray microscope piloted by the Zeiss Scout-and-Scan Control System software(version 11.1.6411.17883) at the Okinawa Institute of Science and

	Technology Graduate University (OIST, Japan). Specimens were stained in a 2 M iodine
	solution for 24 hours before being moved to microtubes filled with 99% ethanol for scanning.
	Scan settings (provided in Table 1) were selected to yield maximal scan quality. Full 360-
	degree rotations were based on 1601 projections. Post-imaging stack reconstruction was done
	using the Zeiss Scout-and-Scan Reconstructor software (version 11.1.6411.17883) and the
	output saved as DICOM file folders.

Ant specimens and scanning protocol C. perpusilla and Melissotarsus sp. were collected during Ant Course in August 2016 in Mozambique (Gorongosa National Park, Sofala Province, 100m elevation).

  Similar observations were made on Carebara soldiers where a similar proportion of the thorax was dedicated to muscle 46 (14.11% in the soldier of C. perpusilla from Chapter 2, 13.52% in Melissotarsus). Melissotarsus and Carebara. Muscle 41 is a small muscle, accounting for less than 1% of the thorax volume in Euponera sikorae (Ponerinae) and Cataglyphis savignyi (Formicinae) queens and workers (Appendix 1). It is apparently reduced in large Myrmicinae (Messor barbarus, personal observation). Its complete loss seems associated with miniaturisation even though it is reduced yet present in some minute ants (Pheidole oculata and Carebara castanea, personal observation). Complete loss of muscle 41 is not a mandatory step during miniaturisation. Interestingly, this muscle has a dorsal and central position in the prothorax, and its absence leaves extra space for additional fibres of muscle 46 in Melissotarsus or the oesophagus in Carebara.

	Interestingly, minute ants had relatively larger levator muscles despite the absence of
	muscle 41. Redundancy between muscles 41, 42 and 43 allowed the loss of muscle 41 in both

Acknowledgements

Acknowledgements .................................................................................................................... 2 I Introduction .............................................................................................................................. 9

Acknowledgements

We are grateful to Peter Hawkes for providing the colony and identifying the Carebara species, and to Romain Péronnet for providing springtails. Many thanks to Evan Economo for hosting AK during a six-months funded research internship at the Okinawa Institute of Science and Technology (OIST), as well as access to the micro-CT scanner. AK thanks Luc Passera for advice on food dyes.

Acknowledgements

AK thanks Johan Billen for providing histological sections of Carebara perpusilla workers and soldiers.

Acknowledgements

We thank Francisco Hita Garcia for his help to choose a medium-sized non-spiny arboreal Tetramorium species, for providing a specimen and assisting AK for scanning. We acknowledge Johan Billen for providing histological sections of Carebara and Melissotarsus and knowledge on labial glands. We thank Christian Peeters for significant early contribution in designing this study but could not see the results and write the article. We honour his memory with this article.

Supplementary Data

Caste

 S1: MicroCT scan parameters for each C. perpusilla specimen. Some parameters were identical for all scans: power = 5W; voltage = 60kV; amperage = 83µA.

Supplementary data

Caste

General Discussion

Summary

During my PhD, I studied the consequences of miniaturisation on the morphology and the behaviour of ant workers.

The first chapter focused on division of labour between minute workers and larger soldiers in Carebara perpusilla, one of the smallest ants in the world and our model species for subsequent morphological studies. We found that workers were in charge of brood care and foraging: they hunted microinsects -even though they cannot sting or spray acid -and scavenged on large insect carcasses that they buried with soil pellets. They carried food pieces back to their nest between their mandibles. They were also able to chew galleries through plaster, drag soil pellets and carcasses hundreds of times their own mass, and lay/follow odour trails. Soldiers sometimes assisted workers to cut through the sclerites of carcasses, but otherwise stayed inside the nest and stored food inside their large abdomen, acting as repletes.

Carebara workers were able to perform a wide variety of tasks, which supports [START_REF] Seid | The Allometry of Brain Miniaturization in Ants[END_REF] in contradiction to the behavioural simplification hypothesis. This hypothesis, stated by [START_REF] Cole | Size and behavior in ants: Constraints on complexity[END_REF], states that miniaturisation should be associated with a simplification of the behavioural repertoire.

The second chapter focused on intraspecific miniaturisation. We investigated the allometric relationships inside the thorax of Carebara workers and soldiers and matched these differences to miniaturisation and division of labour. We discovered that the nervous system filled a larger portion of the thorax in workers than soldiers, providing an empirical evidence of Haller's rule for the thorax. In addition, workers had relatively larger direct head muscles and external trochanter (ET) muscles. These muscles are useful for load-carrying: direct muscles move the head up and down, and ET muscles support the body while walking. In contrast, soldiers invested more space in indirect head muscles and petiole muscles. Indirect head muscles move the head sideways, which provides greater manoeuvrability to the long head of soldiers. Large petiole muscles are useful to support and move their heavy abdomens, used as food reservoirs by the colony.

Appendix 1

Peeters, C., Keller, R. A., Khalife, A., Fischer, G., Katzke, J., Blanke, A., & Economo, E. P. (2020). The loss of flight in ant workers enabled an evolutionary redesign of the thorax for ground labour. Frontiers in Zoology, 17(1), 1-13.

Free access from the editor's website: https://doi.org/10.1186/s12983-020-00375-9

Appendix 2

Khalife, A., Keller, R. A., Billen, J., Garcia, F. H., Economo, E. P., & Peeters, C. (2018). Skeletomuscular adaptations of head and legs of Melissotarsus ants for tunnelling through living wood. Frontiers in Zoology, 15(1), 1-11.

Free access from the editor's website: https://doi.org/10.1186/s12983-018-0277-6