Sébastien Dailly

Julien Formento

Lorenzo Ottaviani

Hugo Lezarme

Kevin Fayet

Perrine Lagarde

Merci

Adeline Bulot

Alexandre Génin

Bastien Roxanne Durand

sourd toujours dans la bonne humeur

Keywords: AFLP, jardin commun, pelouse sèche, exclos, adaptation locale, pelouse méditerranéenne, marqueurs neutres, origine des plantes, poaceae, différenciation entre populations, brûlage dirigé, θST, PST, traits phénotypiques, recrutement des plantules, Thero-Brachypodietea AFLP, common garden, dry grassland, exclosures, local adaptation, Mediterranean grassland, neutral markers, plant origin, poaceae, population differentiation, prescribed burning, θST, PST, phenotypic traits, seedling recruitment, Thero-Brachypodietea

Le brachypode rameux (Brachypodium retusum) est une espèce herbacée pérenne qui domine les pelouses sèches de Méditerranée occidentale. Dans notre zone d'étude du Sud de la France, la recolonisation spontanée est très faible après perturbation du sol. Cette observation ne correspond pas aux résultats d'études menées dans d'autres régions montrant une capacité de colonisation élevée. L'objectif principal de cette thèse était donc de tester les différentes hypothèses pouvant expliquer les différents patrons de colonisation.

Dans un premier chapitre, nous avons testé si ces différents patrons de colonisation résultent d'une différenciation génétique entre les populations. Des marqueurs AFLP ont été utilisés pour analyser la structure génétique de 17 populations de Méditerranée occidentale dont la différenciation neutre entre populations. Dans un sous-échantillon de 13 populations françaises, la différenciation de traits phénotypiques sous sélection a été testée dans un jardin commun et comparée à la différenciation neutre. Dans le deuxième chapitre, nous présentons une étude sur la différenciation adaptative des traits phénotypiques et avons testé une potentielle réponse différentielle à la manipulation de facteurs environnementaux clés. Les deux derniers chapitres de la thèse ont analysé les facteurs environnementaux qui limitent la recolonisation in situ, indépendamment du génotype ou de la population. Dans le troisième chapitre, nous avons testé l'effet du pâturage et du feu sur le recouvrement végétatif ainsi que sur la reproduction sexuée de B. retusum installé. Enfin, dans le quatrième chapitre, nous avons mesuré l'effet de l'arrosage initial et du pâturage sur l'installation de plantules transplantées initialement germées en serre et de plantules issues de semis in situ. Nos résultats ont montré que les populations de B. retusum sont génétiquement différenciées pour les marqueurs neutres mais également pour les traits phénotypiques. Cette différenciation est supérieure à la dérive seule et suggère une adaptation aux conditions environnementales, en particulier aux températures estivales et à la fréquence du gel en hiver. Une réponse différentielle à la manipulation expérimentale de facteurs environnementaux (sol, pâturage, humidité du sol) a confirmé le caractère adaptatif de la différenciation génétique. Les expériences in situ ont montré que le feu a un effet positif sur la reproduction de B. retusum et sur la communauté végétale associée, alors que son recouvrement végétatif n'était pas supérieur à la moyenne de la communauté. L'exclusion du pâturage pendant deux saisons n'a eu d'incidence sur aucun des paramètres mesurés chez les populations adultes. L'arrosage initial a affecté l'installation des plantules au cours de la première saison. Au cours de la deuxième saison, l'effet seul de l'arrosage n'était pas significatif, cependant son effet est resté positif sur la survie uniquement dans les parcelles pâturées. Le pâturage, au début du cycle de vie de B. retusum, a eu un effet négatif sur le recrutement et la croissance des plantules. En conclusion, la différenciation adaptative entre les populations peut avoir contribué aux différences régionales en termes de capacité de colonisation et doit être prise en compte lors du ciblage des populations sources pour l'introduction de plantes en restauration écologique. La réponse positive de B. retusum a indiqué que le feu était une force sélective importante dans le passé qui pourrait être utilisée pour favoriser l'espèce et la communauté végétale associée dans des opérations de conservation et de restauration. L'exclusion du pâturage à court terme est tolérée par les populations adultes de B. retusum, mais l'abandon à long terme entraîne une diminution de son recouvrement par rapport aux graminées pérennes à croissance rapide. Enfin, lors des premiers stades de l'installation des plantules, il est nécessaire d'éviter le pâturage pour garantir le succès de l'introduction, sinon le stress dû au pâturage doit être compensé par un arrosage.

Remerciements-Acknowledgments

Remerciements

Après trois années de travail sur cette thèse, enfin nous y voilà, la touche finale de ce manuscrit afin de remercier toutes les personnes qui de près ou de loin ont contribué à mon parcours de doctorante.

Je voudrais remercier dans un premier temps mes directeurs de thèse Armin Bischoff et Thierry Dutoit pour m'avoir permis de réaliser cette thèse, pour leur soutien, pour avoir résisté à mon obstination et mes centaines de questions ainsi que pour le temps qu'ils m'ont accordé tout au long de ces années.

Merci à Armin pour m'avoir poussée à développer mon esprit critique et scientifique autant sur l'utilisation des concepts théoriques que sur la mise en place pratique des expérimentations.

Merci à Thierry pour m'avoir fait partager sa longue expérience sur la Crau et ses connaissances sur les communautés végétales et la botanique.

Au-delà de tous les aléas qui peuvent se présenter à un doctorant (même une rave party improbable), je suis très heureuse d'avoir eu l'occasion de réaliser cette thèse avec vous, j'ai vraiment beaucoup appris.

I would like to thanks Johannes Kollmann, Rob Marrs, Pierre-Olivier Cheptou and Nathalie Machon who have accepted to read and judge this work.

I am grateful to my thesis committee members for their constructive comments, our discussions beyond the two committees and their involvement in my thesis: Emmanuel Corcket, Alex Baumel and Hans Martin Hanslin.

Je voudrais remercier tous ceux qui ont contribué à une bonne entente et ambiance au sein du labo (l'ordre d'apparition n'est pas une marque de préférence). J'espère n'oublier personne … .

Je souhaite remercier tous ceux qui font me sentir toujours « chez moi » dans mon bureau. Theoretical background

Global biodiversity is often described as species richness [START_REF] May | How many species inhabit the earth?[END_REF]). However, more recent approaches to quantify biodiversity loss and protection use a broader definition including all complexity levels from genes to ecosystems [START_REF] Sutherland | Identification of 100 fundamental ecological questions[END_REF]. Biodiversity related to genes includes intra-species variation in genetic information such as intra-and inter-population differentiation and as species diversity, it is influenced by evolutionary forces. The genetic structure of populations is the result of adaptive (selection) and neutral processes (genetic drift, mutations) but their relative contribution is largely unknown [START_REF] Brousseau | Neutral and Adaptive Drivers of Microgeographic Genetic Divergence within Continuous Populations: The Case of the Neotropical Tree Eperua falcata (Aubl.)[END_REF]) (Figure I).

Selection, genetic drift and gene flow shape diversity, structure and genetic variation of plant populations [START_REF] Loveless | Ecological Determinants of Genetic Structure in Plant Populations[END_REF][START_REF] Lenormand | Gene flow and the limits to natural selection[END_REF][START_REF] Leimu | A Meta-Analysis of Local Adaptation in Plants[END_REF].

Natural selection may result in adaptive population differentiation due to genetically based phenotypic differences that improve local fitness in different environments. Adaptive differentiation occurs when differences among individual heritable traits cause differences in survival and reproduction. Adaptation is an evolutionary process through which traits are modified by natural selection. However, selection is not the only force resulting in population differentiation and may be counterbalanced by other evolutionary forces such as drift, gene flow and mutation (see Appendix I for definitions). Main ecological factors driving biodiversity are abiotic (resource availability, water availability, climate) or biotic (intra species or inter species competition). Plant distribution and biomass are largely determined by climate and locally modified by soil conditions [START_REF] Polis | Why Are Parts of the World Green? Multiple Factors Control Productivity and the Distribution of Biomass[END_REF].

Local adaptation is often the result of natural selection by abiotic factors such as climate and soil [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Hancock | Adaptation to Climate Across the Arabidopsis thaliana Genome[END_REF][START_REF] Manel | Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation[END_REF]). In addition to these major selective factors, grazing, water availability and fire shape populations and communities [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF][START_REF] O'connor | Local Extinction in Perennial Grasslands: A Life-History Approach[END_REF][START_REF] Blondel | The 'Design' of Mediterranean Landscapes: A Millennial Story of Humans and Ecological Systems during the Historic Period[END_REF]) (Figure II). (1975).

Adaptive differentiation is widespread in grassland species, and plants often show adaptation at regional scale [START_REF] Becker | The spatial scale of adaptive population differentiation in a wide-spread, well-dispersed plant species[END_REF][START_REF] Bucharova | Genetic differentiation and regional adaptation among seed origins used for grassland restoration: lessons from a multispecies transplant experiment[END_REF]. In grassland populations, climate is the major large-scale environmental factor driving genetic differentiation, in particular temperature, precipitation and their seasonal distribution [START_REF] Manel | Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation[END_REF][START_REF] Bucharova | Genetic differentiation and regional adaptation among seed origins used for grassland restoration: lessons from a multispecies transplant experiment[END_REF]. Soil conditions often vary at smaller scales resulting in small-scale adaptive differentiation (Van der [START_REF] Van Der Putten | The sustainable delivery of goods and services provided by soil biota[END_REF][START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF]. Soil chemistry, but also soil structure and organisms are potential selective forces [START_REF] Wardle | Communities and ecosystems: linking the aboveground and belowground components[END_REF]. Several studies showed grass species adaptation to soil type including serpentine soils (e.g [START_REF] Freitas | Effects of water stress and soil texture on the performance of two Bromus hordeaceus ecotypes from sandstone and serpentine soils[END_REF] for

Bromus hordeaceus and [START_REF] Meimberg | Patterns of introduction and adaptation during the invasion of Aegilops triuncialis (Poaceae) into Californian serpentine soils[END_REF] for Aegilops triuncialis) or fertility gradients [START_REF] Sherrard | Local adaptation across a fertility gradient is influenced by soil biota in the invasive grass, Bromus inermis[END_REF]. PH is, for example, one key soil chemistry factor to which plant populations may be adapted [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Raabová | The role of spatial scale and soil for local adaptation in Inula hirta[END_REF]. Soil characteristics and climate also influence land management which is a third important driver of adaptive differentiation in grassland systems. In particular, grazing intensity depending on soil quality is known to affect plant performance and may select for genotypes that tolerate aboveground plant damage [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF][START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF].

Apart from genotype-environment interactions, in particular affecting the outcome of plant introduction measures, environmental factors may limit colonisation and seedling recruitment independent of plant genotype or population. Mediterranean dry grasslands are semi-natural habitats that depend on human land management. Sheep grazing is one of the key management factors driving plant species composition and diversity (reviewed by [START_REF] Milchunas | Quantitative Effects of Grazing on Vegetation and Soils Over a Global Range of Environments[END_REF]. Several studies have tried to identify plant traits linked to grazing but few general patterns have been found. In arid and semi-arid environments such as Mediterranean dry grasslands, water availability is known to be a strongly limiting factor for plant species establishment [START_REF] Noy-Meir | Desert Ecosystems: Environment and Producers[END_REF][START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF]). However, an increase in soil humidity may also increase competition to ruderal plant species better adapted to more humid mesophilic conditions. Wildfires and fire management by shepherds were important drivers in the past but their role in maintaining the ecosystem is nowadays less clear because fire prevention has largely reduced the role of fire in the last decades [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF][START_REF] Parr | Patch Mosaic Burning for Biodiversity Conservation: a Critique of the Pyrodiversity Paradigm[END_REF]. All these factors are acting on critical stages of the perennial grass life cycle (Figure II, O'Connor 1991). Competition and drought affect populations mainly during early stages whereas grazing effects are equally strong during the whole life cycle. Additionally, complex interactions between these major factors complicate predictions on plant responses such as plant establishment and colonisation processes.

involving a diversification of habitats has rather increased biodiversity. The Mediterranean region is today considered as a global biodiversity hotspot including a high number of endemic species [START_REF] Medail | Hot-Spots Analysis for Conservation of Plant Biodiversity in the Mediterranean Basin[END_REF][START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF].

Dry grasslands of temperate Europe are well studied since many decades [START_REF] Tansley | British Ecology During the Past Quarter-Century: The Plant Community and the Ecosystem[END_REF][START_REF] Smith | Ecology of the English chalk[END_REF][START_REF] Hillier | Calcareous Grasslands-Ecology and Management[END_REF][START_REF] Poschlod | The historical and socioeconomic perspective of calcareous grasslands-lessons from the distant and recent past[END_REF]. Mediterranean dry grasslands are less well studied but the number of publications is currently increasing [START_REF] Puerto | Variation in Structure and Diversity in Mediterranean Grasslands Related to Trophic Status and Grazing Intensity[END_REF][START_REF] Lahav | Soil bacterial functional diversity in a potato field[END_REF][START_REF] Peco | The effect of grazing abandonment on species composition and functional traits: the case of dehesa grasslands[END_REF]Buisson et al. 2006;Buisson & Dutoit 2006;[START_REF] Fadda | Consequences of the cessation of 3000 years of grazing on dry Mediterranean grassland ground-active beetle assemblages[END_REF][START_REF] Henry | The age and history of the French Mediterranean steppe revisited by soil wood charcoal analysis[END_REF][START_REF] Henry | The age and history of the French Mediterranean steppe revisited by soil wood charcoal analysis[END_REF][START_REF] Peco | Effects of grazing abandonment on functional and taxonomic diversity of Mediterranean grasslands[END_REF][START_REF] Robin | A comparative review of soil charcoal data: Spatiotemporal patterns of origin and long-term dynamics of Western European nutrient-poor grasslands[END_REF]. Due to the recent decline of these grasslands, an increasing number of studies focuses on ecological restoration (Buisson et al. 2006;Buisson & Dutoit 2006;[START_REF] Fadda | Consequences of the cessation of 3000 years of grazing on dry Mediterranean grassland ground-active beetle assemblages[END_REF][START_REF] Henry | The age and history of the French Mediterranean steppe revisited by soil wood charcoal analysis[END_REF][START_REF] Robin | A comparative review of soil charcoal data: Spatiotemporal patterns of origin and long-term dynamics of Western European nutrient-poor grasslands[END_REF]. The ecological restoration of Mediterranean dry grasslands ("steppes") of the Thero-Brachypodietea type (San [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF] based on spontaneous succession is a very slow process [START_REF] Römermann | Influence of former cultivation on the unique Mediterranean steppe of France and consequences for conservation management[END_REF]. Even after several decades of restoration, the species composition is often different from that of the reference community. In particular, key species hardly establish [START_REF] Blondel | Biology and Wildlife of the Mediterranean Region[END_REF].

Brachypodium retusum is a dominant widespread core species of dry Mediterranean grasslands managed by grazing. The species is called key species in this thesis due to its dominance and its number of interactions with other species [START_REF] Saiz | Structure and spatial self-organization of semi-arid communities through plant-plant co-occurrence networks[END_REF]. Approaches to restore such grasslands and to re-establish B. retusum have failed in the greatest French Mediterranean steppe area of the Crau plain. Several previous studies have showed that the restoration of initial environmental conditions is not sufficient to restore the plant community with its dominant species. Even the transfer of B. retusum rich hay has not been successful in re-establishing populations suggesting a low seedling recruitment (Coiffait-Gombault et al. 2012a, b;Dutoit et al., 2013). The absence of the dominant species has a strong effect on the community structure and functioning [START_REF] Raventós | A resprouter herb reduces negative density-dependent effects among neighboring seeders after fire[END_REF].

Surprisingly, studies in other Mediterranean systems have not confirmed the slow recolonisation found in the Crau plain. The species rapidly re-occur after wildfires and is generally frequent in habitats of moderate disturbance [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF][START_REF] Cassagne | Using a fire propagation model to assess the efficiency of prescribed burning in reducing the fire hazard[END_REF]). This may be explained by the strong resprouting capacity from belowground organs representing a high resilience to disturbances and a competitive advantage over annual species. However, a high re-colonisation was even found after soil disturbance by ploughing [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF].

III. Main thesis topics

The PhD project aims to identify the causes for the low establishment of Brachypodium retusum in Mediterranean steppes and in particular in the Crau area. Two main hypotheses are tested (i)

The genetic differentiation explains differences in colonisation capacity and (ii) colonisation depends on environmental conditions such as humidity, grazing or fire that may limit or favour seedling recruitment. We expect that this project will allow us to better understand the ecology of this key species in order to improve the efficiency of restoration measures.

Basic ecology provides useful fundamental knowledge for restoration ecology which consequently provides guidance for ecological restoration measures (Palmer et al. 2006).

Conversely, ecological restoration provides opportunities to set up ecological experiments, which contribute to fundamental knowledge in basic ecology [START_REF] Bradshaw | Restoration: an acid test for ecology[END_REF]) (Figure III).

Human impact affected and still affects several inter-dependent ecological scales varying through space and time [START_REF] Lewis | Defining the Anthropocene[END_REF]. Understanding the colonisation capacity of a species and the restoration of an ecosystem requires a better understanding of limiting factors at several ecological scales.

This thesis uses the concepts of four main thematic fields (Appendix I), population genetics, population ecology, community ecology and restoration ecology to identify potential filters and limits of B. retusum establishment and their consequences for plant introduction. This transdisciplinary approach allows evaluating the limiting factors at several scales and improving restoration success. a.

Genetic differentiation and adaptation

Genetic and phenotypic variation as a function of environmental variation and adaptation are common at geographically large scales often representing strong environmental gradients [START_REF] Etterson | Evolutionary potential of chamaecrista fasciculata in relation to climate change. i. clinal patterns of selection along an environmental gradient in the great plains[END_REF][START_REF] Brouillette | Adaptive differentiation of traits related to resource use in a desert annual along a resource gradient[END_REF][START_REF] Stojanova | Adaptive differentiation of Festuca rubra along a climate gradient revealed by molecular markers and quantitative traits[END_REF]. Spatial variation in abiotic and biotic factors along environmental gradients select for phenotypic traits and thus contribute to genetic structuring and intraspecific differentiation among populations [START_REF] Manel | Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation[END_REF][START_REF] Hsiung | Local Climate Heterogeneity Shapes Population Genetic Structure of Two Undifferentiated Insular Scutellaria Species[END_REF]. Such genetically based phenotypic differences improve the fitness of local populations in different environments resulting in local adaptation. However, genetic structure is also the result of neutral processes (genetic drift, mutations) and the relative contribution of adaptive and neutral processes is still not well understood [START_REF] Brousseau | Neutral and Adaptive Drivers of Microgeographic Genetic Divergence within Continuous Populations: The Case of the Neotropical Tree Eperua falcata (Aubl.)[END_REF].

Adaptive differentiation is widespread in grassland species and plants often show adaptation at regional scale [START_REF] Becker | The spatial scale of adaptive population differentiation in a wide-spread, well-dispersed plant species[END_REF][START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF]. Climatic and edaphic conditions [START_REF] Nevo | Natural selection of allozyme polymorphisms: a microgeographic climatic differentiation in wild emmer wheat (Triticum dicoccoides)[END_REF]) are the main drivers of adaptive population differentiation that may also occur at small spatial scales, as reported for many grass species [START_REF] Hamrick | Microgeographical Variation in Allozyme Frequencies in Avena barbata[END_REF][START_REF] Nevo | Microgeographic edaphic differentiation in allozyme polymorphisms of wild barley (Hordeum spontaneum, Poaceae)[END_REF][START_REF] Gutterman | Temperatures and Ecological -Genetic Differentiation Affecting the Germination of Hordeum Spontaneum Caryopses Harvested from Three Populations: The Negev Desert and Opposing Slopes on Mediterranean Mount Carmel[END_REF][START_REF] Hsiao | Population genetic structure of Yushania niitakayamensis (Bambusoideae, Poaceae) in Taiwan[END_REF][START_REF] Owuor | Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum[END_REF]. Phenotypic traits are typically under directional natural selection related to climate, and the direction and magnitude of this selection varies among local populations [START_REF] Merilä | Comparison of genetic differentiation at marker loci and quantitative traits[END_REF]. Strong directional selection is common in the wild [START_REF] Endler | Natural selection in the wild[END_REF][START_REF] Kingsolver | The strength of phenotypic selection in natural populations[END_REF]) and most traits are moderately to highly heritable [START_REF] Mousseau | Natural selection and the heritability of fitness components[END_REF][START_REF] El-Lithy | Quantitative Trait Locus Analysis of Growth-Related Traits in a New Arabidopsis Recombinant Inbred Population[END_REF].

The adaptation to soil conditions was first detected on soils contaminated with heavy metals indicating that adaptive differentiation may occur quite rapidly [START_REF] Schat | Identical Major Gene Loci for Heavy Metal Tolerances that Have Independently Evolved in Different Local Populations and Subspecies of Silene vulgaris[END_REF][START_REF] Antonovics | Evolution in closely adjacent plant populations X: long-term persistence of prereproductive isolation at a mine boundary[END_REF]. Furthermore, the pH value and related chemical and biotic properties may represent a strong selective force [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Raabová | The role of spatial scale and soil for local adaptation in Inula hirta[END_REF]. Soil humidity is a climate driven soil parameter and a strong limiting factor for plant fitness in arid and semi-arid ecosystem resulting in drought adaptation [START_REF] Shah | Interaction of drought and high temperature on photosynthesis and grain-filling of wheat[END_REF][START_REF] Suriyagoda | Multiple adaptive responses of Australian native perennial legumes with pasture potential to grow in phosphorusand moisture-limited environments[END_REF]). However, an increase in soil moisture often results in higher competition for other resources representing a counteracting selective force [START_REF] Kadmon | Plant Competition along Soil Moisture Gradients: A Field Experiment with the Desert Annual Stipa Capensis[END_REF].

Ecosystem management is a third potential driver of adaptive differentiation in semi-natural systems such as grasslands. Grazing changes site conditions by biomass removal, plant damage, reduced competition and creates heterogeneity in grassland vegetation [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF][START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF]. It seems to be likely that long-term grazing favours genotypes adapted to grazing [START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF]. However, grazing may also obscure adaptation to other environmental factors if this selection pressure has not been consistent in the past [START_REF] Hufford | Local Adaptation and Effects of Grazing among Seedlings of Two Native California Bunchgrass Species: Implications for Restoration[END_REF]. Isolation-by-environment [START_REF] Orsini | Drivers of population genetic differentiation in the wild: isolation by dispersal limitation, isolation by adaptation and isolation by colonization[END_REF]) is an adaptive evolutionary process increasing population differentiation. Thus, reduced establishment success of introduced or immigrating populations may occur due to maladaptation and should be carefully taken into account in restoration ecology.

Gene flow between different environments may, however, homogenise the gene pool and thus counterbalance adaptive differentiation (reviewed in [START_REF] Räsänen | Disentangling interactions between adaptive divergence and gene flow when ecology drives diversification[END_REF]. Gene flow may be reduced due to isolation-by-environment but also due to isolation-by-distance [START_REF] Wright | Evolution in Mendelian Populations[END_REF]. Isolation-by-distance is a neutral evolutionary process depending on geographic distances and physical barriers (i.e. isolation by resistance; [START_REF] Orsini | Drivers of population genetic differentiation in the wild: isolation by dispersal limitation, isolation by adaptation and isolation by colonization[END_REF]) between populations.

b. Environmental drivers of early establishment, vegetative recovery and reproduction following disturbance

Seedling recruitment, establishment, adult resistance to disturbance and seed production are crucial for spontaneous re-colonisation and/or plant introduction. Previous work has shown that seedling recruitment of B. retusum is very low on restoration sites of the Crau plain (Buisson et al. 2006;Coiffait-Gombault et al. 2012a). Drought stress and competition by the established plant community have been identified as key factors limiting seedling recruitment [START_REF] Pywell | Enhancing diversity of species-poor grasslands: an experimental assessment of multiple constraints[END_REF][START_REF] Nuttle | Evaluation of Restoration Practice Based on Environmental Filters[END_REF]. It is therefore necessary to study the response of B. retusum to these factors and their variation to better understand population dynamics and colonisation processes.

Such knowledge is indispensable to improve the success of re-introduction in ecological restoration or re-vegetation approaches. Studies in Spain and France have demonstrated that B.

retusum benefits from wildfires [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF][START_REF] Cassagne | Using a fire propagation model to assess the efficiency of prescribed burning in reducing the fire hazard[END_REF].

The dense belowground rhizome system allows a rapid post-fire recovery representing a competitive advantage compared to other species of the plant community. Additionally, controlled burning was used in the past to improve forage quality of Mediterranean grasslands [START_REF] Stouff | La Crau dans la vie et l'économie d'Arles au bas Moyen Age[END_REF][START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF]. Thus, the absence of fire may explain the poor establishment of the species currently observed in the Crau area following arable use. Post-fire resprouting from belowground organs would not explain a poor seedling recruitment [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]). However, favouring adult, already established plants increases seed production and potentially seed rain arriving at restoration sites.

Grazing was found to positively affect the growth of B. retusum (Coiffait-Gombault et al. 2012a, b). Similarly to fire, grazing indirectly favours the species since the resprouting capacity represent a competitive advantage over other species of the plant community such as annuals.

Humidity is another key factor in seedling recruitment. Soil moisture limits germination of most plant species. The high germination of B. retusum under optimum humidity in the laboratory (Coiffait-Gombault et al. 2012b) suggests that soil moisture may increase seedling recruitment in Mediterranean grasslands. However, an increase in soil humidity may also increase competition to ruderal plant species better adapted to more humid mesophilic conditions.

Wetter parts of the Crau plain are occupied by such mesophilic species and B. retusum is absent there (Molinier & Talon 1950;[START_REF] Masson | Impacts of water stress removal and disturbance regimes on Mediterranean dry grasslands diversity and succession[END_REF]. Nevertheless, higher water availability may still promote the early stages of seedling establishment (germination, seedling survival and growth).

c. Plant introduction and seed provenance question

Adaptive and neutral population differentiation are important issues in ecological restoration involving plant introduction. Whereas almost all plants show phenotypic and genetic differentiation among populations and regions [START_REF] Jay | Forecasting changes in population genetic structure of alpine plants in response to global warming[END_REF]) it is less clear whether and how local adaptation contributes to this differentiation [START_REF] Leimu | A Meta-Analysis of Local Adaptation in Plants[END_REF]. The current debate on where to select seed material for ecological restoration reflects this ambiguity. Many authors advocate the use of local or regional seed sources which are increasingly adopted by practitioners [START_REF] Mckay | How local is local?"-a review of practical and conceptual issues in the genetics of restoration[END_REF][START_REF] Vander Mijnsbrugge | A question of origin: Where and how to collect seed for ecological restoration[END_REF][START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF]. This approach, however, has been questioned because local adaption may not be common enough [START_REF] Bischoff | The limits of local adaptation in plants and consequences for ecological restoration[END_REF]) and local seed sources may frequently be inbred and genetically impoverished [START_REF] Broadhurst | Seed supply for broadscale restoration: maximizing evolutionary potential[END_REF]). Hence, knowledge on genetic diversity and differentiation patterns and on the relation to phenotypic variation and local adaptation is increasingly required in order to improve strategies for revegetation. In spite of the known value for monitoring, evaluation and decision-making, genetic aspects are a frequently overlooked issue in restoration approaches [START_REF] Mijangos | Contribution of genetics to ecological restoration[END_REF].

Observed phenotypic trait variability in the field can be the result of environmental variation, phenotypic plasticity and genetic variation [START_REF] Falconer | Effect of defoliation on grass growth. A quantitative review[END_REF]. Disentangling these ultimate reasons behind trait differentiation among populations and thus gaining insights into mechanisms behind trait differentiation requires integrated approaches that are able to partition the observed trait variability into its respective components. The combination of quantitative and molecular population genetic methods allows such a partitioning even in natural plant communities [START_REF] Stinchcombe | Cross-pollination of plants and animals: wild quantitative genetics and plant evolutionary genetics[END_REF]. It further allows quantifying the contribution of adaptive differentiation and random demographic processes to observed genetic differentiation patterns [START_REF] Leinonen | QST-FST comparisons: evolutionary and ecological insights from genomic heterogeneity[END_REF]). However, adaptive responses of plant populations to local environmental conditions require the existence of genetically based phenotypic variability.

The distribution of such genetic variability within and among populations depends on factors that are also major drivers of micro-evolutionary patterns. A plant species balance between clonal and sexual reproduction is such a factor potentially influencing both the withinpopulation genetic structure and gene dispersal [START_REF] Silvertown | The Evolutionary Maintenance of Sexual Reproduction: Evidence from the Ecological Distribution of Asexual Reproduction in Clonal Plants[END_REF]. Clonality is advantageous in habitats facing strong aboveground but low belowground disturbance such as grasslands.

It may facilitate resource uptake in heterogeneous environments and is increasing with population age and successional stage [START_REF] Silvertown | The Evolutionary Maintenance of Sexual Reproduction: Evidence from the Ecological Distribution of Asexual Reproduction in Clonal Plants[END_REF]. It may, however, be associated with severe fitness costs such as increased selfing, inbreeding depression or reduced mate availability that in turn may reduce within-population genetic diversity [START_REF] Honnay | A meta-analysis of the relation between mating system, growth form and genotypic diversity in clonal plant species[END_REF].

IV. The study species: Brachypodium retusum Brachypodium genus belong to the Poaceae family and comprises about 20 worldwide distributed taxa including 18 species [START_REF] Catalán | Phylogenetic reconstruction of the genus Brachypodium P. Beauv. (Poaceae) from combined sequences of chloroplastndhF gene and nuclear ITS[END_REF][START_REF] Catalán | Evolution and taxonomic split of the model grass Brachypodium distachyon[END_REF]. Three of them are annual and 15 are perennial species [START_REF] Catalán | Evolution and taxonomic split of the model grass Brachypodium distachyon[END_REF]. Brachypodium shows an intermediate evolutionary placement within the grass temperate pooid clade, being closer to the basal than to the recent Pooideae lineages [START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF].

Ramose false brome (Brachypodium retusum P. Beauv.) is a perennial and allopolyploid (presumed hexaploid) species with a chromosomal base number of 2n=38 [START_REF] Wolny | Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives[END_REF][START_REF] Wolny | Compact genomes and complex evolution in the genus Brachypodium[END_REF][START_REF] Betekhtin | Reconstructing the Evolution of Brachypodium Genomes Using Comparative Chromosome Painting[END_REF]. It is a rhizomatous C3 grass species adapted to the Mediterranean summer drought [START_REF] Contu | Brachypodium retusum. The IUCN Red List of Threatened Species[END_REF]). The species is widespread in the Western Mediterranean (Spain, Southern France, and Italy) but also occurs in the Eastern parts [START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF]). At regional Southern French level, it occurs on calcareous soils of lower mountain ranges but also on decarbonated Red Mediterranean soils of former river valleys whereas B.

retusum is absent from lowlands with deep clay and loam soils [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. B. retusum is an outcrossing wind-pollinated species with low self-compatibility ranging from 0 to 30.6% [START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF]. It flowers from April to July and shows high clonal growth but a low clonal spread. Its rhizomes form a dense network close to the soil surface resulting in a high tolerance to above-ground disturbance such as wildfires or grazing [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF]. B.

retusum seeds do not require vernalisation [START_REF] Khan | Breeding relationships in the genus Brachypodium (Poaceae: Pooideae)[END_REF].

Brachypodium retusum is among the first species resprouting after fires and its regeneration is not affected by the frequency or severity of fire [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]). Due to its dense rhizome-root system, the species stabilizes the soil [START_REF] Martinez-Fernandez | Land use and soil-vegetation relationships in a Mediterranean ecosystem: El Ardal, Murcia, Spain[END_REF][START_REF] Cerdà | Soil aggregate stability under different Mediterranean vegetation types[END_REF][START_REF] Baets | Root tensile strength and root distribution of typical Mediterranean plant species and their contribution to soil shear strength[END_REF]). B. retusum may facilitate seedling recruitment of other species by reducing post-fire mortality [START_REF] Raventós | A resprouter herb reduces negative density-dependent effects among neighboring seeders after fire[END_REF]. It may further reduce negative densitydependent effects among neighbouring seeder plants during the first post-fire year by reducing mortality [START_REF] Raventós | A resprouter herb reduces negative density-dependent effects among neighboring seeders after fire[END_REF]. [START_REF] Saiz | Structure and spatial self-organization of semi-arid communities through plant-plant co-occurrence networks[END_REF] found in a dward shrub community of south-eastern Spain ecological community a high number of ecological network links for the species. This number of links is an indicator for interactions with other species suggesting that B. retusum is a highly interacting species [START_REF] Saiz | Structure and spatial self-organization of semi-arid communities through plant-plant co-occurrence networks[END_REF].

Few studies have tested the spatial association between B. retusum and other species. [START_REF] Maestre | Spatial associations and patterns of perennial vegetation in a semi-arid steppe: a multivariate geostatistics approach[END_REF] found a negative small-scale correlation between B. retusum and Stipa tenacissima in a semi-arid steppe and a positive correlation between B. retusum and Anthyllis cytisoides.

The species was found to be highly competitive in Spanish pine woodlands and shrublands [START_REF] Pausas | Fire severity and seedling establishment in Pinus halepensis woodlands, eastern Iberian Peninsula[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF][START_REF] Maestre | Mechanisms underlying the interaction between Pinus halepensis and the native late-successional shrub Pistacia lentiscus in a semi-arid plantation[END_REF]) reducing seedling survival of pine species by strong competition for water [START_REF] Clary | Water relations in competitive interactions of Mediterranean grasses and shrubs[END_REF]) and for soil nitrogen [START_REF] Bonet | Secondary succession of semi-arid Mediterranean old-fields in south-eastern Spain: insights for conservation and restoration of degraded lands[END_REF][START_REF] Casals | Short-term nitrogen fixation by legume seedlings and resprouts after fire in mediterranean old-fields[END_REF].

Although B. retusum showed high regeneration and competitive ability in Spanish studies, seedling recruitment and colonisation was very low in our southern French study area (see III, (Buisson et al. 2006;Coiffait-Gombault et al. 2012a, b). Moreover, B. retusum represents a main nutritional resource for sheep although many annuals show a higher nutritive value [START_REF] Tatin | Écologie et conservation d'une steppe méditerranéenne: La plaine de Crau[END_REF], thus its re-establishment in degraded areas would represent an agro-economic benefits for stockbreeders.

V.

The study system: Dry Mediterranean grasslands

Grasslands are herbaceous communities dominated by grasses (Poaceae) or other graminoids (Cyperaceae, Juncaceae). Species-rich grasslands represent 27 % of the Earth's ecosystems [START_REF] Henwood | An overview of protected areas in the temperate grasslands biome[END_REF]. The total area of Mediterranean basin dry grasslands has considerably declined in the last decades but they still cover 424,371 km 2 [START_REF] Dixon | Distribution mapping of world grassland types (M. Ebach[END_REF]. Increased land use intensity, in particular transformation to arable use and land abandonment followed by shrub encroachment are threatening these highly diverse ecosystems. Mediterranean dry grasslands are under decline and protected by the EU habitats directive. B. retusum is still widespread in the Mediterranean but also suffers from the loss of this habitat (San Miguel

2008).

Grasslands of the Mediterranean basin depend on three major environmental factors: climate, edaphic conditions and human impact, in particular livestock grazing [START_REF] Blondel | The 'Design' of Mediterranean Landscapes: A Millennial Story of Humans and Ecological Systems during the Historic Period[END_REF]. Fire and grazing are common disturbances in Mediterranean grasslands and shape populations and community [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF]. Their strong decrease (fire protection, land abandonment) may partly explain the decline of Mediterranean steppes (San [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF].

a.

Study area

The study and sampling area to analyse genetic differentiation comprises Spain (Andalucía and Catalunya), Italy (Liguria and Sicilia) and southern France (departments Vaucluse, Bouchesdu-Rhône, Gard, Hérault and Aude). The core study area in south-eastern France is characterised by a typical Mediterranean climate with a high amount of sunshine (over 3000 hours/year), a hot and dry summer and a mild winter (mean temperature of 7°C) The annual temperature average is 14 ° C and the maximum rainfall from spring to autumn (500 mm/year).

Within the core study area B. retusum occurs on base-rich calcareous sols (Rendzina) of lower mountain ranges with pH values of 7.5 to 8.0 and on Red Mediterranean Soils (Haplic Cambisol, see below) with pH values of 6.5 to 7.5. The latter soils originate from former river beds of large streams (Rhone, Durance) and are characterised by a high density of stones shaped by these streams (pebbles, cobbles). Many areas have been grazed by sheep since the Neolithic age but recent grazing history is not known for all sites [START_REF] Badan | Les bergeries romaines de la Crau d'Arles. Les origines de la transhumance en Provence[END_REF][START_REF] Leveau | L'herbe et la pierre dans les textes anciens sur la Crau : relire les sources écrites[END_REF]).

b.

In situ experimental field : the Crau plain

The Crau plain with an area of about 600 km 2 is located in south-eastern France (Bouches-du-Rhône department; 43°33'N 4°52'E; 10 m above sea level). The study site was used as a model system for tests on the influence of environmental factors in the field. The plant diversity is high and representative of Mediterranean "steppe" ecosystems [START_REF] Wolff | La Crau, une steppe méditerranéenne unique en France?[END_REF]. The stony surface limits plant cover. Fifty percent of the soil surface is covered by coarse pebbles or cobbles [START_REF] Bourrelly | Dynamique annuelle et production primaire nette de l'écosystème steppique de Crau (Bouches du Rhône)[END_REF]).

The plain is part of the paleo-delta of the Durance River which formerly flowed into the Mediterranean sea and had successively covered the plain with sediments from the prealps 650 000 to 120 000 years ago [START_REF] Devaux | Notice de la carte phyto-écologique de la Crau (Bouches du Rhône)[END_REF][START_REF] Colomb | La Crau, histoire Plio-Pléistocène[END_REF]. The geological history and Mediterranean climate have formed the Red Mediterranean Soil (Haplic Cambisol) with low fertility levels, in particular of phosphorus and potassium [START_REF] Römermann | Influence of former cultivation on the unique Mediterranean steppe of France and consequences for conservation management[END_REF]. The Crau soil is particularly shallow (40-60 cm) with an impermeable layer of "puddingstone" (5 to 40 m deep) which is not the case at other sites of the same soil type [START_REF] Devaux | Notice de la carte phyto-écologique de la Crau (Bouches du Rhône)[END_REF].

The absence of access to groundwater increases drought stress limiting plant growth at the Crau sites.

Sheep grazing in the Crau plain began 4000 years ago during the Roman period and continued during the Middle ages until nowadays [START_REF] Badan | Les bergeries romaines de la Crau d'Arles. Les origines de la transhumance en Provence[END_REF][START_REF] Leveau | L'herbe et la pierre dans les textes anciens sur la Crau : relire les sources écrites[END_REF]). Most of the Crau plain is intensively grazed during spring. A large part of the steppe vegetation was lost due to irrigation (since the middle of the XVI th century) transforming the dry into mesophilic grasslands used for hay making ("La Crau verte"). The remaining steppe vegetation "La Crau sèche" is classified as a nature reserve (since 2001) and a Natura 2000 habitat. In the XX th century, even a part of the dry Crau was transformed into arable fields and orchards. Today only 20% of the initial steppe area is still intact representing 8600 ha of which 5811 ha are belonging to the nature reserve [START_REF] Wolff | La Crau, une steppe méditerranéenne unique en France?[END_REF].

VI. Research questions and thesis structure

My PhD thesis aims to answer to the following major questions:

(1) What are the spatial and environmental scales of genetic differentiation? Is differentiation adaptive? What are the major drivers of differentiation and adaptation? What are the implications for provenance choice in ecological restoration?

(2) Which ecological factors limit seedling recruitment and establishment? In particular, do fire, grazing, water supply affect germination, survival, growth and reproduction and are interactions between these factors significant? How can we use this information to improve the success of introduction and restoration measures?

Genetic differentiation in plant species may result from adaptation to environmental conditions but also from stochastic processes. The drivers selecting for local adaptation and the contribution of adaptation to genetic differentiation are often unknown. Previous restoration and succession studies have revealed different colonisation patterns for Brachypodium retusum, a common perennial Mediterranean grass. In order to understand these patterns, we measured AFLP marker differentiation in 17 Mediterranean populations and this neutral differentiation was compared to differentiation in phenotypic traits (Chapter 1). We further tested adaptation to different environmental factors in a common garden experiment (Chapter 2).

Colonisation processes are often limited by the availability of a sufficient amount of seeds (low seed production and dispersal) and by seedling recruitment as a critical life cycle stage. In Mediterranean dry grasslands, three main environmental factors influence seed production and establishment. Sheep grazing is the dominant type of management largely affecting plant populations. Wildfires and fire management were important drivers in the past but their role in maintaining the ecosystem is nowadays less evident because fire prevention and abandonment of traditional prescribed burning have largely reduced their impact in the last decades. Drought stress may be a third factor limiting seedling recruitment being particularly strong during the dry and hot Mediterranean summer but in our study region also occurring in autumn and spring.

The last two parts of the thesis analyse environmental factors that may limit seedling recruitment, growth and reproduction: the effect of grazing and fire on vegetative regeneration as well as on the sexual reproduction of adult B. retusum and its associated plant community (Chapter 3) and the effect of watering and grazing on early establishment (Chapter 4). In Chapter I, we focused on neutral and quantitative differentiation of B. retusum populations which to my knowledge has never been studied before. We analysed genetic diversity, structure and differentiation in Western Mediterranean populations using AFLP markers. We further compared this neutral variation with variation in phenotypic traits testing a subset of Southern

French populations in order to evaluate the relative contribution of drift, gene flow and selection. We hypothesized that small-scale environmental gradients influence adaptive differentiation in phenotypic traits but not or to a much lesser degree differentiation in neutral genetic markers.

Contribution of the thesis:

-Genetic diversity and structure at Western Mediterranean scale.

-Effect of drift and environmental conditions on genetic differentiation.

-Recommendations for targeting source populations in ecological restoration.

Chapter I

Comparison of neutral and adaptive differentiation in the Mediterranean grass species Brachypodium retusum.

INTRODUCTION

Selection, genetic drift and gene flow shape diversity, structure and genetic variation of plant populations [START_REF] Lenormand | Gene flow and the limits to natural selection[END_REF][START_REF] Leimu | A Meta-Analysis of Local Adaptation in Plants[END_REF]. Adaptive differentiation is widespread in grassland species, and plants often show adaptation at regional scale [START_REF] Becker | The spatial scale of adaptive population differentiation in a wide-spread, well-dispersed plant species[END_REF][START_REF] Bucharova | Genetic differentiation and regional adaptation among seed origins used for grassland restoration: lessons from a multispecies transplant experiment[END_REF]. In grassland populations, climate is the major large-scale environmental factor driving genetic divergence, in particular temperature, precipitation and their seasonal distribution [START_REF] Manel | Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation[END_REF][START_REF] Bucharova | Genetic differentiation and regional adaptation among seed origins used for grassland restoration: lessons from a multispecies transplant experiment[END_REF]. Soil conditions often vary at smaller scales resulting in small-scale adaptive differentiation (Van der Putten et al.

2004; [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF]. Soil chemistry, but also soil structure and organisms are potential selective forces [START_REF] Wardle | Communities and ecosystems: linking the aboveground and belowground components[END_REF]. Several studies showed grass species adaptation to soil type including serpentine soils (e.g Freitas and Mooney 1996 for Bromus hordeaceus and Meimberg et al. 2010 for Aegilops triuncialis) or fertility gradients [START_REF] Sherrard | Local adaptation across a fertility gradient is influenced by soil biota in the invasive grass, Bromus inermis[END_REF]. PH is, for example, one key soil chemistry factor to which plant populations may be adapted [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Raabová | The role of spatial scale and soil for local adaptation in Inula hirta[END_REF]. Soil characteristics and climate also influence land management which is a third important driver of adaptive differentiation in grassland systems.

In particular, grazing intensity depending on soil quality is known to affect plant performance and may select for genotypes that tolerate aboveground plant damage [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF][START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF].

Nevertheless, genetic differentiation is not only the result of adaptation to different environments. Stochastic processes such as genetic drift considerably contribute to population differentiation and may even overwhelm local adaptation [START_REF] Hereford | Limits to local adaptation in six populations of the annual plant Diodia teres[END_REF][START_REF] Hereford | A Quantitative Survey of Local Adaptation and Fitness Trade-Offs[END_REF]. Genetic drift and limited dispersal favour isolation-by-distance effects not related to environmental gradients (Sexton et al. 2014).

Our [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. Second, the major climatic factor varying in the study region is rainfall increasing from the east to the west. In a previous study on a subset of populations, significant differentiation was found between soil types within sites representing the same climate although differentiation between sites of different climate was much stronger [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF].

Classical common garden experiments to test for differentiation do not allow distinguishing drift and adaptation as driving forces [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. Molecular markers provide a powerful tool to study neutral genetic variation and to compare it with potentially adaptive quantitative traits [START_REF] Brommer | Whither Pst? The approximation of Qst by Pst in evolutionary and conservation biology: Whither Pst?[END_REF][START_REF] Leinonen | QST-FST comparisons: evolutionary and ecological insights from genomic heterogeneity[END_REF][START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF]. Meta-analyses have shown that differentiation in quantitative traits is often higher than differentiation in neutral markers since gene flow reduces neutral variation but differentiation in traits that are under strong selection is maintained [START_REF] Leinonen | Comparative studies of quantitative trait and neutral marker divergence: a meta-analysis: QST-FST meta-analysis[END_REF][START_REF] Kort | A meta-analysis of the effects of plant traits and geographical scale on the magnitude of adaptive differentiation as measured by the difference between QST and FST[END_REF]. Thus, comparisons of quantitative traits and neutral markers provide valuable insights into the causes of spatial genetic divergence among populations [START_REF] Leinonen | Comparative studies of quantitative trait and neutral marker divergence: a meta-analysis: QST-FST meta-analysis[END_REF]. They may in particular help to disentangle the contribution of climate and genetic drift to population differentiation often occurring at similar spatial scales [START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF].

Using AFLP markers, we aimed at a better understanding of spatial genetic structure in the Western Mediterranean. Using a subset of Southern French populations (regional scale), we tested whether neutral genetic variation correspond to variation in phenotypic traits measured in a common garden. We correlated differentiation at regional level to climate and soil factors to identify the major environmental drivers.

More specifically, we address the following research questions:

(1) What is the structure of genetic diversity of B. retusum at Western Mediterranean scale?

2) Does the comparison of phenotypic differentiation and neutral genetic differentiation allow the detection of environmental selection pressure at regional scale in Southern France?

(3) What are the major drivers of adaptive differentiation; in particular, what is the role of localscale soil type compared with large-scale climate?

Chapter I

MATERIALS AND METHODS

Study species and sampling

Ramose false brome (Poaceae, Brachypodium retusum P. Beauv.) is a rhizomatous perennial C3 grass species. It is a predominantly outcrossing wind-pollinated species that shows high clonal growth but also sexual reproduction. Its rhizomes form a dense network close to the soil surface resulting in a high resilience to above-ground disturbance such as wildfires or grazing [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF]. B. retusum is a perennial and allopolyploid (presumed hexaploid) species with a chromosomal base number of 2n=38 [START_REF] Wolny | Compact genomes and complex evolution in the genus Brachypodium[END_REF].

Plant material of 17 populations was collected in the Western Mediterranean basin from Northern Italy to Southern Spain focusing on Southern French populations (Table S1). For 15 populations, we collected plant material (seeds or leaves) from twenty patches with a minimum distance of 10 m between patches to avoid that patches belong to the same individual. In each patch, we used only one seed or leave for further analyses resulting in 20 different mother plants per population. Two populations were bulk samples with unknown number of mother plants (SES, ROQ, Table S1). Southern French populations were collected in pairs from base-rich calcareous soils of lower mountain ranges and from close-by decalcified red Mediterranean soils (Haplic cambisols). Calcareous soils are shallow on calcareous bedrock with an average soil pH of 8.4. Red Mediterranean soils show a higher clay content resulting in a better nutrient and water retention (pH 7.3). Because of the higher soil fertility and accessibility, grasslands on red Mediterranean soils were more intensively used in the past (in particular grazing) than those on calcareous soils. In sixteen populations, seeds were sampled at natural sites and later on grown in a common garden (see below) or in a growth chamber. Leaves for AFLP analysis were then sampled from these seed-grown plants. In one population, leaves were directly sampled in the field.

Sample preparation and DNA extraction

Leaves of 20 plants per population (ten for ROQ population) were collected for AFLP analysis resulting in a total of 366 plants were analyzed. Between 10-15 mg of silica-dried and further frozen leaves from the field, greenhouse-cultivated samples were ground in a mixer mill "TissueLyser" (Quiagen-Retsch). Total DNA was extracted using NucleoSpin Plant II Kit (Macherey & Nagel, Germany) (Detailed method description 1). DNA concentrations were measured using a photometer (Biophotometer, Eppendorf, Germany).

Chapter I AFLP genotyping

According to [START_REF] Vos | AFLP: a new technique for DNA fingerprinting[END_REF] 72°C for 1 min, followed by 22 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, 72°C

for 5 min and 4°C for 2h. The fragment length produced by the amplification was separated and quantified by capillary electrophoresis using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, California, U.S.A.) with GS600 LIZ size marker.

Analysis of phenotypic traits in common garden plants

In order to understand genetic differentiation based on quantitative traits, individuals from 13

French populations were grown for two years in a common garden. In December 2015, collected seeds were germinated in peat plugs (4 cm x 4 cm), placed in a growth chamber (temperature 15/20 °C, 12/12h, night/day) and watered regularly. In March 2016, 24 plugs per population (corresponding to 24 patches) were randomly selected and transplanted to a previously ploughed experimental site in Avignon-Montfavet. Two plugs per population were planted to random positions within each of 12 blocks (3 * 3m²). During the first four months, seedlings were moderately watered. Plots were hand-weeded to limit competition. Plant height, diameter, tiller number, leaf number and the length of the longest leaf were measured for two growing seasons.

Chapter I

Data analysis

Large-scale genetic structure using AFLP Peaks were scored in Peak Scanner V 1.0 (Applied Biosystems) as present (1), absent (0), or "no data" (NA) if they were not unambiguously identified. In Raw Geno 2.0 [START_REF] Arrigo | Evaluating the impact of scoring parameters on the structure of intra-specific genetic variation using RawGeno, an R package for automating AFLP scoring[END_REF], only fragments longer than 100 bp and smaller than 500 bp were considered. Maximum binning between peaks was set at 1.75 and minimum at 1.5. The reliability of AFLP markers was checked by repeating the complete analysis from DNA amplification to AFLP screening on 16 samples for each pair of primers. A Bayesian analysis using BAYESCAN [START_REF] Foll | A genome-scan method to identify selected loci appropriate for both dominant and codominant markers: a Bayesian perspective[END_REF] was run to detect loci potentially under selection (outliers) and remove them for further analysis to focus on neutral genetic diversity. Two datasets were used, a global one to understand large-scale genetic structure and a subsample to analyse genetic structure at regional scale using AFLP and quantitative traits.

Diversity indices were estimated using R package poppr. Parameters of genetic diversity (percentage of polymorphic loci, number of multilocus genotypes, Shannon-Wiener Index of MLG diversity, Nei's diversity index of expected heterozygosity and standardized index of association) were calculated for each population. Genetic differentiation among populations was analysed using divergence at neutral marker θST, an analogue of Fst for binary data using GenAlEx 6.5 [START_REF] Peakall | GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research--an update[END_REF]. The significance test of θST was based on 9999

permutations. An UPGMA tree was constructed from the matrix of pairwise population differences (R package phangorn).

Regional scale genetic structure: phenotypic and neutral marker differentiation

For a subset of thirteen Southern French populations, the mean θST was compared to the mean PST based on phenotypic traits. A Bayesian analysis using the software STRUCTURE 2.2 [START_REF] Pritchard | Documentation for Structure software: version 2[END_REF]) was applied to assess genetic structure among AFLP samples.

STRUCTURE uses an iterative approach to cluster genotypes into K populations without a priori knowledge to which population individuals belong. From K=1 to K=13 populations, and for each K solution, ten replicates were run with 100,000 burn-in simulations followed by 2 million simulations per replicate. Using STRUCTURE, direct posterior probabilities for K (loglikelihood) as well as the ad hoc statistic ΔK [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF] were estimated using uniform a priori values of K between one and thirteen (total number of populations).

Chapter In order to evaluate local soil type versus regional-scale differentiation (climate, drift), the two adjacent populations from red Mediterranean and calcareous soils were considered as originating from the same site resulting in a total of six sites and two soil types per site. Thus, each population represented a particular combination of site and soil. For AFLP markers, we applied an analysis of molecular variance (AMOVA) based on θST statistics using GenAlEx 6.0.

An ANOVA was calculated to analyse the effect of site, soil and the soil×site interaction on first PCA axis. A multiple regression on distance matrices (MRM) was used to identify variables (climate distance, geographic distance, genetic distance and pH distance) that best predicted Pst distance among populations. Additionally, partial distance-based redundancy analyses (dbRDA) was applied to evaluate the relationship between divergence in phenotypic traits and environmental variables (Rpackage Vegan). Partial dbRDA were fitted separately for Pst of vegetative and reproductive traits using permutation testing [START_REF] Legendre | Distance-Based Redundancy Analysis: Testing Multispecies Responses in Multifactorial Ecological Experiments[END_REF]. First, a marginal test was performed using only environmental variables as predictors.

Second, a conditional test was run using genetic distance and geographical coordinates as covariates to take into account the potential confounding effects of isolation by neutral genetic Chapter I drift. The genetic distance matrix was transformed into continuous rectangular vectors via principal coordinates analysis. The significance of environmental factors was evaluated using a dbRDA permutation test (9999 permutations). All statistical analyses were run in R (R, version 3.3.1, R Development Core Team (2013)) except for genetic structure.

RESULTS

AFLP analysis allowed genotyping of 366 individuals on 587 loci. After filtering for reliable non-redundant loci (error rate <4.39 %), suppressing unscored individuals and nonpolymorphic loci, we created two datasets. A global dataset (98.48% of polymorphic loci) with 322 individuals was genotyped on 323 loci and a Southern French dataset (97.27% of polymorphic loci) with 258 individuals was genotyped on 320 loci. Only two outliers under selection were found by BAYESCAN analysis and removed from the datasets. No clone was found in any of the datasets.

Large-scale genetic structure using AFLP (global dataset) Average Shannon diversity (H) of tested populations ranged from 2.302 to 3.044, and expected heterozygosity, He, from 0.134 to 0.205 (Table 1). In twelve populations, the standardized index of association was significantly different from zero rejecting the null hypothesis of no linkage between markers of twelve populations. In five populations, no linkage between markers was detected. The significant rbarD values ranging from 0.001 to 0.101, suggest asexual reproduction but occurring at different rate in these twelve populations. In the global data set, the Italian NIT was the only population showing private alleles.

Chapter I

Table1: Genetic diversity estimates in total dataset populations based on AFLP data. In the global dataset, the average θST (0.102; P < 0.001) revealed moderate genetic differentiation among the 17 populations. Pairwise θST between populations ranged from 0.006 to 0.224 and were significantly different from zero except for one population pair (Table S2).

The UPGMA tree calculated on the pairwise θST matrix revealed differentiation of non-French populations but no clear geographical pattern for French populations (Fig. 1). Chapter I Regional scale genetic structure: phenotypic and neutral marker differentiation

In the Southern French dataset, the average θST (0.072; P < 0.001) was lower than in the global data set and pairwise θST between populations ranged from 0.006 to 0.197 (Table S2). The STRUCTURE analysis provided the strongest support when samples were clustered into two groups (K=2) and five groups (K=5) based on ΔK method of Evanno (Fig. S1). At K=5, both assignment ratio by individual and geographical distribution of genetic cluster are indicating a high level of admixture (Fig. 2 and3). Compared to differentiation in neutral AFLP markers, the phenotypic differentiation was higher. The average Pst for vegetative (0.171; P < 0.001) and reproductive traits (0.138; P < 0.001) revealed significant phenotypic differentiation among the 13 populations. Pairwise Pst between populations were 0.062 to 0.432 for vegetative and 0.066 to 0.355 for reproductive traits, respectively (Table S3). Average population differentiation was at least two times greater Site of origin contributed more to the differentiation than soil of origin. The site of origin accounted for 2.33 % of total variation for AFLP markers, 12.85 % for vegetative traits and 8.37 for reproductive traits whereas the soil of origin accounted for 0.62, 1.92 and 0.05 %, respectively (Table 2). Variation between soil types was still significant in AFLP markers (highly significant) and vegetative traits but not in reproductive traits. The significant soil x site interaction in molecular and phenotypic traits indicated that soil type variation depend on site of origin. Multiple regression analysis showed that phenotypic differentiation in vegetative and reproductive traits was correlated to climatic distance between sites but also provided evidence that differentiation in vegetative traits was partly explained by neutral genetic distances (Table 3).

Chapter I

Chapter I The dbRDA results showed a significant correlation between phenotypic trait Pst and climate factors when not corrected for differentiation in neutral markers confirming MRM results (Table 4A). When neutral variation in AFLP markers was taken into account a part of these significant correlations disappeared (Table 4B). However, correlations between phenotypic traits and mean July temperature and number of frost days remained significant (marginally significant for reproductive traits, Table 4B, Fig. S2). Additionally, the correlation between corrected vegetative traits and mean annual temperature was marginally significant.

Chapter I [START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF]. These reproductive characteristics favor high levels of genetic variability [START_REF] Ge | RAPD variation within and between natural populations of the wild rice Oryza rufipogon from China and Brazil[END_REF][START_REF] Nybom | Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants[END_REF]. However, longevity and in particular clonal growth may have reduced diversity compared with other perennial Brachypodium species [START_REF] Young | The population genetic consequences of habitat fragmentation for plants[END_REF]. Indeed, most populations showed linkage disequilibrium, associations between alleles at different loci (rbarD, table 1), indicating a potential effect of asexual reproduction on genetic diversity.

Second, the cluster analysis of genetic differentiation (UPGMA, Fig. 1) revealed that Spanish and Italian populations are well separated from Southern French ones. At regional Southern

French scale, analyses of AFLP data showed a low differentiation between populations (θST of 7.2%) associated to strong admixture and weak geographical pattern (Structure, Fig. 2 and3).

Such a low genetic structuring at regional scale is typical for outcrossing species with longdistance gene flow and was also found for the outcrossing B. pinnatum [START_REF] Bąba | Genetic diversity of the expansive grass Brachypodium pinnatum in a changing landscape: Effect of habitat age[END_REF]. The self-compatible B. sylvaticum showed, however, a higher structuration and differentiation of populations since selfing reduces gene flow between populations [START_REF] Rosenthal | Evidence for multiple sources of invasion and intraspecific hybridization in Brachypodium sylvaticum (Hudson) Beauv. in North America[END_REF].

Regional scale genetic structure: phenotypic and neutral marker differentiation

In Southern French populations, phenotypic differentiation (PST) was higher than neutral molecular differentiation (θST) supporting divergent natural selection and adaptation to local environments [START_REF] Volis | Distinguishing adaptive from nonadaptive genetic differentiation: comparison of QST and FST at two spatial scales[END_REF][START_REF] Leinonen | QST-FST comparisons: evolutionary and ecological insights from genomic heterogeneity[END_REF]. Critical ℎ 2 was low confirming that the difference between neutral molecular and phenotypic differentiation is significant and robust [START_REF] Brommer | Whither Pst? The approximation of Qst by Pst in evolutionary and conservation biology: Whither Pst?[END_REF].

No isolation-by-distance was found in phenotypic traits but a significant effect of θST on vegetative phenotypic traits still showed an influence of neutral processes on phenotypic differentiation. Genetic drift and gene flow are major drivers of population differentiation even in phenotypic traits (e.g. [START_REF] Lande | Natural Selection and Random Genetic Drift in Phenotypic Evolution[END_REF][START_REF] Orsini | Drivers of population genetic differentiation in the wild: isolation by dispersal limitation, isolation by adaptation and isolation by colonization[END_REF]. However, effects of environmental conditions on phenotypic differentiation measured in a common garden experiment remained significant after controlling geographical-distance and genetic distance based on AFLP in the dbRDA analysis (Table 4). This finding supports that divergent directional selection led to adaptive differentiation. The major environmental factors associated to phenotypic differentiation among Southern French populations were summer temperature and number of Chapter I frost days. Summer temperatures are closely related to drought stress strongly affecting plant fitness and survival in Mediterranean systems and in particular limiting seedling recruitment [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF][START_REF] Thomas | The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape[END_REF][START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. Southern French populations are relatively close to the northern range limit of B. retusum [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF][START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF]. Thus, it is likely that frost is a strong selective force contributing to population differentiation (e.g. Kreyling et al. 2012a;b). Frost has been identified as a major driver of population differentiation in trees [START_REF] Savolainen | Genetic variation in cessation of growth and frost hardiness and consequences for adaptation of Pinus sylvestris to climatic changes[END_REF]J. Kreyling et al. 2012a) but also in some herbaceous species [START_REF] Agrawal | Evolution of plant resistance and tolerance to frost damage[END_REF]Kreyling et al. 2012b).

Soil type within sampling sites explained a significant part of variation among populations in neutral markers and in vegetative phenotypic traits but explained variation was much smaller between soils within sites than between sites suggesting that soil was not a major driver of genetic structure. The soil pH as the principal difference between the two tested soil types had no effect on phenotypic differentiation (Pst) when corrected for the influence of genetic drift or geographical distance. Small-scale adaptive differentiation to soil conditions has been shown for sites contaminated with heavy metals [START_REF] Ernst | Population differentiation in grassland vegetation[END_REF]) but environmental gradients were very strong in the cited studies. If gradients are less strong gene flow may prevent adaptive differentiation at smaller scales [START_REF] Leimu | A Meta-Analysis of Local Adaptation in Plants[END_REF][START_REF] Hereford | A Quantitative Survey of Local Adaptation and Fitness Trade-Offs[END_REF]. Thus, adaptation to soil conditions often showing small-scale variation was not always detected or its magnitude was smaller than that of climate acting on larger scales and thus being less sensitive to homogenizing gene flow [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Raabová | Ecological rather than geographic or genetic distance affects local adaptation of the rare perennial herb, Aster amellus[END_REF].

CONCLUSIONS

Brachypodium retusum populations of the Western Mediterranean basin exhibited a high genetic diversity and weak to moderate differentiation between populations. Patterns of differentiation in neutral markers suggest a predominant influence of genetic drift. At regional scale in Southern France, both divergent selection and drift were detected by comparing phenotypic and neutral molecular differentiation. The major driver of adaptive differentiation was climate, in particular summer temperature and winter frost frequency. The two main soil types of the region contributed significantly to population differentiation but explained much less variation than climate and genetic drift at larger scales. Local adaptation is therefore highly probable in B. retusum. Accordingly, plant provenance targeting in ecological restoration needs to consider this spatial and environmental pattern of population differentiation. Local seed Chapter I collected on the same soil type should be preferred but proximity of source populations seems to be more important than corresponding soil type. Chapter I In Chapter I, we showed an adaptive differentiation of French populations related to climate, in particular to mean July temperature and frost day number. Adaptive differentiation was tested regarding principal component axis of vegetative or reproductive traits.

SUPPORTING INFORMATION

ℎ 2 formula ℎ 2 () = 2 2 () () 1-() with 2 () = [1-()] ℎ 2 2 ()
In Chapter II, we tested population differentiation using different subsets in a common garden and in a growth chamber experiment. The major common garden experiment is the same used for the comparison of phenotypic traits and molecular markers in Chapter I. However, in

Chapter II only first-year measurements were included since all data were collected one year earlier than in Chapter I studies. We further included a stone cover treatment not detailed in Chapter I and set up additional experiments to test for grazing (simulated by clipping) and humidity (soil moisture) effects on Southern French B. retusum populations. The major aim was to test differential population responses related to environmental conditions of collection sites. We hypothesised that B. retusum populations are adapted to the main gradients varying among collected populations: such as humidity (soil moisture), stone cover and grazing.

Contribution of the thesis:

-Differentiation of B. retusum populations in phenotypic traits at regional scale in Southern France and between major soil types.

-Adaptation to humidity, stone cover and grazing.

-Recommendations for targeting source populations in ecological restoration.

-Environmental requirements for successful establishment.

INTRODUCTION

Genetic differentiation among populations is an important strategy of plant species to survive under different environmental conditions [START_REF] Linhart | Evolutionary Significance of Local Genetic Differentiation in Plants[END_REF][START_REF] Bischoff | Scales and drivers of local adaptation in Brassica nigra (Brassicaceae) populations[END_REF]. Adaptive differentiation occurs if environmental gradients are strong and gene flow counteracting differentiation is low [START_REF] Kawecki | Conceptual issues in local adaptation[END_REF]. Climate is a major driver of local adaptation and genetic differentiation in plant populations, in particular on large geographic scales [START_REF] Etterson | Evolutionary potential of chamaecrista fasciculata in relation to climate change. i. clinal patterns of selection along an environmental gradient in the great plains[END_REF][START_REF] Leinonen | Local adaptation in European populations of Arabidopsis lyrata (Brassicaceae)[END_REF][START_REF] Bucharova | Genetic differentiation and regional adaptation among seed origins used for grassland restoration: lessons from a multispecies transplant experiment[END_REF]. However, microclimatic gradients may also act on much smaller scales resulting in small-scale adaptation and differentiation [START_REF] Knight | Local adaptation within a population of Hydrocotyle bonariensis[END_REF][START_REF] Lenssen | Local adaptation of the clonal plant Ranunculus reptans to flooding along a small-scale gradient[END_REF][START_REF] Bischoff | Detecting local adaptation in widespread grassland speciesthe importance of scale and local plant community[END_REF].

The adaptation to soil conditions was first detected on soils contaminated with heavy metals indicating that adaptive differentiation may occur quite rapidly [START_REF] Schat | Identical Major Gene Loci for Heavy Metal Tolerances that Have Independently Evolved in Different Local Populations and Subspecies of Silene vulgaris[END_REF][START_REF] Antonovics | Evolution in closely adjacent plant populations X: long-term persistence of prereproductive isolation at a mine boundary[END_REF]. Furthermore, the pH value and related chemical and biotic properties may represent a strong selective force [START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Raabová | The role of spatial scale and soil for local adaptation in Inula hirta[END_REF]. Humidity is also a limiting factor for plant growth and reproduction leading to drought adaptation [START_REF] Shah | Interaction of drought and high temperature on photosynthesis and grain-filling of wheat[END_REF][START_REF] Suriyagoda | Multiple adaptive responses of Australian native perennial legumes with pasture potential to grow in phosphorusand moisture-limited environments[END_REF]. However, an increase in soil moisture often results in higher competition for other resources representing a counteracting selective force [START_REF] Kadmon | Plant Competition along Soil Moisture Gradients: A Field Experiment with the Desert Annual Stipa Capensis[END_REF].

Ecosystem management is a third potential driver of adaptive differentiation in semi-natural systems such as grasslands. Grazing changes site conditions by biomass removal, plant damage, reduced competition and creates heterogeneity in grassland vegetation [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF][START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF]. It seems to be likely that long-term grazing favors genotypes adapted to grazing [START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF]. However, grazing may also obscure adaptation to other environmental factors if this selection pressure has not been consistent in the past [START_REF] Hufford | Local Adaptation and Effects of Grazing among Seedlings of Two Native California Bunchgrass Species: Implications for Restoration[END_REF].

Genetic differentiation is not only the result of adaptation to different environments. Stochastic processes such as genetic drift considerably contribute to population differentiation and may even overwhelm local adaptation [START_REF] Hereford | Limits to local adaptation in six populations of the annual plant Diodia teres[END_REF][START_REF] Hereford | A Quantitative Survey of Local Adaptation and Fitness Trade-Offs[END_REF]. Genetic drift and limited dispersal favor isolation by distance effects not related to environmental gradients [START_REF] Sexton | Genetic Isolation by Environment or Distance: Which Pattern of Gene Flow Is Most Common?[END_REF]. [START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF] found both isolation by distance due to stochastic processes, and isolation by environment in seven tested plant species. The contribution of adaptive differentiation to genetic structure may even be low compared with genetic drift (Michalski andDurka, 2012, Sexton et al. 2013).

Genetic differentiation in quantitative and thus potentially adaptive traits is typically tested in common garden experiments [START_REF] Petit | Gene flow and local adaptation in two endemic plant species[END_REF][START_REF] Leinonen | Local adaptation in European populations of Arabidopsis lyrata (Brassicaceae)[END_REF][START_REF] Bischoff | Testing population differentiation in plant specieshow important are environmental maternal effects[END_REF]. Tests for local adaptation require reciprocal transplantation to original collection sites [START_REF] Blanquart | A practical guide to measuring local adaptation[END_REF]. However, common garden experiments comparing the response to the manipulation of key environmental factors allow conclusions on adaptive differentiation and may reveal more specific information on drivers of local adaptation than reciprocal transplantations in the field [START_REF] Lenssen | Local adaptation of the clonal plant Ranunculus reptans to flooding along a small-scale gradient[END_REF][START_REF] Pahl | No evidence for local adaptation in an invasive alien plant: field and greenhouse experiments tracing a colonization sequence[END_REF].

We used the perennial grass Brachypodium retusum (Pers.) P.Beauv. as a model species to test for neutral and adaptive population differentiation at a regional scale in Southern France. The species is dominant in dry grasslands of the Western Mediterranean basin but also occurs in matorral and open woodlands [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF]. In contrast to studies showing a high colonization capacity of the species [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF][START_REF] Cassagne | Using a fire propagation model to assess the efficiency of prescribed burning in reducing the fire hazard[END_REF], spontaneous natural regeneration of B. retusum was poor in the La Crau nature reserve (Southern France) thirty years after conversion from arable use to traditional sheep grazing complicating ecological restoration operations (Coiffait-Gombault et al., 2012).

We hypothesize that genetic differentiation contributes to the different colonization patterns and that this differentiation results from adaptation to major environmental factors distinguishing habitats. In the study region, B. retusum occurs in lower calcareous mountain ranges and on more or less decalcified red Mediterranean soils of former riverbeds such as the Crau area. The latter habitat is also characterized by stones (pebbles, cobbles) shaped by the former rivers. These stones were found to favor growth and survival of B. retusum because rhizomes and roots can grow underneath benefitting from higher soil moisture during the hot and dry Mediterranean summer [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. Apart from soil characteristics and stone cover, these two habitats show also large differences in soil moisture and grazing intensity. The information on population differentiation and adaptation to environmental factors is crucial to predict colonization processes but also to improve seed sourcing in ecological restoration [START_REF] Mckay | How local is local?"-a review of practical and conceptual issues in the genetics of restoration[END_REF][START_REF] Vander Mijnsbrugge | A question of origin: Where and how to collect seed for ecological restoration[END_REF].

In our study, we tested B. retusum populations from calcareous and closest red Mediterranean soils replicated at different geographical distances in order to evaluate whether local-scale differentiation along strong environmental gradients can be as important as large-scale differentiation driven by macro-climate or genetic drift [START_REF] Bischoff | Detecting local adaptation in widespread grassland speciesthe importance of scale and local plant community[END_REF][START_REF] Hereford | Limits to local adaptation in six populations of the annual plant Diodia teres[END_REF][START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF]. We further analyzed differential responses of these populations to grazing simulated by clipping, to stone cover and to soil moisture. We specifically wanted to answer the following research questions:

(1) At which scales does population differentiation occur: what is the role of local-scale soil type compared with large-scale variation? (2) Is this differentiation adaptive in terms of soil conditions (stone cover), management (grazing simulated by clipping) and humidity (soil moisture, germination)?

MATERIALS AND METHODS

Study species

Ramose false brome (Poaceae, Brachypodium retusum P. Beauv.) is a rhizomatous perennial C3 grass species. Its rhizomes form a dense network under the soil surface, resulting in a high resilience to above-ground disturbance such as wildfires or grazing [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF].

Vegetative growth is characterized by tillering at the stem base but also by lateral branching at upper stem parts. B. retusum is an outcrossing wind-pollinated species. It flowers from April to

July and shows high clonal growth and reproduction when rhizome connections disintegrate.

However, rhizomes are short and just allow a slow clonal spread (<5 cm *a -1 in our experiments).

Collection sites and sampling

The study and sampling sites were situated in the central part of the French Mediterranean climate zone from Montpellier to the lower Durance valley. Seeds were collected from six populations on base-rich calcareous soils (Calcaric cambisol) of lower mountain ranges with pH values of 7.8 to 8.9 (Table 1, Fig. 1). Close to each of these six populations on calcareous grasslands, six populations were also collected from usually decalcified red Mediterranean soils (Haplic cambisols) with pH values of 6.7 to 8.0. Three additional populations were collected in the Crau nature reserve as the focal site of Mediterranean dry grassland restoration (Buisson and Dutoit, 2006) in order to obtain a better representation of this largest red Mediterranean soil area of the study region. For further analysis, the four Crau populations (distance: 4-11km) were pooled to the red Mediterranean soil population of Saint-Martin-de-Crau (SM, Table 1).

Red Mediterranean soils show a higher clay content than calcareous soils resulting in a better nutrient and water retention. Due to the higher soil fertility, grasslands on red Mediterranean soils were more intensively used (in particular grazing, less frequently mowing) than those on calcareous soils (Table 1). They are also rich in hydrated oxides of iron and aluminum and in silica whereas calcareous soils are poorer in these elements. Up to 50% of red Mediterranean soil surface is covered by cobbles and pebbles deposited by former rivers. Calcareous soils are less deep on calcareous bedrock and contain numerous angular brittle stones.

The study area is characterized by a typical Mediterranean climate with hot and dry summers and mild and humid winters. The six sampling sites differ in climatic conditions (Table 1)

whereas differences between soil types within sites are small. Annual precipitation depends on the proximity to mountain ranges and is generally higher in the western than in the eastern part of the study area. Close to the Mediterranean Sea, winter temperatures are higher resulting in a lower frost frequency.

Seed collection and preparation

In each population, all inflorescences of each of fifty 1 m² patches were collected. The minimum distance between patches was 10 m and sampling was done in one or several parallel lines depending on population shape (linear versus polygon). Due to clonal growth of the species, the real patch size of individuals is unknown. A distance of 10 m was considered as sufficient to avoid that different patches belong to the same individual [START_REF] Montalvo | Transplantation of the Subshrub Lotus scoparius: Testing the Home-Site Advantage Hypothesis[END_REF].

On average, one patch comprised forty inflorescences resulting in a total of 2000 collected inflorescences per population. In germination experiments, the number of seed families may be higher than the number of patches because one patch may comprise more than one individual.

This pooling at patch level reduces intra-population variation compared to half-sib designs based on individual seed families. In all other experiments, only one seedling per patch was kept for planting corresponding to classical half-sib designs.

Population differentiation under common conditions

Population differentiation was tested in a growth chamber germination experiment and in a common garden study. Inflorescences were shaken in tubes with glass beads using a vortex mixer to separate seeds from glumes. Transparency under transmitted overhead projector light was used as a criterion to remove empty seeds without embryo. In December 2016, seeds were germinated in peat plugs (4 cm x 4 cm). Four seeds of each sampled patch were sown per plug and altogether forty patches of each population were randomly selected for a total of 160 seeds and 40 plugs per population. In April 2016, half of the seedlings were clipped 2 cm above the soil.

We measured the same traits as in the differentiation study. Additionally, the number of inflorescences per plant was counted at the end of the season. This was not possible in the population differentiation study under common conditions since plants rarely (<10%) flower in the first year.

The response to soil moisture during germination was analyzed in a controlled experiment manipulating moisture levels. The major objective was to test adaptive differentiation in germination response under dry conditions. Five moisture levels from 0.3 to 20% soil moisture were adjusted by adding water to plastic boxes filled with 1.15 kg of sand and using a Theta Probe ML2x sensor (Delta-T Devices Ltd, Cambridge, United Kingdom). A subset of ten populations was chosen for this experiment. Six seeds of each population (four for calcareous population of SM due to limited seed number) were sown to each of 12 boxes of each of the five soil moisture levels resulting in a total of 360 seeds per population assigned to 60 boxes.

Boxes were closed by a translucent lid to keep initial moisture levels. The boxes were placed in a growth chamber with a 12-hours cycle of light and temperature (day: 25°C; night: 12°C).

Germination and moisture were measured every three days for a period of six weeks.

Germinated seeds were removed. At the end of the experiment, the water potential of the sand was measured using WP4C Dewpoint Water Potentiometer (Decagon Devices, Pullman, Washington, United States of America). Water potentials were close to 0 (saturation) for the highest and -0.18 MPa for the lowest moisture level (Appendix S1, see Supplemental Data with this article).

Statistical analysis

Linear models were used to analyze population and treatment effects on germination and growth traits. Population was divided in a soil and site component. The two adjacent populations from red Mediterranean and calcareous soils were considered as originating from the same site resulting in a total of six sites and two soil types per site. Thus, each population represented a particular combination of site and soil. Site, soil and their interaction were included as fixed effects in the models. The effect size of site of origin, soil of origin and the site x soil interaction was calculated as 100*factor sum of square/total sum of squares.

The common garden studies were designed as completely randomized block experiments. Thus, block was included as an additional random factor. The adaptation treatments (stone cover, clipping) were included as fixed factor for measurements taken after treatment start. In some cases log10(x+1) transformation was required to comply with assumptions of linear models (normality, homoscedasticity).

In the germination experiments, final germination percentage in plugs (population differentiation study) and in boxes (soil moisture test) was calculated as response variable.

Germination percentage was arcsin (sqrt(x/100)) transformed prior to analysis [START_REF] Quinn | Experimental Design and Data Analysis for Biologists[END_REF]. Since position of plugs was completely randomized before transplantation to the field, no block effect was considered (germination in the population differentiation study).

The soil moisture test on germination was analyzed in a split-plot model. The box effect was included as a random factor nested in moisture level. Accordingly, moisture was tested against box whereas soil, site and all interactions were tested against the model error.

A Tukey HSD posthoc test was calculated to analyze differences between treatment levels more in detail if the treatment main effect or interactions were significant. Posthoc tests were preferred over a priori linear contrasts because we had no clear a priori hypothesis on specific adaptation to tested environmental factors. All statistical analyses were run in R (R, version 3.3.1, R Development Core Team ( 2013)).

RESULTS

Population differentiation under common conditions

A high variation in germination capacity was found between populations ranging from 10%

(Ca, red Mediterranean soil) to 75% (Ni, calcareous soil, (Fig. 2). Site and soil of origin as well as their interaction had a significant influence on germination (Table 2). Populations from calcareous soils and from the westernmost sites germinated significantly better than those from red Mediterranean soils and eastern parts of the study area, respectively. Differences between soil types depended on site of origin, resulting in a significant interaction.

Population differentiation in plant growth was smaller than in germination but still significant (Table 2, Fig. 2). Differentiation in growth traits did not decrease during the growing season (Appendix S2). Site of origin contributed more to the differentiation than soil of origin. Effect size of site of origin was between 3% of total variation for leaf length and 14% for leaf number whereas effect size of soil of origin was always lower than 1% (Fig. 3). The site x soil interaction explained 1% and 7.7 % of the model variation. In agreement with the germination results, the westernmost populations from Ni and Mon showed a higher performance in early (leaf length, cumulative leaf length) and late growth traits (tiller length and number, cumulative tiller length, tussock diameter). The soil type effect was only significant for leaf length being higher in populations from red Mediterranean soils than in those from calcareous soils. In several traits, in particular in leaf length, tiller length and plant diameter, a significant soil x site of origin interaction was detected. For the westernmost sites of origin, growth was higher in populations from red Mediterranean soils whereas no differences or even a higher performance on calcareous soils were found for populations from the eastern and central parts of the study area. Figure 3: Percentage of variation explained by collection site, soil type within collection site and site x soil interaction for growth traits (May).

Adaptation to differences in soil conditions, management and soil moisture

Stone cover around the seedlings significantly increased plant height, tiller number and thus cumulative tiller length but not cumulative leaf length (Table 2, Fig. 4). The effect of stone cover was the same for plants from the two soil types, while stone cover had contrasting effects on leaf number and cumulative leaf length as well as on tiller number and cumulative tiller length of plants from the different sites. The populations from SR including the SM population showed a consistent positive response to stone cover whereas the response of the Me populations was negative or neutral. Simulated grazing (clipping) significantly reduced plant height, leaf number, tiller number, cumulative tiller length, cumulative leaf length, and inflorescence number (Table 3, Fig. 5).

The negative effect on tiller number was the result of cutting underneath lateral branches that were also counted as tillers. While site x treatment interactions were not significant, several significant soil x treatment interactions were detected. Populations from red Mediterranean soils showed significantly higher compensatory growth resulting in smaller cumulative tiller length, and inflorescences number between clipped and non-clipped plants. The test of soil moisture effects on germination in different B. retusum populations revealed high germination rates even under dry conditions (0.3% volumetric water content) and a significant decrease of germination under maximum soil moisture (at saturation level: 19.5%, Fig. 6). The highest germination was obtained for intermediate soil moisture levels. With 50% to 80% on average, germination rates were higher than in peat plugs used for the germination test on population differentiation. In agreement with the peat plug germination test, site and soil type effects were significant with a higher germination rate in populations from calcareous soils and from the westernmost sites, respectively (Fig. 6).

The interaction between soil type of origin and soil moisture was not significant and the one between site and soil moisture was only marginally significant suggesting similar adaptation to dry conditions. The marginally significant interaction was explained by a stronger negative response of the Ni populations to drought stress compared with populations from the three other sites.

DISCUSSION

Population differentiation under common conditions

Our common garden study demonstrated genetic differentiation among populations at a regional scale. We found significant differences between populations for germination capacity and several morphological traits such as leaf length, tiller number and height whereas variation Site of origin (climatic differences) better explained this differentiation than soil type.

Independent of soil type, the two eastern populations showed significantly lower germination rates and growth than western populations. The significant effect of site is likely the result of climatic differences between eastern and western parts of the study area although our design does not allow to distinguish random genetic drift from climatic differences as drivers of differentiation. The sites of the western populations (Ni, Mon) are closer to the Cévennes Mountains and receive more rainfall than those of the Eastern populations [START_REF] Ceresetti | Space-time structure of heavy rainfall events: application to the Cévennes-Vivarais region[END_REF].

Climate is known to be a major driver of population differentiation and adaptation [START_REF] Linhart | Evolutionary Significance of Local Genetic Differentiation in Plants[END_REF][START_REF] Etterson | Evolutionary potential of chamaecrista fasciculata in relation to climate change. i. clinal patterns of selection along an environmental gradient in the great plains[END_REF][START_REF] Ravenscroft | Intraspecific functional differentiation suggests local adaptation to long-term climate change in a calcareous grassland[END_REF] Water availability and temperature have a particularly strong effect on plant fitness leading to population differentiation in the case of spatial variation [START_REF] Shah | Interaction of drought and high temperature on photosynthesis and grain-filling of wheat[END_REF][START_REF] Xu | Combined effects of water stress and high temperature on photosynthesis, nitrogen metabolism and lipid peroxidation of a perennial grass Leymus chinensis[END_REF]. [START_REF] Yuan | Environmental conditions and genetic differentiation: what drives the divergence of coexisting Leymus chinensis ecotypes in a large-scale longitudinal gradient?[END_REF] found for example that population differentiation in the grass species Leymus chinensis was driven by precipitation, temperature and soil water content. A mainly soil moisture driven differentiation was also shown for Boechera stricta [START_REF] Lee | Quantifying Effects of Environmental and Geographical Factors on Patterns of Genetic Differentiation[END_REF]. However, the authors also found that isolation by distance due to stochastic processes such as genetic drift considerably contribute to population differentiation. Adaptive differentiation and isolation-by-distance effects often act at the same time on genetic structure of populations and their contribution to differentiation may be difficult to distinguish [START_REF] Michalski | Assessment of provenance delineation by genetic differentiation patterns and estimates of gene flow in the common grassland plant Geranium pratense[END_REF][START_REF] Durka | Genetic differentiation within multiple common grassland plants supports seed transfer zones for ecological restoration[END_REF].

Nevertheless, shoot and leaf growth (number and length) were lower in populations from dryer Eastern sites rather selecting for growth forms with smaller and less numerous leaves and shoots that are better protected against drought stress [START_REF] Liancourt | Competition and a short growing season lead to ecotypic differentiation at the two extremes of the ecological range[END_REF][START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF]. The significant site effects on the cited growth traits were stable throughout the whole growing season and did not decrease with time suggesting a minor influence of environmental maternal effects on our results. Environmental maternal effects may contribute to population differentiation but particularly affect early traits and vanish during plant development [START_REF] Roach | Maternal Effects in Plants[END_REF][START_REF] Donohue | Completing the cycle: maternal effects as the missing link in plant life histories[END_REF].

Soil type is also known to select for population differentiation in plants [START_REF] Linhart | Evolutionary Significance of Local Genetic Differentiation in Plants[END_REF]Raabova et al., 2011). In our study, we detected a significant soil type of origin effect on differentiation at a small scale of 10 to 20 km but only for germination capacity and leaf length.

Germination is under stronger control of environmental maternal effects than growth traits affecting population differentiation in such early traits [START_REF] Roach | Maternal Effects in Plants[END_REF][START_REF] Bischoff | Testing population differentiation in plant specieshow important are environmental maternal effects[END_REF]. However, it is also possible that selection for clonal growth under higher grazing pressure of red Mediterranean soils has reduced allocation of resources to seeds compared with less grazed calcareous sites. Although providing some evidence for soil of origin effects, our results do not confirm earlier studies showing a high small-scale differentiation along strong environmental gradients [START_REF] Lenssen | Local adaptation of the clonal plant Ranunculus reptans to flooding along a small-scale gradient[END_REF][START_REF] Bischoff | Detecting local adaptation in widespread grassland speciesthe importance of scale and local plant community[END_REF][START_REF] Raabová | Ecological rather than geographic or genetic distance affects local adaptation of the rare perennial herb, Aster amellus[END_REF][START_REF] Bischoff | Scales and drivers of local adaptation in Brassica nigra (Brassicaceae) populations[END_REF]. Since no specific barriers prevented pollen dispersal at this scale, gene flow may have counteracted local adaptation to soil type [START_REF] Kawecki | Conceptual issues in local adaptation[END_REF].

Adaptation to differences in soil conditions, management and soil moisture

Brachypodium retusum populations showed a differential response to stone cover and clipping, and to a lesser degree to soil moisture in the germination phase.

Our study demonstrated a clearly positive effect of stone cover on the growth of B. retusum, in particular on plant height, tiller number and plant diameter. Stone cover increases soil moisture by shading and water condensation and protects lower stem parts and roots from strong solar radiation and heat [START_REF] Peters | Nurse rocks are more important than nurse plants in determining the distribution and establishment of globose cacti (Mammillaria) in the Tehuacán Valley, Mexico[END_REF][START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. A differential response of Aizoaceae species demonstrated that plants might be locally adapted to type and density of stone cover [START_REF] Ellis | Coexistence and differentiation of 'flowering stones': the role of local adaptation to soil microenvironment[END_REF]. In our study, however, the differential response was not related to the dominant soil types but to site of origin. Although the texture and depth of calcareous soils and red Mediterranean soils are different, limestone layers and pieces close to the soil surface may have similar effects on microclimate. Thus, the differential response to stone cover cannot be related to differences to local environmental conditions at collection sites.

As expected, clipping largely reduced plant height, tiller number and leaf number throughout the whole season resulting in a highly reduced reproduction. The effect was less strong in populations from red Mediterranean soils than in populations from calcareous sites suggesting an adaptation to higher grazing intensity. Grazing exerts strong directional selection and may result in genetic differentiation and local adaptation to long-term differences in grazing intensity [START_REF] Linhart | Evolutionary Significance of Local Genetic Differentiation in Plants[END_REF][START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF]. Higher compensatory growth may be one important adaptation of grasses to higher grazing intensity [START_REF] Sarmiento | Adaptive strategies of perennial grasses in South American savannas[END_REF].

Although we do not exactly know whether our collection sites show such long-term differences, red Mediterranean soils were more grazed in the past than calcareous soils because slopes are less steep and rocky, and soils are more fertile both favoring sheep grazing [START_REF] Verheye | Mediterranean soils. Land Use[END_REF].

Drought stress reduces germination and usually plant species show a positive correlation between soil moisture and germination rates [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF][START_REF] Thomas | The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape[END_REF]. The ability to germinate fast under moderate to low soil moisture may be crucial for species colonizing dry environments [START_REF] Schütz | Germination requirements and seedling responses to water availability and soil type in four eucalypt species[END_REF][START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF]. All tested B. retusum populations showed a high germination capacity even under the lowest soil moisture level (-0.18 MPa=0.3 % volumetric water content), but a considerable drop under saturated conditions.

The observed germination pattern differs from the results of [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF] who found a strong decrease in B. retusum germination already at water potentials of -0.01 and -0.05 MPa.

Although the results may depend on age of seeds and specific experimental conditions this difference suggests genetic variability in germination response to soil moisture. Environmental maternal effects may contribute to population differentiation in germination but there is little evidence for a contribution to adaptive responses through transgenerational plasticity [START_REF] Galloway | Transgenerational Plasticity Is Adaptive in the Wild[END_REF]Etterson 2007, Bischoff and[START_REF] Bischoff | Testing population differentiation in plant specieshow important are environmental maternal effects[END_REF]. In our study, the response to soil water potential was not much different among populations. Only the population of one of the two sites with the most humid climate (Ni) showed a stronger decline under dry conditions suggesting weak adaptation to climate-mediated soil moisture.

CONCLUSIONS

Significant population differentiation was found at a regional scale of 30 to 200 km.

Differentiation between major soil types representing strong environmental differences were found at even smaller scales (10-20 km). However, differentiation between soil types was weaker than differentiation among sites suggesting a major influence of climate and/or genetic drift on population differentiation. In Chapter III, we analyse in situ growth and reproduction in adult B. retusum populations to evaluate environmental factors limiting the availability of propagules in the study area.

Although decreased in importance due to fire protection measures, wildfires and pastoral fires used by shepherd to improve nutritional value have shaped plant populations and communities.

Grazing is required to maintain species composition of the ecosystem by preventing shrub encroachment. However, grazing also represents a major stress limiting growth and reproduction. We hypothesize that B. retusum benefits from fire and grazing and that sexual reproduction increases after burning. The study focuses on B. retusum but also include the surrounding plant community and its biodiversity.

Contribution of the thesis:

-Knowledge on grazing and fire as major factors influencing vegetative growth and reproduction compared to the associated plant community.

-Effect of fire and grazing on the associated plant community.

-Recommendations for conservation management and ecological restoration.

INTRODUCTION

Mediterranean dry grasslands are semi-natural habitats that depend on human land use such as grazing and related fire management shaping plant populations and communities [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF][START_REF] Bond | Fire and plants[END_REF]. Both increased land use intensity, in particular transformation to arable use, and land abandonment followed by shrub encroachment are threatening these highly diverse ecosystems [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. As in other European grassland ecosystems, diversity is linked to traditional extensive land use [START_REF] Poschlod | The historical and socioeconomic perspective of calcareous grasslands-lessons from the distant and recent past[END_REF][START_REF] Pywell | Enhancing diversity of species-poor grasslands: an experimental assessment of multiple constraints[END_REF][START_REF] Gazol | Landscapeand small-scale determinants of grassland species diversity: direct and indirect influences[END_REF]. Sheep grazing is one of the key land use factors driving plant species composition and diversity (reviewed by [START_REF] Cingolani | Grazing Effects on Rangeland Diversity: A Synthesis of Contemporary Models[END_REF]. Wildfires and fire management by shepherds in the Mediterranean region were also important drivers until the industrial revolution but their role in maintaining the ecosystem is nowadays less clear because fire suppression and abandonment of traditional pastoral fires have largely reduced their impact in the last decades [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF][START_REF] Parr | Patch Mosaic Burning for Biodiversity Conservation: a Critique of the Pyrodiversity Paradigm[END_REF]).

Many plant species and communities are adapted to fire and depend on specific fire regime [START_REF] Bond | Fire and plants[END_REF]. Changes in such disturbance regime often lead to shifts in plant community composition [START_REF] Bond | Fire as a global 'herbivore': the ecology and evolution of flammable ecosystems[END_REF]. In Mediterranean "Matorral" (shrublands), an increase in fire frequency favors seeder shrubs and resprouter grasses such as Brachypodium retusum [START_REF] Vilà-Cabrera | Effects of fire frequency on species composition in a Mediterranean shrubland[END_REF]. Accordingly, fire suppression may change community structure and diversity of such ecosystems (Bond et al. 2005). At species level, plants with different adaptive strategies to survive fires have been selected over centuries [START_REF] Buhk | The challenge of plant regeneration after fire in the Mediterranean Basin: Scientific gaps in our knowledge on plant strategies and evolution of traits[END_REF]). Resprouting from protected tissues and/or from undamaged below-ground organs are common strategies but also increased seed production, fire-resistant seeds, fire-stimulated germination or flowering [START_REF] Clarke | Resprouting as a key functional trait: how buds, protection and resources drive persistence after fire[END_REF][START_REF] Lamont | Fire-Proneness as a Prerequisite for the Evolution of Fire-Adapted Traits[END_REF][START_REF] Pyke | Fire-Stimulated Flowering: A Review and Look to the Future[END_REF]. For example, some fynbos and chaparral geophyte species only start flowering after fire and disperse seeds on bare soil to benefit from reduced competition and higher nutrient availability [START_REF] Keeley | Smoke-induced flowering in the fire-lily Cyrtanthus ventricosus[END_REF][START_REF] Tyler | Reproduction and growth of the chaparral geophyte, Zigadenus fremontii (Liliaceae), in relation to fire[END_REF].

As fire, grazing favors resprouting either from belowground organs or from the stem base. This may be combined with other avoidance strategies such as small growth, early germination and flowering, and a short flowering period [START_REF] Landsberg | Grazing response groups among understorey plants in arid rangelands[END_REF][START_REF] Díaz | Can grazing response of herbaceous plants be predicted from simple vegetative traits?[END_REF]. In grasslands, seedling recruitment is often quite low because grazing and dense grassland swards may hamper seed production, germination and seedling recruitment [START_REF] Drobnik | Adaptation of plant functional group composition to management changes in calcareous grassland[END_REF]). However, Mediterranean steppe vegetation is a quite open grassland type and the grazing period is short allowing the survival of many annual species [START_REF] Briske | Plant responses to grazing: a comparative evaluation of annual and perennial grasses[END_REF]. Similar to fire, grazing increases mineralization of organic matter and nutrient availability during the seedling recruitment period [START_REF] Bond | Fire as a global 'herbivore': the ecology and evolution of flammable ecosystems[END_REF]. Although several studies have shown that grazing is needed to maintain the unique species composition of Mediterranean steppe communities, the consequences of short-term and long-term grazing abandonment are not precisely known [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]. There is also a lack in studies analyzing the interaction of fire and grazing in Mediterranean grasslands. Annual fire may increase the productivity of ungrazed sites but not of recently grazed sites [START_REF] Buis | Controls of Aboveground Net Primary Production in Mesic Savanna Grasslands: An Inter-Hemispheric Comparison[END_REF]). The fire-by-grazing interaction was also found to depend on the annuals-perennials ratio of plant communities (Noy-Meir 1995).

In our study, we analyzed fire and grazing effects in a Mediterranean steppe (Thero-Brachypodietea) dominated by the resprouter grass Brachypodium retusum. This steppe community is under decline and protected by the EU habitats directive (San Miguel 2008). B. restusum is still widespread in the Mediterranean but also suffers from the loss of this habitat.

In ecological restoration, B. retusum shows poor re-establishment (Coiffait-Gombault et al. 2012;[START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF] and it is obvious that restoration remains incomplete if the dominant species of pristine steppes is lacking. Thus, a better knowledge on the ecology of the species and effects of major management factors is needed to better conserve this plant community. Previous studies have demonstrated that B. retusum benefits from fire [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF][START_REF] Cassagne | Using a fire propagation model to assess the efficiency of prescribed burning in reducing the fire hazard[END_REF]. Wildfires particularly occur in undergrazed or abandoned areas but also prescribed burning was used in the past to improve forage quality [START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF][START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. The dense below-ground rhizome network of B. retusum allows a fast post-fire regrowth [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]). Fire may increase reproductive stalk number [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF] but it is unknown whether fire also promotes seed set and viability. We further do not know whether fire season influences population and community dynamics of Mediterranean steppes and their dominant species. Wildfires mainly occur during the dry and hot summer period whereas shepherds burned grasslands at the end of winter (pastoral fires) to remove standing litter and favor resprouting [START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF]). Both fire types may also promote annual species by providing favorable conditions for autumn and spring germination.

Similarly, grazing was found to have a positive effect on the growth of B. retusum (Coiffait-Gombault et al. 2012) and on the diversity of its associated plant community [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]). However, grazing may reduce sexual reproduction due to vegetative biomass and/or inflorescence removal [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. We were particularly interested in combined grazing and fire effects on the dominant species Brachypodium retusum and on its associated plant community. We hypothesize that both fire and grazing favor B. retusum relative to other species of the plant community. We further hypothesize that other resprouting species but also annual species benefit from fire and grazing increasing biodiversity and changing plant species composition. Our last hypothesis is that fire effects are stronger in ungrazed situations and that prescribed burning may be an efficient strategy to restore steppe communities in undergrazed or abandoned grasslands. We set up two experiments to specifically analyze the following research questions:

(1) What is the effect of fire and grazing on the vegetative growth and sexual reproduction of B. retusum compared with co-occurring species cover of the Mediterranean steppe?

(2) How do fire and grazing affect plant species composition and diversity?

(3) Do fire effects depend on season and can fire help to maintain diversity of grazed or abandoned grasslands?

MATERIALS AND METHODS

Study area

The study was set up in the "Crau" Mediterranean grassland ("steppe", EUNIS habitat E1.311, Natura 2000 habitat type 6220) in South-Eastern France. The study region is characterized by hot/dry summers and mild/humid winters with an annual precipitation of 517 mm and a mean temperature of 15.3 °C (Meteo-France, meteorological station of Saint-Martin-de-Crau, Appendix S1). Summer drought is common but the summer 2016 was even drier than average (55 mm instead of 100 mm on average). The soils of the study area are decalcified Red Mediterranean Soils (Haplic cambisol) with a mean pH of 6.8. The vegetation has been shaped by a long history of grazing back to the Neolithic [START_REF] Henry | The age and history of the French Mediterranean steppe revisited by soil wood charcoal analysis[END_REF] and by former winter fires [START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF][START_REF] Chalvet | La vulnérabilité de la forêt provençale face aux incendies : naissance d'une notion (fin XIXe siècle)[END_REF]). The steppe is still annually grazed by itinerant sheep during spring.

Due to risks for inhabitants and land users, winter fires are no longer used as a management strategy since the end of the 19th century [START_REF] Chalvet | La vulnérabilité de la forêt provençale face aux incendies : naissance d'une notion (fin XIXe siècle)[END_REF]). However, wildfires still occur accidentally during summer, in particular close to roads and field tracks. No fire was observed at the study site since 1973 (Prométhée fire database).

The grassland is species-rich, dominated by annual plants and the perennial grass Brachypodium retusum (Buisson & Dutoit 2006, Appendix S2). In long-term exclosures (since 2001), 40% of initially occurring grassland species disappeared and have been replaced by highgrowing herbaceous ruderal species [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]).

The study species, Brachypodium retusum (Pers.) P. Beauv., (ramose false brome, Poaceae), is a perennial grass species. Its rhizomes form a dense network close to the soil surface allowing resprouting after destruction of aboveground organs by fire or grazing [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF]. The species shows tillering at the stem base but also lateral branching at upper stem parts. Clonal growth is common but slow. B. retusum usually flowers from April to July but grazed or cut plants may also flower later in the season. At grazed sites, the species is highly dominant.

Long-term grazing exclosure results in an increase of B. retusum height and the development of other competitive grass perennial species such as Dactylis glomerata [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF].

Design

Two experiments testing the effect of late-winter fires (= winter fires) simulating pastoral fires, summer wildfires (= summer fires) and sheep grazing were set up in March 2016 and run for two seasons. The winter fire treatment was applied once in March 2016 and the summer fire treatment once in July 2016. In the first experiment, fire treatments were applied to the traditionally grazed steppe (Appendix S3, A). In the second experiment, fire treatments were applied to long-term exclosures (abandoned grassland, Appendix S3, B).

Effect of fire and grazing in a traditionally managed steppe area

The experiment was set up at two different sites comprising three blocks each (Negreiron 43°32'18.28"N / 4°49'0.75"E, Collongue 43°31'37.55"N / 4°49'55.82"E) to reflect heterogeneity in grazing but the site effect was finally pooled into block for statistical analysis in order to increase parsimony.

Blocks were split into two parts (half-blocks) of which one was fenced to prevent grazing. The three fire treatments (summer fire, winter fire, no fire) were applied to three plots of 4 m² within each of the twelve half-blocks (split-plot design) in order to test combined effects of fire and grazing (36 plots in total). The distance between plots within half-blocks was 1 m, between half-blocks within blocks 50 m and between blocks at least 200 m. The position of fire by total vegetation cover to obtain weighted cover. The cover of May 2016 and 2017 was finally used for analyses representing maximum vegetative development before aboveground organs partially die due to summer drought. The regeneration of B. retusum was measured by comparing May 2017 and October 2016 cover. In order to evaluate sexual reproduction, the number of inflorescences was counted in each plot during flowering peak in July 2016 and 2017. At the same time, twenty inflorescences per plot were harvested to count seeds. If number of inflorescences per plot was lower than twenty all inflorescences were sampled. Seeds were prepared by crushing the inflorescences in a plastic tube with glass beads using a vortex, then spreading the separated spikelets on an overhead projector in order to check whether spikelets are filled. Empty seeds or spikelets were removed from seed counts. Seeds were stored at room temperature and germinated in peat plugs (4 x 4 cm). Four seeds of each experimental plot (block x treatment combination) were sown to one plug. The plug trays were placed in a growth chamber under optimum germination conditions (temperature 15/20 °C, 12/12h, night/day, [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]) and watered regularly. Coleoptile emergence was used as a proxy of germination and was recorded every 2-3 days for seven weeks.

Vegetation survey

Species composition of the associated plant community was analyzed in May 2016 and May 2017 during the peak of flowering. The cover of all vascular plant species was estimated using a modified Braun-Blanquet scale (Braun-Blanquet 1932: + = one individual per plot, 1 < 5% cover, 2: 5 -25 %, 3: 26 -50 %, 4: 51 -75 %, 5:> 75%). Contrary to the B. retusum survey, the sum of individual species cover may be higher than 100%.

Data analysis

In the combined fire and grazing experiment, a split-plot ANOVA was performed to analyze fire treatment and grazing effects on individual response variables (individual species cover, B. retusum inflorescence, seed number and germination rate, species richness, diversity indices).

Grazing (whole-plot factor) was tested against the block x grazing interaction. Fire (split-plot factor), the fire x grazing interaction and block were tested against the model residuals. In the long-term exclosure experiment, a reduced model was applied only including the fire treatment and block. When necessary log10(x+1) and square root transformations were used to comply with assumptions of linear models (normality, homoscedasticity). A Tukey HSD posthoc test was calculated to analyze differences between treatment levels if treatment main effects or interactions were significant.

In order to analyze plant community data, Braun-Blanquet scores were transformed into percentage cover: + = 0.5%; 1 = 2.5%; 2 =15.0%; 3 = 37.5%; 4 = 62.5%; 5 = 87.5%. Species richness (S), evenness (J') and Bray-Curtis index (BC) were calculated using vegan v2.4-2 package. J' evenness was calculated as H'/ln(S), with H' being the Shannon diversity index [START_REF] Pielou | An introduction to mathematical ecology[END_REF]. Species composition was compared using NMDS (Non-Metric Multidimensional Scaling) based on the similarity index of Bray-Curtis [START_REF] Borcard | Unconstrained Ordination[END_REF] in order illustrate changes in plant species composition as well as the species most correlated with each treatment. NMDS analyses were run using 30 random starting configurations in 1 to 15 dimensions. The run with the lowest stress value was finally applied. A permutation multivariate analysis of variance (PERMANOVA, Anderson 2001) with 9999 permutations was used to test whether the community composition was significantly different between the fire and grazing treatments. A pairwise posthoc test with Bonferroni adjustment was calculated to analyze differences between levels of the fire treatment. All statistical analyses were run in R (R, version 3.3.1, R Development Core Team ( 2013)) including multivariate analyses using the vegan package.

RESULTS

B. retusum survey

In the combined fire and grazing experiment, fire significantly reduced B. retusum cover in the following two years (Tab. 1, Appendix S5). Several months after fire treatments (October 2016), even cover relative to neighboring plants (weighted cover) was lower (Tab.1).

Accordingly, inflorescence and seed production were significantly reduced but a higher germination rate was found in seeds produced in winter fire plots compared with control plots (Tab.1). In the second post-fire year (2017), the fire main effect on weighted cover of B. retusum was not significant any more (Fig. 1A, Tab.1). B. retusum showed a rapid regeneration of its vegetative cover in the burned plots (Fig. 1C, Tab.1) compared with control plots in which its cover decreased from 2016 to 2017. At the end of the survey period in July 2017, inflorescence number was higher in burned plots but differences were only significant between summer fire treatment and control (Fig. 2A, Tab.1). We also found a significantly higher seed number per inflorescence in summer and winter fire plots (Fig. 2C, Tab.1) whereas germination rate was no longer different between seeds from the different treatments.

The effect of grazing was not significant for any of these variables (Tab. 1). Only a significant fire x grazing interaction occurred in 2017 for B. retusum cover. Grazing reduced absolute and weighted cover in unburned controls.

In the long-term exclosures, fire effects were similar to those in the combined grazing and fire experiment (Tab. 1). Cover (weighted and non-weighted), inflorescence number and seed number per inflorescence decreased in the beginning of the first year, but recovered rapidly in the second post-fire year (Tab. 1, Fig. 1B,1D). Although absolute B. retusum cover was still lower in 2017, relative (weighted) cover was not different any more to the control whereas inflorescence number was even higher in the summer fire treatment compared with the unburned control (marginally significant). Due to the low seed set in all treatments, differences in seed number per inflorescence were not significant.

Plant community

In the combined fire and grazing experiment, fire significantly reduced species richness three months after treatments in 2016 but one year later, in 2017, species richness was significantly higher in burned plots than in unburned controls (Tab. 2, Fig. 4). The second year differences in species richness were significant between summer fire and control plots whereas winter fire species richness was not different from the other treatments. In both years, evenness and Bray Curtis dissimilarity were significantly higher in the fire treatments. Regardless of the year, grazing effect and the fire x grazing interaction were not significant.

In the long-term exclosures, species richness was not significantly affected three months after fire treatment (May 2016) but in the second year, a significant increase was measured between summer fire and control. As in the first experiment, evenness (marginally) and Bray-Curtis dissimilarity were significantly higher in burned plots compared to the controls. However, the fire effect on Bray-Curtis dissimilarity was not significant any more in the second post-fire year. In 2017, one year after fire treatments, NMDS ordination showed a clear separation between the plant community of the combined fire and grazing experiment in the steppe area and the fire experiment in long-term exclosures (Fig. 4). Along axis 1 of the NMDS, B. retusum and ruderal species such as Sonchus oleraceus, Avena sterilis and Avena barbata characterize the vegetation of the long-term exclosures whereas short annual and stress-tolerant species such as

Polycarpon tetraphyllum, Evax pygmea, Euphorbia falcata, Galium murale are more abundant in the traditionally managed steppe. In the long-term exclosure, fire changed the plant community more than in the steppe (Fig. 4). The burned long-term exclosures were closer to the traditionally managed steppe than the unburned controls. The PERMANOVA testing differences in species composition between treatments showed a significant effect of fire in the combined fire and grazing experiment (F2,35=2.865*) and in long-term exclosures (marginally significant, F2,17=1.78 ( * ) ). In the combined fire and grazing experiment, the effect of summer fire was stronger than that of winter fire whereas no such differences occurred in the long-term exclosures (Appendix S6). The effect of grazing and the grazing x fire interaction were not significant (grazing: F1,35=1.512 NS , fire × grazing: F2,35=0.212 NS ).

In both experiments, the cover of annual species such as Medicago monspeliaca, Euphorbia falcata, and Bromus rubens as well as the perennial grass Poa bulbosa particularly benefitted from fire treatments (Appendix S7). In particular, summer fire had a strong positive effect on the cover of M. monspeliaca and B. rubens whereas E. falcata and P. bulbosa also benefitted from winter fire. The grazing effect was not significant for these species. Chapter III

DISCUSSION

In the combined fire and grazing experiment, the fire effect was much stronger than the grazing effect on both the performance of the dominant species B. retusum and plant species composition. In our case, grazing reduced aboveground biomass by 40% but did not significantly influence the majority of measured B. retusum traits and community parameters.

Fire had a similar effect in the combined fire and grazing experiment at traditionally managed sites and in the long-term exclosures allowing a fast recovery of typical steppe species such as

B. retusum.
Effects of fire and grazing on B. retusum B. retusum can survive fire by resprouting from rhizomes favored by axillary buds [START_REF] Clarke | Resprouting as a key functional trait: how buds, protection and resources drive persistence after fire[END_REF]. Adapted plant species such as rhizomatous resprouters may even benefit, since fire increases resource availability [START_REF] Neary | Fire effects on belowground sustainability: a review and synthesis[END_REF], stimulates re-growth from below-ground buds (Fidelis et al. 2014) and opens the canopy reducing competition for light, water and nutrients [START_REF] Everson | Factors Affecting the Timing of Grassland Regrowth after Fire in the Montane Grasslands of Natal[END_REF]. In our study, B. retusum cover increased rapidly after fire whereas controls showed a decrease in cover due to the summer drought in 2016. In particular, reduced competition for water resulting from biomass removal was likely the reason for the better post-fire regeneration [START_REF] Everson | Factors Affecting the Timing of Grassland Regrowth after Fire in the Montane Grasslands of Natal[END_REF]. However, weighted cover of Brachypodium retusum relative to total vegetation cover was in the beginning significantly lower than in fire treatments indicating that post-fire regeneration was slower than in other species of the plant community. Thus, our results do not confirm studies in Eastern Spain showing a faster recovery of B. retusum compared to accompanying species resulting in a rapid increase of weighted B. retusum cover [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF].

Regeneration was faster in the summer than in the winter fire treatment suggesting that B.

retusum is better adapted to natural summer fire. The period of fire management by shepherd may not have been long enough for an evolutionary response whereas summer fire disturbance is much older allowing adaptive processes. The stronger summer fire effect on B. retusum regeneration may also be the result of a higher standing biomass and a lower water content (dry leaves and stems) corresponding to higher fire temperatures and biomass loss compared with winter fire [START_REF] Hobbs | Fire and Grazing in the Tallgrass Prairie: Contingent Effects on Nitrogen Budgets[END_REF][START_REF] Trabaud | Etude du comportement du feu dans la garrigue de chêne kermès à partir des températures et des vitesses de propagation[END_REF]). Nevertheless, the traditional winter fire management is already well-known to improve pasture quality and vigor by allowing the regeneration of B.

retusum aboveground biomass [START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF]).

In the long-term exclosures, fire effects on B. retusum cover were similar to those of the combined grazing and fire experiment in the traditionally managed steppe. However, the species reached cover values of the unburned control already in the second post-fire year indicating a faster recovery. Long-term exclosure from grazing results in a strong biomass increase mainly due to the growth of perennial grass species [START_REF] Bond | Beyond the forest edge: Ecology, diversity and conservation of the grassy biomes[END_REF]. Abandonment of grazing also increases litter biomass, C:N ratios and decreases soil nutrient content [START_REF] Peco | Effects of grazing abandonment on functional and taxonomic diversity of Mediterranean grasslands[END_REF][START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]). Thus, fire temperatures are higher and therefore fire has a stronger effect on plant species than in grazed systems with lower living and dead biomass [START_REF] Hobbs | Fire and Grazing in the Tallgrass Prairie: Contingent Effects on Nitrogen Budgets[END_REF]. The high recovery of B. retusum in our exclosures suggests that wildfire or prescribed burning may help to maintain the typical species composition and community structure in long-term absence of grazing.

In addition to vegetative resprouting, fire increased allocation of resources to reproductive organs in the second post-fire year favoring seed production and quality. The production of inflorescences was significantly higher in both fire treatments, but in particular after summer fire. Fire-stimulated flowering is already known for several species in the Mediterranean regions of Australasia and South Africa with peak flowering occurring five to eighteen months after fire [START_REF] Lamont | Fire-stimulated flowering among resprouters and geophytes in Australia and South Africa[END_REF][START_REF] Pyke | Fire-Stimulated Flowering: A Review and Look to the Future[END_REF]. Flowering may be triggered by fire-specific cues such as chemical compounds of smoke and ash or scorching of below-ground storage organs [START_REF] Keeley | Smoke-induced flowering in the fire-lily Cyrtanthus ventricosus[END_REF][START_REF] Tyler | Reproduction and growth of the chaparral geophyte, Zigadenus fremontii (Liliaceae), in relation to fire[END_REF] but also by a fire-induced increase in light and temperature [START_REF] Fidelis | Does fire induce flowering in Brazilian subtropical grasslands?[END_REF]. As resprouting, inflorescence production may be favored by a higher resource availability due to reduced competition and higher nutrient or water availability (Coates & Duncan 2009).

Fire also increased the seed set measured as seed production per inflorescence in the second post-fire year whereas high fire damage to photosynthetically active tissues (leaves, stems) reduced seed set in the first year. A higher post-fire seed set in the following years was also measured for two Australian perennials [START_REF] Lamont | Fire May Stimulate Flowering, Branching, Seed Production and Seedling Establishment in Two Kangaroo Paws (Haemodoraceae)[END_REF]. The effect on ex-situ germination was less strong being only positive in the same year of the fire treatments. In the second season, when the increase in inflorescence production and seed set occurred, differences in germination were not significant any more. Germination is an important trait under selection for adaptation to fire [START_REF] Keeley | Fire in Mediterranean Ecosystems: Ecology, Evolution and Management[END_REF]. Many species from a wide range of taxa show a rapid and increased germination after fire [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF][START_REF] González-Rabanal | Effects of high temperatures, ash and seed position in the inflorescence on the germination of three Spanish grasses[END_REF]. However, B.

retusum seeds were produced after fire excluding a direct exposure to fire cues. Our germination experiment was set up in a growth chamber not taking into account modified conditions in burned plots (nutrient availability, destruction of organic matter, presence of ashes, etc.).

Moreover, in some species of fire-prone ecosystems, there is no evidence that fire triggers germination (Le [START_REF] Stradic | Diversity of germination strategies and seed dormancy in herbaceous species of campo rupestre grasslands[END_REF].

The grazing main effect on vegetative growth and on reproduction was not significant neither in the first nor in the second season. Although statistical power to test grazing as the whole-plot factor of the split-plot design was lower than for fire as split-plot factor the small differences between grazing treatment means confirm that grazing impact is much less strong than fire impact. B. retusum tolerates the short-term absence of grazing even though grazing is an important factor in maintaining population cover (Coiffait-Gombault et al. 2012). However, a significant fire x grazing interaction was measured during the second season with plant cover being lower only in unburned plots. The result confirmed our hypothesis that fire impact is stronger in ungrazed situations. Higher fire temperatures due to higher litter biomass already cited for abandoned grasslands [START_REF] Peco | Effects of grazing abandonment on functional and taxonomic diversity of Mediterranean grasslands[END_REF][START_REF] Hobbs | Fire and Grazing in the Tallgrass Prairie: Contingent Effects on Nitrogen Budgets[END_REF]) may have increased the impact of fire in ungrazed control plots. However, this significant interaction was also visible in winter fire plots that were burned before grazing. This effect could be explained by the behavior of sheep avoiding recently burned B. retusum plants, thus increasing the positive effect of fire by reducing post-fire biomass consumption.

Effects of fire and grazing on the plant community

In the combined fire and grazing experiment, fire reduced species richness three months after treatments but in the second year, the fire effect was already strongly positive. Fire also increased evenness by removing biomass of the dominant B. retusum and community dissimilarity. Infrequent and/or irregular fire disturbance may result in species loss and community composition changes [START_REF] Laurance | Slow burn: the insidious effects of surface fires on tropical forests[END_REF]. However, in fire-prone ecosystems, such disturbance maintains species richness and community structure (Bond et al. 2005[START_REF] Fidelis | Short-term changes caused by fire and mowing in Brazilian Campos grasslands with different longterm fire histories[END_REF]. Accordingly, fire suppression may considerably reduce plant diversity of fire-prone systems [START_REF] Sparks | Effects of late growing-season and late dormant-season prescribed fire on herbaceous vegetation in restored pine-grassland communities[END_REF][START_REF] Leach | Ecological Determinants of Species Loss in Remnant Prairies[END_REF].

Again, the effect of summer fire was stronger than that of winter fire. Fire season was found to affect plant communities. Fire during the growing season may, for example, increase plant diversity, in particular the number of annuals and cool-season perennials whereas no such effect was observed for dormant season (fall, spring) fire [START_REF] Biondini | Seasonal fire effects on the diversity patterns, spatial distribution and community structure of forbs in the Northern Mixed Prairie[END_REF][START_REF] Howe | Response of Early-and Late-Flowering Plants to Fire Season in Experimental Prairies[END_REF]. The growing season of B. retusum and most other species of the community starts in late winter and lasts until July corresponding to the two fire dates. The stronger summer fire effect may be the result of a higher standing biomass and a lower water content (dry leaves and stems) corresponding to higher fire temperatures and biomass loss [START_REF] Hobbs | Fire and Grazing in the Tallgrass Prairie: Contingent Effects on Nitrogen Budgets[END_REF][START_REF] Trabaud | Etude du comportement du feu dans la garrigue de chêne kermès à partir des températures et des vitesses de propagation[END_REF]).

The fire effect on long-term exclosures was similar to that on traditionally managed steppe plots. Fire generally increased diversity in the second season. Contrary to the traditionally managed steppe, there was no decrease in species richness confirming the particularly beneficial effect of fire in abandoned grassland for conserving species-rich dry grasslands [START_REF] Bond | Beyond the forest edge: Ecology, diversity and conservation of the grassy biomes[END_REF]. Plant species composition of burned long-term exclosures was closer to the traditionally managed steppe than that of the unburned control.

In both experiments and systems, differences in species composition were mainly explained by an increase in native annual seeder species whereas fire reduced the cover of resprouter grasses in the first post-fire year. Similar to the results on the dominant species B. retusum, fire-induced community changes do not correspond to those of earlier studies in Spanish B. retusum grasslands [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]). The La Crau steppe is a more open and less nutrient-rich grassland type than the Spanish field sites allowing the development of a higher diversity of annual species (Buisson & Dutoit 2006). Apart from resprouting, seedling recruitment is therefore a second successful strategy of post-fire regeneration as it is already known for other dry and nutrient-poor grasslands [START_REF] Valone | Fire and grazing in a shrub-invaded arid grassland community: independent or interactive ecological effects?[END_REF]. Although the short study period does not allow conclusions on the duration of facilitative effects, we were able to demonstrate that fire helps annual species to develop. Further research is needed to identify the optimum frequency of fire disturbance to maintain such positive effects on plant diversity.

Grazing was expected to have positive but less strong effects on plant diversity of the steppe community. Such positive effects are mediated by sward opening, biomass reduction and soil surface disturbance (Coiffait-Gombault et al. 2012;[START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]). In our study, the effects of grazing and the interaction between fire and grazing were not significant. Again, the small differences between treatment means confirmed that grazing has a much smaller effect than fire in spite of lower statistical power for testing whole-plot effects. The hypothesis that fire effects are stronger in ungrazed situation was not supported at community level. As already demonstrated for the dominant species B. retusum, the plant community tolerated the shortterm absence of grazing without species loss or differences in diversity indices. These findings confirmed results of a previous study in the same system showing only small changes in the first five years after grazing abandonment [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF].

CONCLUSIONS

We showed a concomitant positive effect of fire on the reproduction of the dominant grass B.

retusum and the species diversity of its associated plant community. Contrary to our expectations, fire did not favor perennial resprouters such as B. retusum over annuals confirming that both strategies resprouting from below-ground organs and seedling recruitment are successful in short term post-fire vegetation recovery. Short-term grazing exclusion had only small effects on B. retusum and associated plant community. However, the long-term exclosures abandoned 17 years before our experiments showed large differences to the traditionally managed steppe such as a lower B. retusum reproduction and a lower plant diversity. In such abandoned or undergrazed systems, fire has a particularly positive effect on B. retusum performance and plant diversity and may thus compensate for long-term grazing absence to conserve this unique Mediterranean grassland ecosystem.

Petrorhagia prolifera (L.) P.W.Ball & Heywood Phleum pratense L. ssp nodosum (L.) Dumort.

Plantago bellardii All.

Plantago coronopus L.

Plantago lagopus L.

Plantago lanceolata L.

Poa annua L.

Poa bulbosa L.

Polycarpon tetraphyllum (L.) L.

Psilurus incurvus (Gouan) Schinz & Thell.

Ranunculus paludosus Poir.

Reichardia picroides (L.) Roth

Rostraria cristata (L.) Tzvelev

Ruta montana (L.) L.

Sagina apetala Ard.

Salvia verbenaca L.

Sanguisorba minor Scop.

Scabiosa atropurpurea L.

Schedonorus arundinaceus (Schreb.) Dumort.

Scorzonera laciniata Jacq.

Senecio vulgaris L.

Seseli tortuosum L.

Sherardia arvensis L.

Sideritis romana L.

Silene gallica L.

Silene nocturna L.

Silene otites (L.) Wibel

Sonchus asper (L.) Hill

Sonchus oleraceus (L.) L.

Stipa capillata L.

Taeniatherum caput-medusae (L.) Nevski Teucrium chamaedrys L.

Teucrium polium L.

Thymus vulgaris L.

Tolpis barbata (L.) Gaertn. ssp umbellata (Bertol.) Jahand. & Maire Torilis nodosa (L.) Gaertn.

INTRODUCTION

European semi-natural grasslands are under constant decline and belong to the most threatened habitats today [START_REF] Poschlod | The historical and socioeconomic perspective of calcareous grasslands-lessons from the distant and recent past[END_REF][START_REF] Pywell | Enhancing diversity of species-poor grasslands: an experimental assessment of multiple constraints[END_REF]. Grasslands have been frequently converted to arable fields because improved drainage and irrigation techniques allow nowadays crop production at sites previously limited to extensive grassland use [START_REF] Bischoff | Succession of floodplain grasslands following reduction in land use intensity: the importance of environmental conditions, management and dispersal[END_REF][START_REF] Tatin | Écologie et conservation d'une steppe méditerranéenne: La plaine de Crau[END_REF]. Under

Mediterranean climate, strong summer drought limits standing biomass, and the combination of grazing and climate has resulted in particularly species-rich grasslands also called pseudosteppes [START_REF] Blondel | The Mediterranean Region: Biological Diversity in Space and Time[END_REF][START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. Ecological restoration of Mediterranean grasslands based on spontaneous succession is a slow process. In particular, characteristic species that are limited to this type of grasslands slowly establish or even remain absent [START_REF] Blondel | Biology and Wildlife of the Mediterranean Region[END_REF]Coiffait-Gombault, Buisson, & Dutoit, 2012;[START_REF] Römermann | Influence of former cultivation on the unique Mediterranean steppe of France and consequences for conservation management[END_REF].

Water availability is well-known to be a limiting factor of plant establishment [START_REF] Noy-Meir | Desert Ecosystems: Environment and Producers[END_REF][START_REF] Ronnenberg | Contrasting effects of precipitation and fertilization on seed viability and production of Stipa krylovii in Mongolia[END_REF]. Drought stress reduces germination and increases seedling mortality [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF][START_REF] Thomas | The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape[END_REF]. Our test species Brachypodium retusum showed relative high germination under drought but still germination increased with higher soil moisture [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. However, an increase in water availability also increases competition for light and other resources [START_REF] Kadmon | Plant Competition along Soil Moisture Gradients: A Field Experiment with the Desert Annual Stipa Capensis[END_REF]. In our study area, higher soil moisture favoured undesired ruderal species such as the commonly high-growing grass Brachypodium phoenicoides that outcompete typical pseudo-steppe species [START_REF] Masson | Impacts de différents régimes de perturbations et niveaux de ressource hydrique pour contrôler une espèce proliférante dans un écosystème pseudo-steppique: le cas de Rubus ulmifolius Schott. dans la plaine de la Crau (Bouches-du-Rhône, France)[END_REF].

Mediterranean dry grasslands depend on human land management, in particular on grazing (San [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF][START_REF] Apostolova | Dry Grasslands of Southern Europe: Syntaxonomy, Management and Conservation[END_REF]. Land abandonment results in shrub encroachment which is apart from land use intensification the major threat to this type of ecosystem [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. Grazing was expected to have positive but less strong effects on plant diversity of the steppe community. Grazing animals remove biomass reducing above-ground competition and create gaps by trampling both shaping plant communities and favouring the co-existence of different species [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF]Coiffait-Gombault et al., 2012;[START_REF] Drobnik | Adaptation of plant functional group composition to management changes in calcareous grassland[END_REF]. Thus, grazing may have positive effects on seedling establishment [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF]. However, grazing is also known to increase seedling mortality hampering establishment [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. Although showing a relatively low palatability, young seedlings of our study species B. retusum are negatively affected by grazing [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF][START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF].

Brachypodium retusum is the dominant perennial grass species of dry grasslands in the Western Mediterranean basin but also occurs in shrublands (matorral, garrigue) and open woodlands [START_REF] Catalán | Phylogeny and Evolution of the Genus Brachypodium[END_REF][START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF]. The species is still abundant in the Mediterranean but B. retusum grasslands (Thero-Brachypodietea) are under decline and protected by the EU habitats directive [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. Due to its high cover and numerous interactions with other species of the community [START_REF] Saiz | Structure and spatial self-organization of semi-arid communities through plant-plant co-occurrence networks[END_REF], successful restoration of plant community structure of such grasslands depends on the re-establishment of B. retusum. In our study area, the "La Crau" nature reserve in South-Eastern France, B. retusum was not able to re-colonise restoration sites within thirty years although pristine dry grasslands with a high population density of the species occur adjacent to these sites (Coiffait-Gombault et al., 2012). Apart from low seed production [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF] and dispersal limitation [START_REF] Bischoff | Dispersal and re-establishment of Silaum silaus (L.) in floodplain grassland[END_REF], conditions of establishment after arrival of seeds may represent a strong environmental filter [START_REF] Nuttle | Evaluation of Restoration Practice Based on Environmental Filters[END_REF][START_REF] O'connor | Local Extinction in Perennial Grasslands: A Life-History Approach[END_REF][START_REF] Pywell | Enhancing diversity of species-poor grasslands: an experimental assessment of multiple constraints[END_REF]. We hypothesize that water availability and grazing are key factors limiting seedling establishment. We further hypothesize that initial watering has a positive effect whereas initial grazing has a negative effect on seedling recruitment. We were particularly interested in the interaction of both factors since the joint effect of water and grazing stress on seedling establishment has rarely been tested so far [START_REF] Ibáñez | Positive and negative interactions between environmental conditions affecting Cercocarpus ledifolius seedling survival[END_REF][START_REF] Soliveres | Temporal dynamics of herbivory and water availability interactively modulate the outcome of a grass-shrub interaction in a semi-arid ecosystem[END_REF].

We present results of a full-factorial field experiment manipulating water availability and grazing on an ex-arable field converted to traditional dry grassland management in 1985 [START_REF] Römermann | Influence of former cultivation on the unique Mediterranean steppe of France and consequences for conservation management[END_REF][START_REF] Buisson | The implications of seed rain and seed bank patterns for plant succession at the edges of abandoned fields in Mediterranean landscapes[END_REF]. Since germination and initial seedling growth are particularly critical steps in plant establishment [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF][START_REF] Thomas | The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape[END_REF], we tested effects on seedlings grown from fieldsown seeds and on seedlings pre-grown under greenhouse conditions. We specifically asked the following questions: (1) What is the effect of initial watering and grazing on B. retusum field germination? (2) What is the effect of these factors on survival and growth of sown and transplanted B. retusum seedlings? (3) Does the effect of watering depend on grazing?

MATERIALS AND METHODS

Study area

The study site is part the of "La Crau" Nature reserve in South-Eastern France. The area is characterized by hot/dry summers and mild/humid winters with an annual precipitation of 517 mm (1980-2010) and a mean temperature of 15.3 °C (Figure S1). The soils of the study area are decalcified Red Mediterranean Soils (Haplic cambisol) with a mean pH of 6.8. The sites are grazed by itinerant sheep from February to June.

The experiment was set up on a restoration site in "Peau de Meau" (43°34'09N / 4°49'55E;

Figure S2). The site was managed as an arable field between 1960 and 1985 to grow melon and cereal crops (Buisson et al., 2006). Afterwards, the site has been traditionally grazed as the surrounding pristine grasslands. These surrounding reference sites belong to the Mediterranean Thero-Brachypodietea habitat "Pseudo-steppe with grasses and annuals" (EUNIS habitat E1.311, Natura 2000 habitat type 6220, San [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. Although management was the same as on reference site B. retusum has not re-occurred on the restoration site since 1985.

Brachypodium retusum (Pers.) P. Beauv., (ramose false brome, Poaceae), is the dominant species of this habitat in our study area. The rhizomes of this perennial grass form a dense network close to the soil surface allowing resprouting after destruction of above-ground organs [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF]. The species shows tillering at the stem base but also lateral branching at upper stem parts. Clonal growth is common but slow. B. retusum usually flowers from April to July and seeds germinate after autumn rainfalls or during early spring when humidity is sufficient [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF].

Experimental design

In autumn 2016, we set up a split-plot experiment testing the effects of grazing, initial watering and its interaction on germination rate, survival, seedling height, leaf length, tiller number, leaf number and seedling diameter for (i) transplanted seedlings and (ii) sown seedlings (Figure 1).

Blocks were split into two parts (half-blocks) of which one was fenced to prevent sheep grazing. The two watering treatments (watering, no watering), were applied to two plots of 9 m² within each halfblock (split-plot design) in order to test combined effects of watering and grazing (20 plots in total). A distance of 40 m between half-blocks within blocks was chosen to avoid any influence of exclosures on grazed plots since grazing is often much stronger close to exclosures (Saatkamp, and that of grazing treatment was randomized within blocks. Local seeds collected in the previous year were used in the experiment. In each plot, 18 seedlings previously germinated and grown for 2 months in peat plugs (4 cm x 4 cm) in a growth chamber (temperature 15/20 °C, 12/12h, night/day) were transplanted in October 2016. At the same time, 20 seeds were sown to each of 18 pockets between transplanted seedlings.

Transplanted seedlings and one emerging seedling per pocket were marked for continuous survival and growth measurements.

In autumn 2016 (late October to early December) and in spring 2017 (early April to early July), plots of the initial watering treatment were irrigated every seven to fourteen days in order to add 60 l/m² of water per month. Thus irrigation doubled the average monthly precipitation during this period. The irrigation treatment was interrupted in winter and early spring since soils were water saturated. The grazing exclosures was realised using fences with stretched, nonelectrified nets (1 m high).

Data collection

Field final germination was measured by counting the number of emerging coleoptiles in May 2017. Growth and survival were measured at the end of autumn, after the summer drought and the autumnal rainy season to be sure to identify the living individuals from the dead ones (December 2016 and 2017), during the grazing period (May 2017 and2018) and after the end of the grazing period (July 2017). The growth measurements included plant height, diameter, tiller number, leaf number and the length of the longest leaf to characterise plant performance [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF].

Data analysis

Linear models were run to analyse the effects of watering (yes/no), grazing (yes/no) and planting type (field sown/greenhouse sown). The full model included the three factors, all twoway interactions, the three-way interaction watering x grazing x planting type and block. In order to consider the split-plot design of the experiment grazing was tested against the grazing x block interaction. Watering and the watering x grazing interaction were tested against the grazing x watering x block interaction. Finally, planting type, block and all interactions with planting type were tested against the model residuals. A repeated measures analysis based on this model was calculated for survival since more data points were available than for growth traits. A reduced model only including grazing, watering, their interaction and block was applied to germination data. As in the full model grazing was tested against the grazing x block interaction. Watering, the watering x grazing interaction and block were, however, tested against the model residuals. When necessary log10(x+1) and square root transformations were used for count and proportion data, respectively, to comply with assumptions of linear models (normality, homoscedasticity). A Tukey HSD posthoc test was calculated to analyze differences between treatment levels if treatment main effects or interactions were significant.

For growth traits, the effect size of grazing, watering, planting type, and of their interactions was calculated as 100* sum of square/total sum of squares. All statistical analyses were run in R (R, version 3.3.1, R Development Core Team (2013)). Sown seedlings showed significantly lower survival rates than planted seedlings, an effect that became stronger during the study period (Table A.1, Fig. 3). Watering had no significant effect on survival rates whereas grazing had a significantly negative effect. A significant grazing x watering interaction indicated that the watering effect depended on grazing. A positive effect of watering was only apparent in grazed plots. The significant grazing x watering x date interaction showed that this grazing-dependent watering effect also changed with time (Table A.1). In December 2017 and May 2018, watering had a significantly negative effect on survival in ungrazed plots. The watering effect was not significant at the end of the study period (Table 1, Fig. 1). However, watering significantly increased tiller number, cumulative tiller length and plant diameter in at least two of the previous three observation dates (Table 1; Table A .2). Grazing significantly reduced leaf length, plant height, cumulative leaf length and cumulative tiller length (Table 1, Fig. 4). Nevertheless, it increased significantly tiller number and plant diameter (Table S1, Fig. 4). However, a high recovery was measured after the grazing period 2017 resulting in nonsignificant grazing effects on all measured variables in December 2017 (Table A.2). Significant interactions (watering x grazing and watering x grazing x seedling type) were only found at the earliest dates in May and July 2017 and disappeared later on. Thus the growth measurements do not confirm the grazing-dependent increase in survival rates

Chapter IV At the end of the study period, in May 2018, only a seedling type and grazing factors were significant (Table 1, Fig. 4). Transplanted seedling still showed a higher growth than sown seedlings except for leaf number. B. retusum for grazing plots show lower leaf length, plant height, cumulative leaf length and cumulative tiller length (Table 1, Fig. 4).

Effect size of watering was always lower than that of grazing (Figure 6). In July 2017, watering explained between 0.004 % for leaf length and 1.43 % for plant diameter. Grazing explained from 0.73 % for cumulative tiller length to 21.78% for tiller number. The site x soil interaction explained 0.02 % for leaf number and 2.13 % of the model variation for cumulative leaf length.

In May 2018, watering explained between 0.001 % for plant height and 1.43 % for cumulative leaf length. Grazing explained from 4.23 % for plant diameter to 40.85 % for plant height. The site x soil interaction explained 0.04 % for plant diameter and 6.94 % of the model variation for tiller number.

DISCUSSION

Field germination

In our study, watering had no significant effect on the germination of Brachypodium retusum.

The result confirmed a previous growth chamber experiment showing that already at low to moderate moisture levels the response to water addition is not significant [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF].

However, it does not correspond to other growth chamber studies in dry or Mediterranean ecosystems showing a very sensitive germination response to soil moisture [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF][START_REF] Thomas | The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape[END_REF]. Additionally, pre-emergence mortality increases with drought resulting in a stronger drought effect on seedling emergence than on germination (Tobe et al. 2005). Our field germination data correspond to seedling emergence since we did not analyse germination in the soil. The typical Mediterranean autumn and early-spring rainfall may have been sufficient

to avoid seedling mortality in the soil explaining the absence of watering effects on field germination in our study.

Grazing had a negative effect on field germination. Gap creation by trampling livestock increases field germination [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF]) but trampling, urine and faeces excretion may have the opposite effect [START_REF] Bilotta | The Impacts of Grazing Animals on the Quality of Soils, Vegetation, and Surface Waters in Intensively Managed Grasslands[END_REF]. Trampling is known to damage seeds or emerging coleoptiles [START_REF] James | Demographic processes limiting seedling recruitment in arid grassland restoration[END_REF][START_REF] Rother | Demographic bottlenecks in tropical plant regeneration: A comparative analysis of causal influences[END_REF]. Dung pads may reduce light required by many seeds to germinate [START_REF] Milotić | Reduced germination success of temperate grassland seeds sown in dung: consequences for post-dispersal seed fate[END_REF]. Furthermore, grazing sheep may have damaged seedlings before recording germination in our study.

Survival and growth

The main watering effect on seedling survival was not significant neither in field sown seedlings nor in seedling pregrown in a greenhouse confirming that the species is well adapted to drought [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. Brachypodium retusum is able to store water in rhizomes and respond fast to rainfall [START_REF] Clary | Water relations in competitive interactions of Mediterranean grasses and shrubs[END_REF]. However, the watering effect depended on grazing and the watering × grazing interaction was also significantly affected by observation date. Watering had a positive effect in grazed but not in ungrazed plots. This effect was stronger during and after the grazing season representing a particularly stressful period for seedlings.

Watering allowed a higher resilience to grazing whereas watering may increase competition to ruderal species when not stressed by grazing. In similar Mediterranean dry grasslands, higher soil moisture favoured ruderal species such as the high-growing grass Brachypodium phoenicoides outcompeting typical pseudo-steppe species [START_REF] Masson | Impacts de différents régimes de perturbations et niveaux de ressource hydrique pour contrôler une espèce proliférante dans un écosystème pseudo-steppique: le cas de Rubus ulmifolius Schott. dans la plaine de la Crau (Bouches-du-Rhône, France)[END_REF]. Water addition to ungrazed plots may also increase soil pathogen infestation reducing plant performance and influencing interspecific competition [START_REF] Van Der Putten | How Soil-Borne Pathogens May Affect Plant Competition[END_REF][START_REF] Van Der Putten | Linking above-and belowground multitrophic interactions of plants, herbivores, pathogens, and their antagonists[END_REF].

Contrary to germination and survival, watering treatment had a significantly positive effect on growth and the effect varied among seasons. In ecosystems with seasonal aridity, water is a limiting factor for growth [START_REF] Noy-Meir | Desert Ecosystems: Environment and Producers[END_REF]) mainly due to a trade-off between drought tolerance and growth capacity [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF].

Very young B. retusum plants allocate resources to tillering and leaf growth. Later, tillers elongate and older leaves die [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. Watering increased leaf growth and tillering in the beginning and tiller growth up to seven months after treatment stop.

Grazing had a negative effect on B. retusum survival, in particular in the beginning of the study period. Grazing is known to increase early seedling mortality hampering establishment of the species [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. Although palatability is low compared with dicots and several other grasses, young seedlings are still attractive to sheep [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF] and play an important role in sheep nutrition (Meuret et al. 2013). Apart from biomass removal by grazing sheep, trampling, urine and faeces excretion may also reduce survival as already detailed in the germination section [START_REF] Bilotta | The Impacts of Grazing Animals on the Quality of Soils, Vegetation, and Surface Waters in Intensively Managed Grasslands[END_REF]. Negative grazing effects on survival disappeared six months after the end of the grazing season confirming a high resilience of B.

retusum to above-ground disturbance [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]. Additionally, negative grazing effects were mitigated by watering resulting in a significant grazing x watering interaction as discussed above.

Grazing significantly reduced leaf growth and plant height but increased tillerering and plant diameter after the first gazing period. These significantly positive grazing effects disappeared at the end of the study period. Reduced growth of defoliated grasses caused by the loss of photosynthetic active tissues has been widely observed [START_REF] Falconer | Effect of defoliation on grass growth. A quantitative review[END_REF][START_REF] Van Staalduinen | Differences in the compensatory growth of two co-occurring grass species in relation to water availability[END_REF]. However, different compensatory growth mechanisms may limit the damage such as stimulating photosynthesis, removing old and dead tissues, changed resource allocation and tiller production [START_REF] Mcnaughton | Compensatory Plant Growth as a Response to Herbivory[END_REF][START_REF] Oesterheld | Intraspecific variation in the response of Themeda triandra to defoliation: the effect of time of recovery and growth rates on compensatory growth[END_REF][START_REF] Andersen | Dispersal distance as a benefit of myrmecochory[END_REF][START_REF] Zhao | Compensatory growth responses to clipping defoliation in Leymus chinensis (Poaceae) under nutrient addition and water deficiency conditions[END_REF]. Six months after the end of the first grazing period, grazing effects were not significant any more confirming again a high resilience to above ground perturbations [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF].

As expected, seedlings pre-grown in a greenhouse established better than field-germinated seedlings as it was demonstrated by other studies [START_REF] Godefroid | How successful are plant species reintroductions?[END_REF][START_REF] Menges | Restoration demography and genetics of plants: when is a translocation successful?[END_REF].

Greenhouse grown seedlings showed already a well-developed root and rhizome network that was transplanted in peat plugs. Rhizome establishment in B. retusum is a key factor in resistance and tolerance to environmental stress [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF]. A lower mortality of established seedling is, however, also apparent in other species resulting in a mortality decrease with size and age of seedlings [START_REF] Gilbert | Effects of seedling size, El Niño drought, seedling density, and distance to nearest conspecific adult on 6-year survival of Ocotea whitei seedlings in Panamá[END_REF][START_REF] Quintana-Ascencio | Sapling survival and growth of coniferous and broad-leaved trees in successional highland habitats in Mexico[END_REF]. Although significant, the magnitude of the seedling type effect on survival was relatively low (10%) thus not strongly advocating expensive pre-growth and transplanting. Differences in growth were much higher than differences in survival. In particular, plant height and diameter were greater in pre-grown seedlings even two seasons after planting. The large differences in tiller number and cumulative tiller length in the beginning became, however, much smaller ate the end of the experiment suggesting a catch-up in these growth traits. The response of pre-grown and field-germinated seeds to the water and grazing treatments was similar resulting in non-significant interactions.

CONCLUSIONS

Initial watering had a positive effect on early seedling establishment but not on germination indicating that in Mediterranean dry grasslands, the second step of seedling recruitment is limited by water availability. However, the watering effect disappeared after two growing seasons except for a significantly higher survival in grazed and watered plots. Grazing had a generally negative effect on field germination and seedling establishment during the first two years. In ecological restoration, initial fencing (at least two years) increases thus germination, survival and growth of introduced populations [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. Fencing may also be sufficient to avoid water limitation and the need to irrigate grazed plots. Alternatively, watering may help to mitigate grazing stress if fencing is not possible. Seedlings pre-grown under optimum conditions established better than field-sown seedlings. However, the effect magnitude was relatively low for seedling survival, and seedling recruitment of field-sown seed was higher than expected from previous studies showing a very low re-colonisation of restored sites with adjacent source populations (Coiffait-Gombault 2012, Helm et al. in press). Further observation is required to evaluate whether a long-term fitness advantage justifies the higher costs of pre-growth and planting compared with simple sowing.

Chapter synthesis_Transition to the general discussion

The thesis has focused on intrinsic, extrinsic factors and their interaction limiting the reestablishment of the common Mediterranean species Brachypodium retusum. In Chapter I, we showed that B. retusum Western Mediterranean populations exhibit a high neutral genetic diversity despite asexual reproduction indicating a high gene flow between populations. French populations showed a significant differentiation due to conjoint action of drift and selective adaptation related to mean July temperature and frost day number. Differentiation was also shown at local scale between populations originating from different soil types but its magnitude was much smaller than at regional scale between populations from the same soil type. In

Chapter II, we focused on quantitative traits and confirmed this finding on the magnitude of local soil and regional climate/drift effects on differentiation. We further found differential population responses to stone cover and grazing manipulated in common garden experiments and to a much lesser degree to soil moisture tested in a growth chamber. In Chapter III, we Gene flow is a random process that can constrain evolution by preventing adaptation to local conditions but it may also increase evolutionary potential by spreading new genes [START_REF] Kawecki | Conceptual issues in local adaptation[END_REF]. Its effects depend on the distance between populations, breeding system, pollination agents, seed dispersal mechanisms and physical boundaries. Previous studies have shown that gene flow is common in perennial outcrossing species [START_REF] Hamrick | Effects of life history traits on genetic diversity in plant species[END_REF]. In

Chapter I, we found that gene flow is quite high and limit population differentiation in the Western Mediterranean basin.

common under near-natural conditions (e.g. [START_REF] Endler | Natural selection in the wild[END_REF][START_REF] Kingsolver | The strength of phenotypic selection in natural populations[END_REF]) and most traits appear to be moderately or highly heritable (e.g. [START_REF] Mousseau | Natural selection and the heritability of fitness components[END_REF][START_REF] El-Lithy | Quantitative Trait Locus Analysis of Growth-Related Traits in a New Arabidopsis Recombinant Inbred Population[END_REF].

b. Main drivers of adaptive adaptation

Genetic variation within and among populations is strongly affected by ecological factors [START_REF] Nevo | Natural selection of allozyme polymorphisms: a microgeographic climatic differentiation in wild emmer wheat (Triticum dicoccoides)[END_REF][START_REF] Macel | Climate Vs. Soil Factors in Local Adaptation of Two Common Plant Species[END_REF][START_REF] Manel | Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation[END_REF]. Climatic and edaphic conditions are the main causes for differences among populations and can result in small-scale differentiation as reported for many grass species [START_REF] Hamrick | Microgeographical Variation in Allozyme Frequencies in Avena barbata[END_REF][START_REF] Nevo | Microgeographic edaphic differentiation in allozyme polymorphisms of wild barley (Hordeum spontaneum, Poaceae)[END_REF][START_REF] Gutterman | Temperatures and Ecological -Genetic Differentiation Affecting the Germination of Hordeum Spontaneum Caryopses Harvested from Three Populations: The Negev Desert and Opposing Slopes on Mediterranean Mount Carmel[END_REF][START_REF] Hsiao | Population genetic structure of Yushania niitakayamensis (Bambusoideae, Poaceae) in Taiwan[END_REF][START_REF] Owuor | Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum[END_REF]. In Chapters I and II, we showed that adaptive differentiation is more related to regional-scale climatic conditions than to local-scale soil type. Mean July temperature and winter frost frequency were identified as major climatic drivers of differentiation. The significant effect of site was likely the result of climatic differences between eastern and western parts of the study area. The sites of the western populations (Ni, Mon) are closer to the Cévennes Mountains and receive more rainfall than those of the Eastern populations [START_REF] Ceresetti | Space-time structure of heavy rainfall events: application to the Cévennes-Vivarais region[END_REF]. In Chapter II, we specified which phenotypic traits are the most differentiated and tested environmental factors to which populations may be adapted. We found significant differences between populations for germination capacity and several morphological traits such as leaf length, tiller number and height whereas variation in leaf number per tiller was low. In particular, germination and tiller number are closely linked to seedling recruitment and establishment supporting our hypothesis that different colonisation patterns observed at different Mediterranean sites (high: [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]low: Coiffait-Gombault et al. 2012a) may result from genetic differentiation.

Soil type within sampling sites explained a significant part of variation among populations in neutral markers and in vegetative phenotypic traits but explained variation is much smaller between soils within sites than between sites suggesting that soil was not a major driver of genetic differentiation. Small-scale adaptive differentiation to soil conditions has been found

for grasses to heavy metal soil contamination [START_REF] Rozema | Ecophysiological adaptations of coastal halophytes from foredunes and salt marshes[END_REF][START_REF] Ernst | Population differentiation in grassland vegetation[END_REF]. However, at a small scale, gene flow may be stronger than selective pressure and thus counteract selection [START_REF] Kawecki | Conceptual issues in local adaptation[END_REF][START_REF] Leimu | A Meta-Analysis of Local Adaptation in Plants[END_REF][START_REF] Hereford | A Quantitative Survey of Local Adaptation and Fitness Trade-Offs[END_REF]). Gene flow is high for epizoochorous species [START_REF] Fischer | Experimental Studies on the Dispersal of Plants and Animals on Sheep in Calcareous Grasslands[END_REF]. Southern French population were connected in the past due to transhumance involving spring grazing in the lowlands and summer grazing in the mountains. This is particularly well documented for the exchange between our focal study site, the Crau plain, and the French Alps lasting from Roman period to 1962 (date of the last pedestrian transhumance, [START_REF] Gardelle | La transhumance ovine entre les régions méditerranéennes et les Alpes en 1964[END_REF][START_REF] Badan | Les bergeries romaines de la Crau d'Arles. Les origines de la transhumance en Provence[END_REF][START_REF] Congés | La fortune éphémère de Glanum : du religieux à l'économique (à propos d'un article récent)[END_REF]). However, the grazing period of our Mediterranean B. retusum collection sites is early spring which is far before the seed production and maturation period limiting the effect of epizoochorous dispersal by sheep.

We further were able to demonstrate in Chapter II that Brachypodium retusum populations respond differently to stone cover and clipping -whereas the differential germination response to soil moisture was relatively small. Stone cover had a generally positive effect on the growth of B. retusum, in particular on plant height, tiller number and plant diameter probably related to the more favourable microclimate. Stones were found to favour growth and survival of B.

retusum because rhizomes and roots can grow underneath and thus benefitting from higher soil moisture during the Mediterranean summer [START_REF] Lahav | Soil bacterial functional diversity in a potato field[END_REF][START_REF] Peters | Nurse rocks are more important than nurse plants in determining the distribution and establishment of globose cacti (Mammillaria) in the Tehuacán Valley, Mexico[END_REF][START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. We were not able to relate the differential response to stone cover to major soil type. Thus, populations from Red Mediterranean soils were not specifically adapted to the presence of pebbles and cobbles of such soils used in the experiment. A more detailed analysis of stone density, shape and sites combined with an experiment combining stones from red Mediterranean and calcareous soils may help to better explain the differential response.

Clipping largely reduced plant height, tiller number and leaf number resulting in a highly reduced reproduction. We also found a differential response of populations that could be linked to grazing intensity at collection sites. The grazing effect was less strong in populations from red Mediterranean soils which are under higher grazing intensity than in populations from calcareous sites. Grazing is known to exert strong directional selection and may result in genetic differentiation and local adaptation to long-term differences in grazing intensity [START_REF] Linhart | Evolutionary Significance of Local Genetic Differentiation in Plants[END_REF][START_REF] Hufford | Local Adaptation and the Effects of Grazing on the Performance of Nassella pulchra: Implications for Seed Sourcing in Restoration[END_REF]. Higher compensatory growth may be one important tolerance mechanism as an adaptation of grasses to higher grazing intensity [START_REF] Sarmiento | Adaptive strategies of perennial grasses in South American savannas[END_REF].

Drought stress reduces germination, and usually plant species show a positive correlation between soil moisture and germination rates [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF][START_REF] Thomas | The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape[END_REF][START_REF] Walder | Temperature and drought drive differences in germination responses between congeneric species along altitudinal gradients[END_REF]). The ability to germinate fast under moderate to low soil moisture confer to species an advantage to colonize dry environments [START_REF] Schütz | Germination requirements and seedling responses to water availability and soil type in four eucalypt species[END_REF][START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF].

All tested B. retusum populations showed a high germination capacity even under the lowest soil moisture level but a considerable drop under saturated conditions. The response to variation in soil moisture was similar among populations. Only one population (Ni) showed a strong decline under dry conditions which may be due to the lower drought tolerance of the relatively humid collection site. These results suggest a weak adaptation to climate-mediated soil moisture. A strong decrease in germination under drought was, however, found in another study including B. retusum populations from Spain [START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF]. Although other factors such as seed age and quality or experimental conditions may influence germination results, such a strong difference to our results still suggests genetic differentiation in the response to soil moisture. In my thesis, the high stability of germination under drought was also confirmed in situ (Chapter IV) as the watering treatment did not increase germination.

II.

Plant regeneration, reproduction and early establishmenteffects and interactions with environmental factors [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. Fire is closely related to climate but has specific effects on the vegetation (Bond & Keeley 2005a). In Mediterranean grasslands, characterized by summer drought, fire is common and thus known to be one of the major factors shaping plant populations and communities [START_REF] Naveh | The evolutionary significance of fire in the mediterranean region[END_REF].

a.

Fire does not favour vegetative recovery compared to other dry grassland species but increases sexual reproduction

Fire is an important ecological and evolutionary factor in several worldwide ecosystems such as boreal forests, eucalypt woodlands, shrublands, grasslands and savannahs. Although fire disturbance may reduce species richness in non-adapted systems with low fire frequency [START_REF] Laurance | Slow burn: the insidious effects of surface fires on tropical forests[END_REF], fire effects on plant communities of fire-prone systems are positive (Bond & Keeley 2005b). In fire-prone grasslands, anthropogenic fire suppression has reduced species richness [START_REF] Leach | Ecological Determinants of Species Loss in Remnant Prairies[END_REF]. Fire suppression may even result in a complete biome switch to non-adapted forest and a subsequent loss of animal and plant species [START_REF] Bond | Fire and plants[END_REF].

Fire has an effect on both plant species traits and community but effects are rarely analysed together. The fire impact on species and the associated community is, however, well documented for Pinus species [START_REF] Richardson | Ecology and Biogeography of Pinus[END_REF]. Similarly, we analysed the effect of fire on the population dynamics of one species (vegetative recovery, reproduction of B. retusum) and on the associated plant community.

In Chapter III, we showed a positive effect of winter and in particular of summer fire on B.

retusum on reproduction and to a lesser degree also on regeneration of vegetative cover.

However, vegetative regrowth of the species was not higher than community average and in the beginning, other species showed a faster regeneration. In particular, annual seeder species benefitted from fire, increasing the diversity of the associated community. B. retusum is a resprouter showing increased vegetative growth after injury or a strong change in growing conditions [START_REF] Del Tredici | Sprouting in temperate trees: A morphological and ecological review[END_REF]. Resprouting is a well-known tolerance strategy allowing regeneration after biomass removal [START_REF] Clarke | Resprouting as a key functional trait: how buds, protection and resources drive persistence after fire[END_REF]. In several perennial plants, fire can also trigger flowering [START_REF] Lamont | Fire-stimulated flowering among resprouters and geophytes in Australia and South Africa[END_REF]. For example, in several fynbos and chaparral geophyte species, flowering occurs soon after fire and seeds are dispersed on bare soil providing a fitness advantage over species not responding to fire [START_REF] Keeley | Seed Production, Seed Populations in Soil, and Seedling Production After Fire for Two Congeneric Pairs of Sprouting and Nonsprouting Chaparal Shrubs[END_REF][START_REF] Keeley | Smoke-induced flowering in the fire-lily Cyrtanthus ventricosus[END_REF][START_REF] Borchert | Patterns of post-fire flowering and fruiting in Chlorogalum pomeridianum var. pomeridianum (DC.) Kunth in southern California chaparral[END_REF].

At the community level, species richness and diversity increased. Such positive effects of fire on community structure have also been reported for pine-grassland communities [START_REF] Sparks | Effects of late growing-season and late dormant-season prescribed fire on herbaceous vegetation in restored pine-grassland communities[END_REF]. Fire effects at species and community scales may vary depending on fire regime [START_REF] Bond | Fire and plants[END_REF][START_REF] Davies | The role of fire in UK peatland and moorland management: the need for informed, unbiased debate[END_REF]. Changes in such regimes with specific recurrence and period often result in plant community shifts [START_REF] Bond | Fire as a global 'herbivore': the ecology and evolution of flammable ecosystems[END_REF]. Furthermore, an increase in fire frequency may change the proportions of functional groups. In another Mediterranean grassland, fire increased the abundance of herbaceous species and reduced the abundance of both seeder and resprouter shrubs. Brachypodium retusum was dominant in all fire frequencies [START_REF] Vilà | Positive fire-grass feedback in Mediterranean Basin woodlands[END_REF]).

In our study, fire increased herbaceous annual seeder species and reduced the cover of resprouter grasses in the first post-fire year. This result does not correspond to studies in Spanish B. retusum grasslands showing a faster recovery of resprouter species [START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF]). This difference may be explained by site and soil differences as the Crau steppe is a more open and less nutrient-rich grassland type than the Spanish field sites allowing the development of a higher diversity of annual species (Buisson & Dutoit 2006). Fire may favour annual species through a higher post-fire seedling recruitment which was demonstrated in other dry and nutrient-poor grasslands [START_REF] Valone | Fire and grazing in a shrub-invaded arid grassland community: independent or interactive ecological effects?[END_REF]. However, longer-term studies are necessary to identify the optimum fire regime to maintain such positive effects on plant diversity.

b. Grazing reduces early growth and establishment but is well tolerated in adult plants

Contrary to fire that has decreased in importance, grazing is still a strong driver of population and community dynamics.

In Chapter IV, we showed that grazing had a negative effect on early stages of establishment including reduced germination, survival and growth. However, the effect of grazing varies depending on developmental stage with high compensatory growth of B. retusum after grazing periods. Trampling, urine and faeces excretion may change the soil micro-environment (i.e., soil loss, reduced shade, [START_REF] Bilotta | The Impacts of Grazing Animals on the Quality of Soils, Vegetation, and Surface Waters in Intensively Managed Grasslands[END_REF]) and therefore modify conditions that influence germination, survival and growth. Defoliation and the related loss of photosynthetic have been identified as the most important grazing effect [START_REF] Falconer | Effect of defoliation on grass growth. A quantitative review[END_REF][START_REF] Van Staalduinen | Differences in the compensatory growth of two co-occurring grass species in relation to water availability[END_REF][START_REF] Zhao | Compensatory growth responses to clipping defoliation in Leymus chinensis (Poaceae) under nutrient addition and water deficiency conditions[END_REF]. Compensatory growth following defoliation may compensate for such losses by stimulating photosynthesis, removing old and dead tissue, altering mass allocation, increasing growth rate, and producing more tillers in defoliated plants [START_REF] Mcnaughton | Compensatory Plant Growth as a Response to Herbivory[END_REF][START_REF] Oesterheld | Intraspecific variation in the response of Themeda triandra to defoliation: the effect of time of recovery and growth rates on compensatory growth[END_REF][START_REF] Oesterheld | Intraspecific variation in the response of Themeda triandra to defoliation: the effect of time of recovery and growth rates on compensatory growth[END_REF][START_REF] Zhao | Compensatory growth responses to clipping defoliation in Leymus chinensis (Poaceae) under nutrient addition and water deficiency conditions[END_REF].

In Chapter III, we showed that grazing did not negatively affect adult B. retusum populations and the associated community. However, long-term abandonment of grazing may have negative effects on B. retusum since sexual reproduction decreases and other perennial species may outcompete B. retusum and annual species [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]).

At community level, grazing effects are contrasting including positive, negative and nonsignificant effects on plant diversity [START_REF] Huston | Biological Diversity: The Coexistence of Species[END_REF][START_REF] Proulx | Reversal of Grazing Impact on Plant Species Richness in Nutrient-Poor Vs. Nutrient-Rich Ecosystems[END_REF]. Grazing tends to favour small and prostrate plants over tall erect types and annuals over perennials [START_REF] Noy-Meir | Responses of Mediterranean Grassland Plants to Grazing and Protection[END_REF][START_REF] Mcintyre | Plant functional types and disturbance dynamics -Introduction[END_REF][START_REF] Díaz | Plant trait responses to grazing ? a global synthesis[END_REF]. Grazing favours the co-existence of different species by biomass removal reducing above-ground competition and creating gaps (Bullock et al. 2001;[START_REF] Drobnik | Adaptation of plant functional group composition to management changes in calcareous grassland[END_REF]Coiffait-Gombault et al. 2012a).

c. Initial watering has a positive effect in the beginning largely disappearing after one year

In arid and semi-arid environments such as dry grasslands, water availability is known to be a limiting factor for plant establishment, productivity and performance [START_REF] Noy-Meir | Desert Ecosystems: Environment and Producers[END_REF][START_REF] Bochet | Soil water availability effects on seed germination account for species segregation in semiarid roadslopes[END_REF]. Drought stress has been identified as a key factor limiting seedling recruitment [START_REF] Pywell | Enhancing diversity of species-poor grasslands: an experimental assessment of multiple constraints[END_REF][START_REF] Nuttle | Evaluation of Restoration Practice Based on Environmental Filters[END_REF]. In Chapter II, we showed a high germination even under low moisture. In Chapter IV, initial watering had no effect on seed germination and seedling survival confirming that the species is well adapted to low soil moisture [START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF].

B. retusum rhizome has a high capacity to retain water and respond quicker to water additions after drought than shrub species [START_REF] Clary | Water relations in competitive interactions of Mediterranean grasses and shrubs[END_REF]). The watering effect was only positive (increased survival) in grazed plots suggesting that watering may compensate for additional grazing stress.

Watering increased growth at early stages of establishment but the effect disappeared in the following season. In ecosystems with periodic drought, water is a limiting factor for growth [START_REF] Noy-Meir | Desert Ecosystems: Environment and Producers[END_REF] and there is a trade-off between the capacity to tolerate drought and growth [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF]. Our study showed that initial watering had no long-term effects on growth of surviving plants but the higher survival in traditionally grazed grasslands still demonstrated a long-term positive effect on establishment of B. retusum.

III.

Implications for ecological restoration -plant introduction and population management Fragmentation and reduced population size also need to be considered since they directly affect genetic characteristic structure of populations. Habitat fragmentation may reduce population size and increases spatial isolation between populations. Although populations may respond differently to fragmentation, often a decrease in genetic diversity and an increase in population differentiation is observed due to increased random genetic drift, inbreeding and reduced gene flow [START_REF] Young | The population genetic consequences of habitat fragmentation for plants[END_REF][START_REF] Cheptou | Adaptation to fragmentation: evolutionary dynamics driven by human influences[END_REF]).

b. Fire increases sexual reproduction and has positive effect on plant community species-richness

Fire is already used as a restoration tool in Europe and particularly in North America, South Africa and Australia [START_REF] Heinlein | Development of ecological restoration experiments in fire adapted forests at Grand Canyon National Park[END_REF][START_REF] Holmes | Recovery of South African fynbos vegetation following alien woody plant clearing and fire: implications for restoration[END_REF][START_REF] Carrick | Comparing ecological restoration in South Africa and Western Australia: the benefits of a 'travelling workshop[END_REF]. Several studies showed that restoration of such fire-prone ecosystems needs a specific knowledge of the appropriate frequency and period [START_REF] Bond | Fire and plants[END_REF][START_REF] Bond | Fire as a global 'herbivore': the ecology and evolution of flammable ecosystems[END_REF].

In Mediterranean ecosystems, the use of fire use is more controversial mainly due to the higher human population density. Consequently, there is a knowledge gap on the effect of different fire regimes [START_REF] Stouff | La Crau dans la vie et l'économie d'Arles au bas Moyen Age[END_REF][START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF]. Fire management has been abandoned since more than a century in our study region [START_REF] Stouff | La Crau dans la vie et l'économie d'Arles au bas Moyen Age[END_REF][START_REF] Stouff | Arles à la fin du Moyen-Age[END_REF]). In Chapter III, we demonstrated that B. retusum recovers quickly after fire and that fire even increases sexual reproduction.

Additionally, species-richness and diversity of the associated plant community increase. These results suggest that fire is a key and currently missing ecological factor in preserving and restoring steppe ecosystems. Although B. retusum does not recover faster than other species of the plant community, the increase in seed production helps to increase seedling recruitment in burned areas and thus genetic diversity of the populations usually showing clonal growth. Our results also showed that fire may replace grazing as a conservation measure since the species composition of abandoned grasslands showed a post-fire community shift towards typical steppe communities. Since fire-specific effects on ecosystems are not well known, the use of fire for restoration and conservation should be carefully surveyed. In particular, potential negative effects on organisms of higher trophic levels such as animals and microorganisms need to be considered.

c.

Young seedlings need grazing protection or additional watering whereas adult plants benefit from grazing.

Mediterranean dry grasslands depend on human land management, in particular on grazing [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF][START_REF] Apostolova | Dry Grasslands of Southern Europe: Syntaxonomy, Management and Conservation[END_REF]. In Chapter IV, we showed that grazing reduced survival if plants are not watered in the beginning. Thus grazing should be excluded in the beginning.

Alternatively, irrigation may be used to increase establishment if grazing exclusion is not possible.

Grazing may have positive effects on seedling establishment if competition is strong [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF]. However, grazing is also known to increase seedling mortality hampering establishment in less competitive systems [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. Although showing a relatively low palatability, young seedlings of our study species B. retusum are negatively affected by grazing [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF][START_REF] Vidaller | Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum: The role of soil conditions, grazing, and humidity[END_REF]. In Chapter III, we showed that short-term grazing exclosure is tolerated. However, long-term grazing exclosure results in an increase of more competitive grass species such as Dactylis glomerata and a decrease of B. retusum cover relative to other species [START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]. In conclusion, B. retusum benefits from grazing in the long run. Moreover, grazing may promote the coexistence of different functional groups and favour the annual species, thus increasing species richness [START_REF] Bullock | Restoration of biodiversity enhances agricultural production[END_REF][START_REF] Drobnik | Adaptation of plant functional group composition to management changes in calcareous grassland[END_REF]Coiffait-Gombault et al. 2012a, b). Grazing management needs therefore to be considered in restoration [START_REF] Papanastasis | Restoration of Degraded Grazing Lands through Grazing Management: Can It Work?[END_REF] but it is necessary to determine the responses of different functional plant groups to grazing [START_REF] Hellström | Use of sheep grazing in the restoration of semi-natural meadows in northern Finland[END_REF].

d. B. retusum establishes well from seed

Greenhouse-grown transplants usually provide greater initial success than sown seeds [START_REF] Maschinski | The Continuum of Plant Responses to Herbivory: The Influence of Plant Association, Nutrient Availability, and Timing[END_REF][START_REF] Godefroid | How successful are plant species reintroductions?[END_REF]. Seeds need to survive, germinate and emerge before reaching seedling stage resulting in a lower establishment compared with transplanting of seedlings. Thus, seedling mortality often decreases with time [START_REF] Gilbert | Effects of seedling size, El Niño drought, seedling density, and distance to nearest conspecific adult on 6-year survival of Ocotea whitei seedlings in Panamá[END_REF][START_REF] Quintana-Ascencio | Sapling survival and growth of coniferous and broad-leaved trees in successional highland habitats in Mexico[END_REF]) resulting in a decreasing slope of survivorship curves [START_REF] Li | Seedling demography in undisturbed tropical wet forest in Costa Rica[END_REF].

In Chapter IV, we showed that survival and growth were lower in field-sown seedlings than in transplanted seedlings. However, mean survival was still 38% for sown seedlings at the end of the experiment indicating a successful establishment from seeds. The difference between sown and transplanted seedlings (48 % survival) was significant but small. The differences in growth traits diminished during the study period. Thus, we believe that the small fitness advantage of transplanting pre-grown seedlings to restoration sites does not compensate for higher effort and costs of this method. Tests on the effect of seed density are required to improve information on optimum conditions in the field and to provide practical guidance on seed-number limitation [START_REF] Kirchner | Modelling the dynamics of introduced populations in the narrow-endemic Centaurea corymbosa: a demo-genetic integration[END_REF].

Conclusionsanswers to hypotheses

The AFLP marker analysis demonstrated a relatively low but still significant population differentiation at Western Mediterranean and regional Southern French scale. Genetic diversity is high suggesting a high gene flow among populations. A part of this differentiation was found to be adaptive demonstrated by the comparison of neutral AFLP markers and phenotypic traits. We confirmed the strong and predominantly positive effect of fire on B. retusum populations.

Although vegetative recovery was not faster than community average, the species reached prefire cover already after two seasons and in particular increased seed production. Fire may thus favour refreshment of older populations by resprouting but also by higher seedling recruitment

on the more open post-fire sites. When sites are undergrazed or abandoned, fire may replace grazing as a conservation measure to maintain dry grassland community structure and species richness. In ecological restoration, fire may support natural colonisation from still existing local populations through increased seed production and corresponding higher seed rain at restoration sites.

The effect of grazing was less positive than expected. Young seedlings showed a lower survival and growth at least if grazing stress was not compensated by initial watering. Two years of grazing exclosure did not significantly affect vegetative cover and reproduction of adult populations. However, long-term abandonment seems to have a negative effect on B. retusum since 17-year old exclosures showed a lower relative cover and a lower seed production than traditionally grazed plots. Initial watering has a positive effect on early growth traits whereas differences were not visible any more in the second growing season. However, in grazed plots a positive watering effect on survival was still significant at the end of the study period indicating that watering can compensate for grazing stress. Thus watering may improve seedling recruitment and establishment under strong grazing pressure and drought. If drought stress is not strong during the establishment periodwhich was the case during our experiment one or two-year grazing exclusion may be the more cost-effective strategy to improve establishment success.

Perspectives I. Genetic diversity and clonality

With our sampling design, we tried to guarantee that different individuals do not belong to the same clone. Accordingly, we cannot directly measure clone size and in situ clonal growth. Such information would be complementary in terms of colonisation capacity and genetic diversity of founder populations. In order to obtain more detailed information on clonality, small-scale sampling at distances of 0.5 to 5 m is required. Genetic marker analysis applied separately to all small-scale samples allows an identification of clone size, clonal diversity and architecture [START_REF] Douhovnikoff | Clonal growth: Invasion or stability? A comparative study of clonal architecture and diversity in native and introduced lineages of Phragmites australis (Poaceae)[END_REF]. Moreover, clone age can be estimated based on maximum clone diameter since clonal spread by rhizomes is slow and regular (<5 cm *a -1 measured in Chapter II common garden experiments, [START_REF] Steinger | Long-term persistence in a changing climate: DNA analysis suggests very old ages of clones of alpine Carex curvula[END_REF].

Sampling of different ramets in founder populations and tests in common garden experiments would help to better understand the relationship between population fitness and genetic diversity. Ramet senescence and its effect on survival and sexual reproduction can be studied using this approach. Clonal growth may not reduce intrinsic senescence. In iteroparous perennial plants, senescence was found to be positively correlated to time since first reproduction and mean reproductive lifespan [START_REF] Silvertown | Evolution of senescence in iteroparous perennial plants[END_REF]). However, fragmented clones (clonal reproduction) may more likely escape from senescence at the genet level than clones that remain physiologically integrated [START_REF] Silvertown | Evolution of senescence in iteroparous perennial plants[END_REF].

II. Fire effects on seedling recruitment and restoration management

Reproduction is a key factor in B. retusum re-establishment and is enhanced by summer wildfire. However, we do not know how long such positive fire effects last. A medium or longterm experiment would be needed to understand post-fired reproduction dynamics, in particular the return period to a steady state without fire. We also did not measure fire effects on seedling recruitment. Several species better germinate and establish after fire due to favourable site modifications such as reduced competition, chemical compounds released from smoke and ash and a higher nutrient availability [START_REF] Bond | Fire and plants[END_REF]. Experiments on such fire effects may be set up in traditionally managed grasslands but also on degraded sites in order to obtain information on fire as a restoration tool.

I am aware that fire may have negative effects on the epigeic and hypogeic fauna and soil microorganisms. The response of the fauna depends on fire characteristics such as intensity or propagation and animal species exhibit a large range of responses from negatively affected to fire-tolerant or even dependant [START_REF] Swengel | A literature review of insect responses to fire, compared to other conservation managements of open habitat[END_REF]. Before recommending fire as a restoration tool to maintain or restore Mediterranean dry grassland communities, potential negative effects on other organisms need to be evaluated.

III. B. retusum migration and dispersal

Seed dispersal is a key process that often limits the re-establishment of plant species. A better understanding of dispersal is needed to evaluate colonisation capacity based on seedling recruitment. Several studies on the ecological restoration of grassland ecosystems have shown that low dispersal represents a very strong environmental filter [START_REF] Bakker | Seed banks and seed dispersal: important topics in restoration ecology[END_REF][START_REF] Bischoff | Dispersal and re-establishment of Silaum silaus (L.) in floodplain grassland[END_REF][START_REF] Donath | The impact of site conditions and seed dispersal on restoration success in alluvial meadows[END_REF][START_REF] Bischoff | Succession of floodplain grasslands following reduction in land use intensity: the importance of environmental conditions, management and dispersal[END_REF]. Species without specific adaptation to wind dispersal are rarely dispersed more than 5 m per year [START_REF] Jongejans | Field experiments on seed dispersal by wind in ten umbelliferous species (Apiaceae)[END_REF][START_REF] Coulson | Colonization of grassland by sown species: dispersal versus microsite limitation in responses to management[END_REF]. So far, there is no accurate analysis of the dispersal of B. retusum. The fact that this very abundant species of intact steppe communities was not able to colonise adjacent areas of restoration sites within 30 years (Coiffait-Gombault et al. 2012a) suggests a low dispersal capacity. More knowledge on dispersal is crucial to better understand the role of this filter in steppe ecosystem restoration. Two main dispersal vectors have to be considered in our study region. Wind dispersal probably plays a major role (anemochory) because most of the steppe is exposed to strong winds from the north ("mistral"). Animal dispersal is probably the second important vector. Grassland species seeds can be transported by livestock (zoochory). [START_REF] Fischer | Experimental Studies on the Dispersal of Plants and Animals on Sheep in Calcareous Grasslands[END_REF] found the seeds of many species on grazing sheep (wool, hooves). However, the Crau plain is mostly grazed in spring and never during the Brachypodium seed production period in summer [START_REF] Débit | Ressource pastorale et sécheresse[END_REF]. It is therefore unlikely that sheep contribute to the dispersal of the species. As the second group of animals that disperse seeds in grassland ecosystems, ants are very abundant in the Crau (myrmecochory). The seed-eating ant Messor barbarus disperses over 60% of plant species [START_REF] Cerdan | Les fourmis moissonneuses et la végétation de la Crau (Bouches-du-Rhône)[END_REF]. Dispersal by ants is generally limited to a few meters [START_REF] Andersen | Dispersal distance as a benefit of myrmecochory[END_REF][START_REF] Retana | Dual role of harvesting ants as seed predators and dispersers of a non-myrmechorous Mediterranean perennial herb[END_REF]) but some seeds may also be transported much further (77 m, [START_REF] Gomez | Myrmecochorous dispersal distances: a world survey[END_REF]. Thus, it would be of high interest to test the efficiency of wind and ants as dispersal vectors. The wind speed can be manipulated in the laboratory to test the distance of dispersal by wind. In situ tests using platforms corresponding to the inflorescence position in the natural habitat may be used as an additional method to test for wind dispersal. Then the position of the seeds after wind transport indicates in situ dispersal distances. Myrmecochoric dispersal can be analysed by direct observation of seed transport by dominant granivorous ant species (Messor barbarus). Alternatively, seed deposits may be installed at different distances from the nest allowing a direct measurement of transport distances.

IV. Links between B. retusum and associated plant community B. retusum is highly interacting with other species as shown for dense dwarf matorral communities in Spain [START_REF] Saiz | Structure and spatial self-organization of semi-arid communities through plant-plant co-occurrence networks[END_REF]. In general, it is the dominant species of the dry Mediterranean Thero-Brachypodietea grasslands (San [START_REF] Miguel | Management of Natura 2000 habitats. 6220* Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea[END_REF]. Therefore, it seems to be likely that the species strongly affects its associated plant community but such plant-plant interaction have not been tested so far (Coiffait-Gombault 2011).

Plant-plant interactions may strongly influence the establishment of other species during plant succession and ecological restoration through mutualistic (facilitation) and competitive effects [START_REF] Pywell | Enhancing diversity of species-poor grasslands: an experimental assessment of multiple constraints[END_REF][START_REF] Brooker | Facilitation in plant communities: the past, the present, and the future[END_REF]. In particular, facilitation effects are currently discussed as a restoration tool improving seedling recruitment and establishment of target species [START_REF] Padilla | The role of nurse plants in the restoration of degraded environments[END_REF]. Thus, it would be interesting to compare the establishment of characteristic steppe species under B. retusum cover and at non-occupied control sites to evaluate facilitative effects.

V. Global change perspective

Current climate models predict a decrease in spring and summer precipitations in the Mediterranean climate zone resulting in stronger summer drought stress [START_REF] Gibelin | Anthropogenic climate change over the Mediterranean region simulated by a global variable resolution model[END_REF]. Our experiments demonstrated a high drought tolerance of the species but stronger summer drought may still limit B. retusum populations and result in a retreat of corresponding dry grassland communities resulting in a further decline. Experimental tests simulating summer drought in the field or specifically manipulating soil moisture and temperature in growth chambers may help to better understand the climatic limits of the species and its associated plant community (e.g. [START_REF] Jentsch | A new generation of climate-change experiments: events, not trends[END_REF][START_REF] Martin-Stpaul | The temporal response to drought in a Mediterranean evergreen tree: comparing a regional precipitation gradient and a throughfall exclusion experiment[END_REF].

Selection

Natural selection was considered by [START_REF] Darwin | On the origins of species by means of natural selection[END_REF] Adaptation is both an evolutionary process and a product of natural selection. Adaptation is a process of evolution in which traits are modified by natural selection and is a phenotypic trait shaped by natural selection.

However, it is currently known that selection is not the only force leading to evolution and could be counterbalanced by other evolutionary forces.

Drift

Genetic drift is another evolutionary force that could lead to population differentiation. Genetic drift is a change in the gene pool strictly due to chance fixation of alleles and is considered as the null model of population differentiation. It is the result of random processes in finite populations and its strength strongly depends on the effective size of a population, gene flow from other populations and the time scale of observation. In populations with an effective size larger than 500 individuals and over ecological timescale, drift is considered to be of limited importance [START_REF] Hartl | Principles of population genetics[END_REF]. Nevertheless, in small and isolated populations with limited gene flow, drift can lead to significant variation of allele frequencies, the fixation of alleles even over short time scales and thus to rapid differentiation [START_REF] Ellstrand | Population genetic consequences of small population size: implications for plant conservation[END_REF].

Gene flow

Gene flow, also called gene migration, is the transfer of genetic material from one population to another. It can counterbalance the effects of selection and drift by introducing new alleles into a population. Gene flow generally depend on the landscape and environment barriers [START_REF] Manel | Ten years of landscape genetics[END_REF]. However, differentiation can occur even in the presence of gene flow depending on the relative strength of the evolutionary forces.

-Population ecology Population ecology study the environmental factors that influence abundance, distribution and variation in space and time of populations. Several factors impact size, birth, growth, reproduction, death of a population and their variations. Main factors could be separated in abiotic (resource availability, water availability, climate) and biotic factors (intra species or inter species competition) (Neal 2004).

Community ecology

Community ecology began to develop in the beginning of the XX th century with a debate on community definition [START_REF] Clements | Plant succession: an analysis of the development of vegetation[END_REF][START_REF] Gleason | The individualistic concept of the plant association[END_REF]. Later, an ongoing debate arose, [START_REF] Macarthur | Geografical ecology: patterns in the distribution of species[END_REF] suggested that community ecology main goal is to find general rules whereas [START_REF] Lawton | Are there general laws in ecology?[END_REF] suggested that "community ecology is a mess". More recently, [START_REF] Simberloff | Community Ecology: Is It Time to Move On? (An American Society of Naturalists Presidential Address)[END_REF] added that general rules cannot be achieved due to the complex nature of communities. et al. (2006) responded that general laws can be found focusing on four themes which are traits, environmental gradients, interaction milieu and performance currencies.

McGill

For this thesis, we retained that a community is a group of populations of different species living in the same area [START_REF] Krebs | The experimental analysis of distribution and abundance[END_REF]. Community ecology is the study of patterns in the diversity, abundance, and composition of species in communities, and the processes underlying these patterns [START_REF] Vellend | Conceptual synthesis in community ecology[END_REF].

Restoration ecology

Ecological restoration preceded restoration ecology and is practised since centuries by indigenous communities [START_REF] Stevens | Conservation through cultural survival: Indigenous peoples and protected areas[END_REF]. Ecological restoration is "The process of assisting the recovery of an ecosystem that has been degraded, damaged, or destroyed." (Society for Ecological Restoration International Science and Working Policy Group, 2004).

It is only in the later XX th century, that emerged restoration ecology which is the science that develops and tests a body of theory focused on repairing damaged ecosystems [START_REF] Palmer | Ecological theory and community restoration ecology[END_REF]. Restoration ecology first focused on plant community restoration [START_REF] Young | Restoration ecology and conservation biology[END_REF]. Indeed, restoration operations often involve a focus on multi-species assemblages. Later on, population biologists working in a restoration framework focused on population restoration to a level that will allow them to persist over the long term within a dynamic landscape and include the ability to undergo adaptive evolutionary change. Indeed, common or dominant species removal, one whose total biomass (the sum of the biomass of all individuals) is greater than the average total A strong influence of the maternal environment and a high phenotypic plasticity were demonstrated for seed set. Most populations showed a very low in situ seed set at natural sites and there was considerable differentiation among populations which may be a major constraint in ecological restoration based on seedling recruitment (Coiffait-Gombault et al. 2012a, b).

However, in the common garden, differences completely disappeared due to more favourable growing conditions, and seed set was much higher. B. retusum produces flowers but allocates only resources to seeds if conditions for seed production are favourable. Such a reduction in seed production may be explained by abortion which is a widespread bet hedging strategy to concentrate resources to remaining seeds (Lovett Doust & Lovett Doust 1988). Furthermore, sterile florets may still have a positive function since they increase the dispersal of seeds with spikelets as dispersal units in grass (e.g. [START_REF] Monty | Diaspore heteromorphism in the invasive Bromus tectorum L. (Poaceae): Sterile florets increase dispersal propensity and distance[END_REF] for Bromus tectorum).

  Cette thèse a été financée par la région PACA et par le projet Excellence de l'Université d'Avignon et des Pays de Vaucluse avec le soutien du Pôle « Cultures et Patrimoine » d'Arles et du Conservatoire d'Espaces Naturels PACA.

  Merci à EliseBuisson pour son accueil, le partage de son bureau, notre séjour botanique sur Bagaud et nos discussions statistiques et diverses. Merci pour la super ambiance exotique du Introduction I.

Figure I :

 I Figure I: Evolutionary forces and their effect on population differentiation.

Figure II :

 II Figure II: Main factors driving population dynamics at several life cycle stages of a perennial grass after seed input. Modified from O'Connor (1991). Fire factor added according to Naveh

Figure III :

 III Figure III: The relationship between ecological theory, restoration ecology, and ecological restoration. Modified from Palmer et al., (2006).

Figure

  Figure IV: Ecological disciplines of the different chapters illustrating the transversal approach.

Figure 1 :

 1 Figure 1: UPGMA dendrogram showing the relationship between global dataset populations based on θST.

Figure 2 :Figure 3 :

 23 Figure 2: Genetic structure according to STRUCTURE 2.2 for K=2 and K=5 (optimum values according to Evanno method). The y-axis indicates the assignment ratio of each individual and individuals are assorted by population ranked from east (left) to west (right). Different colours correspond to clusters.

  than for neutral markers. Confidence intervals do not overlap. Moreover, critical ℎ 2 values for vegetative and reproductive traits are low: 0.3 and 0.4 (Fig 4A and 4B).

Figure 4 :

 4 Figure 4: Pst simulation (±c.i.) for continuous varying ℎ 2 and upper θST comparison. A: Vegetative traits, B: Reproductive traits.

  400 μL PL1 + 10 μL RNase A  30 '/ 65°C in oven Centrifugation 5 '/ 8000 rpm 3-Filtration / clarification of lysate Transfer the supernatant to a new tube on a NucleoSpin® Filter (violet ring) Centrifugation 2 '/ 8000 rpm Transfer lysate in a 2mL tube avoiding the pellet 4-Adjust DNA binding conditions Add 450 μL PC and mix pipetting up and down -5 times 5-Bind DNA Place a NucleoSpin® Plant II Column (green ring) into a new Collection Tube (2 mL) and load a maximum of 700 μL of the sample. Centrifugation 1

  Figure S1: Delta K, calculated according to Evanno et al. (2005) is plotted against the number of modeled gene pools (K).

Figure S2 :

 S2 Figure S2: Distance-based redundancy analysis (dbRDA) showing the influence of environmental variables on pairwise vegetative traits Pst among populations. The vectors illustrate correlations between the original environmental variables and the dbRDA-axes.

Figure 2 :

 2 Figure 2: Population differentiation decomposed in collection site and soil type within collection site (mean values ±SE, see Table 2 for statistics). A: Final germination, B: Cumulative leaf length, C: Cumulative tiller length and D: Plant diameter. Different lower case letters indicate significant differences (P<0.05).

Figure 4 :

 4 Figure 4: Differential response to stone cover (mean values +SE) between Brachypodium retusum populations in southern France. A: Plant height, B: Cumulative leaf length, C: Cumulative tiller length, D: Plant diameter.

Figure 5 :

 5 Figure 5: Differential response of a subset of populations to simulated grazing (mean values ±SE). Since site x clipping interaction was never significant only soil type x clipping combinations are presented. A: Plant height, B: Cumulative leaf length, C: Cumulative tiller length, D: Inflorescence number by soil type.

Figure 6 :

 6 Figure 6: Differential germination response of a subset of populations to soil moisture (mean values ±SE). Since the soil of origin x soil moisture interaction was never significant only site of origin effects are presented. Soil moisture (F4,55 = 53.71, P < 0.001), site of origin (F3,505 = 27.96, P < 0.001), soil of origin (F1,505 = 33.86, P < 0.001), site of origin x soil of origin x moisture interaction (F12,505 = 1.726).

  in leaf number per tiller was low. In particular, germination and tiller number are closely linked to seedling recruitment and establishment supporting our hypothesis that different colonization patterns observed at different Mediterranean sites (high:[START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF] low: Coiffait-Gombault et al., 2012) may result from genetic differentiation.

  Transition to Chapter IIIIn Chapters I and II, we showed a contribution of drift and adaptation to major environmental factors to B. retusum population differentiation. Adaptive population differentiation in germination and early growth may have contributed to different colonisation patterns observed in previous studies. We further demonstrated under favourable conditions in a common garden and in growth chambers that germination seedling recruitment and sexual reproduction are quite high suggesting a potentially high colonisation capacity (for seed production see Appendix 2).

Fire

  and grazing are two key environmental/management factors that have largely decreased (fire protection, land abandonment) potentially explaining the decline of Mediterranean steppes (San Miguel 2008).

Figure 1 :

 1 Figure 1: Effects of fire and grazing on vegetative growth of B. retusum in the combined fire and grazing experiment (left) and the fire experiment in long-term exclosures (right). A, B: weighted cover one year after fire treatments (May 2017, mean ± SE); C, D: Post-fire regeneration of cover (mean difference between October 2016 and May 2017, ± SE).

Figure 2 :

 2 Figure 2: Effects of fire and grazing on the reproduction of B. retusum (mean ± SE) in the combined fire and grazing experiment (left) and the fire experiment in long-term exclosures (right) in the second post-fire year (July 2017). A, B: Inflorescence number; C, D: Seed number per inflorescence.

Figure 3 :

 3 Figure 3: Effects of fire and grazing on the plant community (mean ± SE) one year after fire treatments (May 2017) in the combined fire and grazing experiment (left) and the fire experiment in long-term exclosures (right). A, B species richness; C, D: evenness.

Figure 4 :

 4 Figure 4: Effect of fire on plant species composition in the combined fire and grazing experiment set up in traditionally managed steppe and in the fire experiment set up in long-term exclosures using NMDS on May 2017 data. Polygons indicate the position of the outmost plots in each treatment (NMDS stress: 0.1471418).

Figure 1 :

 1 Figure 1: Experimental design of the experiment. The figure shows one out of five experimental blocks.

Figure 2 :

 2 Figure 2: Effects of initial watering and grazing on final germination of Brachypodium retusum (mean values ±SE).

Figure 3 :

 3 Figure 3: Effects of initial watering and grazing on survival rate of B. retusum transplanted seedlings (A) and sown seedlings (B) (mean values ±SE).

Figure 4 :

 4 Figure 4: Effects of initial watering and grazing on sown and planted B. retusum seedlings growth (mean values ±SE) at the end of the first grazing period (left side of the panel, July 2017) and at the end of the study period (right side of the panel, May 2018),. A, B: Cumulative leaf length; C, D: Cumulative tiller length; E, F: Tiller number; G, H: Plant height. Different lower case letters indicate significant differences (P<0.05).

Figure 6 :

 6 Figure 6: Percentage of variation in plant growth explained by watering, grazing and their interaction (July 2017 and May 2018).

Figure S2 :

 S2 Figure S2: Satellite image of the study area (May 2017). Darker spots correspond to the grazing exlosures.

  Figure IX: Evolutionary forces and their effect on population differentiation of B. retusum. + increase; -decrease; arrow width represents the relative contribution of each evolutionary force.

Figure X :

 X Figure X: Main factors driving B. retusum populations at different life cycle stages in fieldsown seedlings, established adults and the associated community. ↑ increase; ↓ decrease; ─ no effect

Figure

  Figure XI: Contribution of the thesis to basic and applied ecology.

Factors

  driving differentiation were climate, soil type, stone cover, grazing (simulated by clipping) and to a lesser degree soil moisture. Adaptive differentiation was particularly found in phenotypic traits that are crucial for early establishment such as germination and tiller number. The results support our hypothesis that different colonisation patterns observed at different Mediterranean sites (high: Caturla et al. 2000, Luis et al. 2004; low: Coiffait-Gombault Conclusions-answers to hypotheses et al. 2012a) result at least partially from genetic differentiation among populations. Plant introduction measures in ecological restoration should therefore take into account plant provenance including geographical and environmental distance to restoration sites.

  as the most powerful of the evolutionary forces. It occurs when differences among individual heritable traits cause differences in survival and reproduction. Depending of trait evolution direction, three types of natural selection can be distinguished. Directional selection drives a phenotype (observable trait) to one extreme rather the intermediate phenotype or the opposite extreme phenotype. Stabilizing selection occurs when an intermediate phenotype is favoured. Disruptive selection occurs when extreme phenotypes have an advantage over more intermediate phenotypes. Moreover, sexual selection is a part of natural selection and could be distinguished from it when selection acts on traits involved in reproductive success.

Figure A :

 A Figure A: Differences in seed set of parental plants (in situ) and F1 plants grown in a common garden (mean values ±SE). Sites of origin: SM: Saint Martin-de-Crau, SR: Saint Rémy-de-Provence, Me: Mérindol, Ca: Caumont, Ni: Nîmes, Mo: Montpellier. Different lower case letters indicate significant differences (P<0•05).

  , 100 ng of DNA was digested using the restriction enzymes Eco

	RI and Tru 9I (Fisher Scientific, France) for 3 h at 37°C and then for 3h at 65°C in a total
	volume of 25 µl (15 μL + 10µl of DNA). Digestion products were immediately ligated to 0.5
	μL Eco and 25 μL Mse adaptors for 3 h at 37°C and treated with T4 DNA Ligase and 0.1 μL
	of 100 mM ATP to a final volume of 25 μL (5 μL + 20 µl of restriction products). Ligation
	products were diluted eight times and pre-selective PCR amplification was performed using
	EcoR1+A, Mse+C primers and Taq DNA polymerase in a 44.5 μL volume. The pre-
	amplification thermocycle profile was 94°C for 2 min, followed by 20 cycles at 94°C for 45 s,
	56°C for 45 s, 72°C for 1 min and 72°C for 10 min. Four primer combinations were chosen for
	the selective amplification PCR: ASI: EcoR1-AAC/MseI-CAA, ASII: EcoR1-AGG/ MseI -
	CGG, ASIII: EcoR1-AGC/ MseI -CAG, ASIV: EcoR1-ATG/ MseI -CTA dyed with 6-FAM
	fluorescence at 5′ Eco end (Eurofins Genomics, Ebersberg, Germany). Hundred times diluted
	pre-amplification products were used to perform selective amplification in a final volume of 20
	μL (15 μL + 5 μL of diluted pre-amplification products). As selective amplification thermocycle
	profile, we used 94°C for 2 min, 10 cycles of 94°C for 30 s, 65°C for 30 s (step -1°C per cycle),

  I

	Separate PCA were run on vegetative (plant height, diameter, tiller number, leaf number and
	length) and reproductive phenotypic traits (inflorescence and spikelet number). We used first
	axis PCA scores as a synthetic response variable considering several phenotypic traits at the
	same time to calculate PST values (divergence in quantitative traits). Calculations were run using
	R package Pstat with 9999 bootstraps (Da Silva and Da Silva 2018) but sum of squares (SS)
	were used as variance estimates instead of mean sum of squares in order to compare PST to SS
	based θST. PST is analogous to QST and is used if heritability estimates cannot be measured
	(Brommer 2011)
		=	2 2 +2ℎ 2	2 =	ℎ 2	2 2 +2 ℎ 2	2
	with	2 being the phenotypic variance between populations,	2 the phenotypic variance
	within populations, c the proportion of the total variance explained by additive genetic effects
	and h 2 the heritability, proportion of phenotypic variance explained by additive genetic effects.
	The null assumptions were chosen for	ℎ 2 parameters with c=h 2 =1 (Brommer 2011, Detailed
	method description 2).

  Populations are ranked according to longitude from east (top of the table) to west. Pop:

	Population name, N: Sample size, H: Shannon-Wiener Index of MLG diversity (Shannon,
	2001), He: Nei's diversity index of expected heterozygosity, rbarD: standardized index of
	association, p.rbarD: p.value of rbarD.
	Country Pop	N	H	He	rbarD	p. rbarD
	Italy	NIT	19	2.944 0.205 0.101	0.001
	France	ME_C	20	3.044 0.166 0.001	0.038
	France	ME_R	19	2.944 0.172 0.003	0.003
	France	CA_C	20	2.995 0.164 0.004	0.001
	France	CA_R	20	3.044 0.165 0.002	0.021
	France	SR_C	18	2.890 0.159 0.000	0.180
	France	SR_R	20	2.995 0.166 0.007	0.001
	France	SM_C	20	2.995 0.158 0.004	0.001
	France	SM1_R 20	2.995 0.165 0.006	0.001
	France	SM2_R 20	2.995 0.147 0.008	0.001
	France	NI_C	20	2.995 0.168 0.002	0.009
	France	NI_R	20	2.995 0.159 0.000	0.433
	France	MO_C	19	2.944 0.150 0.000	0.634
	France	MO_R	20	2.995 0.176 0.002	0.019
	France	ROQ	10	2.302 0.165 0.003	0.059
	Spain	NES	18	2.890 0.142 0.001	0.152
	Spain	SES	17	2.833 0.134 0.007	0.001
		Average 18.941 2.929 0.162 0.009	0.091

Table 2 :

 2 Analysis of variance on AFLP (based on AMOVA) and phenotypic vegetative and reproductive traits (based on ANOVA performed on first axis PCA scores). MSD: Mean squared deviation and percentage of variation explained by collection site, soil type within collection site and site x soil interaction.

		df	MSD	% of total variance
	AFLP
	Among sites	5	85.370	2.33 ***
	Among soils	1	52.063	0.62 ***
	Among soil*site	5	56.310	5.00 ***
	Within populations	245	26.927	93.06
	Vegetative traits
	Among sites	5	29.534	12.85 ***
	Among soils	1	19.676	1.92 *
	Among soil*site	5	14.531	6.76 **
	Within populations	242	4.138	65.25
	Reproductive traits
	Among sites	5	26.671	8.37 ***
	Among soils	1	0.775	0.05 NS
	Among soil*site	5	16.122	5.23 **
	Within populations	242	6.028	80.38

Table 3 :

 3 Multiple regression analysis (MRM). Standardized regression coefficients of multipleregression on distance matrices, with climatic distance, geographic distance and genetic distance in AFLP markers (θST) as explanatory and pairwise Pst distances as response variables. R 2 : multiple correlation coefficient.

		Pst vegetative traits	Pst reproductive traits
	Variables	Coefficient P-value	Coefficient P-value
	Climate	0.18	0.04 *	0.07	0.05 *
	Geographic distance 0.10	0.20 NS	0.03	0.06 .
	θST	1.65	0.04 *	1.69	0.13 NS
	pH	0.03	0.60 NS	0.01	0.70 NS
	R 2	0.57	0.02 *	0.52	0.04 *

Table 4 :

 4 Partial distance-based redundancy analyses (dbRDA) testing for effects of environmental factors on divergence in vegetative and reproductive traits (Pst). A: marginal test of individual sets of predictor variables and B: partial (conditional) test including geographic distance (latitude, longitude) and genetic distance as covariates. F-values, significance levels of ANOVA-like permutation tests and percentage of variation explained by each environmental factor. . P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.

			Pst vegetative traits		Pst reproductive traits
		F-values	Explained variation (%)	F-values	Explained variation (%)
	A-Marginal tests
	Precipitation	6.606 *	18.12	3.883 *	15.48
	Mean annual temperature 4.007 *	8.29	2.072 NS	8.27
	Mean January temperature 5.336 *	13.84	3.651 *	13.61
	Mean July temperature	5.048 *	13.04	3.669 *	14.20
	Days T<0	6.181 *	16.95	3.796 *	15.15
	pH	4.001 *	13.20	2.507 NS	9.94
	B-Conditional tests
	Precipitation	0.803 NS	3.30	0.905 NS	7.21
	Mean annual temperature 3.000 NS 10.20	1.318 NS 11.50
	Mean January temperature 2.142 NS	8.21	1.120 NS 10.81
	Mean July temperature	5.362 *	22.60	2.961 .	26.20
	Days T<0	6.345 *	25.80	3.488 .	31.00
	pH	2.839 NS 11.31	2.124 NS 19.42

DISCUSSION

Structure of genetic diversity revealed by neutral molecular markers

In this study, we significantly extend the knowledge on the genetic diversity structure of Brachypodium retusum, the key grass species of a vulnerable grassland habitat in the Western Mediterranean of Europe. First, B. retusum showed a high level of genetic variability and diversity within populations. The number of polymorphic loci was higher than in other Chapter I perennial Brachypodium species (B. pinnatum, B. sylvaticum) but its genetic diversity was lower

[START_REF] Bąba | Genetic diversity of the expansive grass Brachypodium pinnatum in a changing landscape: Effect of habitat age[END_REF][START_REF] Mo | Characterization of Microsatellite Markers and Their Application to Genetic Diversity Analysis of Brachypodium sylvaticum var. breviglume from Yunnan, China[END_REF]

. B. retusum is a widespread outcrossing windpollinated species with high self-incompatibility

Table S1 :

 S1 Soil characteristics, annual precipitation, mean annual air temperature (including January and July mean in brackets), and the number of frost days (T<0) at collection sites. Sites are ranked from east to west. Climate data are based on daily averages from the nearest meteorological stations. Soil: C = calcareous soil, R = red Mediterranean soil, * Soil on granite bedrock, not red Mediterranean soil but chemical properties closer to F than to

		C.
	Country Site	Soil	pH	x	y	Precipitation (mm*a -1 )	Temperature (°C) [Jan/Jul]	Days T<0
	IT	NIT	C	8.1	43.852352	7.955755	469	16.6 [10.1/23.4]
	FR	ME	C	8.0	43.756456	5.199526	583	14.5 [ 6 /23.7]
	FR	ME	R	8.0	43.750425	5.210495	583	14.5 [ 6 /23.7]
	FR	CA	C	8.2	43.898827	4.941070	676	14.6 [ 5.9/24.2]
	FR	CA	R	6.9	43.898319	4.932395	676	14.6 [ 5.9/24.2]
	FR	SR	C	7.8	43.768047	4.831898	625	15.1 [ 6.6/24.3]
	FR	SR	R	6.8	43.798952	4.841659	625	15.1 [ 6.6/24.3]
	FR	SM	C	8.1	43.705354	4.797561	517	15.3 [ 7.4/24.4]
	FR	SM	R	6.8	43.523843	4.832804	517	15.3 [ 7.4/24.4]
	FR	SM	R	6.8	43.633055	5.019083	517	15.3 [ 7.4/24.4]
	FR	NI	C	8.0	43.784700	4.259389	701	14.9 [ 6.7/24.1]
	FR	NI	R	7.8	43.722868	4.339264	701	14.9 [ 6.7/24.1]
	FR	MO	C	8.9	43.645416	3.691638	870	14.1 [ 6.4/23.2]
	FR	MO	R	6.7	43.607580	3.449269	870	14.1 [ 6.4/23.2]
	FR	ROQ C	8.1	42.981988	2.763333	652	15.1 [ 7.7/23.8]
	SP	NES	R*	7.4	41.706568	2.863655	666	16.1 [ 9 /24.2]
	SP	SES	C	-	37.563303	-4.481613	493	17.1 [ 8.6/27.6]

Table S2

 S2

: Pairwise θST among populations. . P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.

Table S3 :

 S3 Pairwise Pst between Southern French populations for vegetative (A) and reproductive (B) traits. All pairwise comparisons were significant (P < 0.001).

		Chapter I
	A	ME_C ME_R CA_C CA_R SR_C SR_R SM_C SM1_R SM2_R NI_C NI_R MO_C
	ME_R 0.079
	CA_C 0.131 0.160
	CA_R 0.083 0.088 0.146
	SR_C 0.089 0.121 0.112 0.100
	SR_R 0.160 0.164 0.075 0.176 0.112
	SM_C 0.128 0.146 0.071 0.141 0.095 0.078
	SM1_R 0.116 0.129 0.071 0.101 0.062 0.095 0.081
	SM2_R 0.139 0.145 0.099 0.143 0.096 0.078 0.089 0.089
	NI_C 0.130 0.098 0.271 0.148 0.200 0.267 0.283 0.223 0.242
	NI_R 0.127 0.091 0.218 0.125 0.176 0.237 0.221 0.175 0.200 0.062
	B	ME_C ME_R CA_C CA_R SR_C SR_R SM_C SM1_R SM2_R NI_C NI_R MO_C
	ME_R 0.075
	CA_C 0.098 0.097
	CA_R 0.150 0.113 0.087
	SR_C 0.082 0.079 0.072 0.080
	SR_R 0.155 0.138 0.121 0.082 0.115
	SM_C 0.091 0.087 0.101 0.160 0.074 0.167
	SM1_R 0.170 0.089 0.084 0.098 0.080 0.114 0.091
	SM2_R 0.096 0.096 0.124 0.098 0.072 0.098 0.095 0.087
	NI_C 0.135 0.134 0.178 0.198 0.149 0.235 0.165 0.199 0.178
	NI_R 0.136 0.132 0.187 0.211 0.142 0.239 0.138 0.193 0.164 0.073
	MO_C 0.116 0.093 0.089 0.085 0.084 0.098 0.098 0.066 0.123 0.192 0.177
	MO_R 0.219 0.236 0.298 0.339 0.333 0.355 0.285 0.318 0.268 0.198 0.201 0.331

MO_C 0.098 0.119 0.085 0.099 0.089 0.088 0.083 0.074 0.125 0.225 0.198 MO_R 0.288 0.245 0.412 0.297 0.351 0.432 0.415 0.362 0.400 0.221 0.189 0.376

Table 1 :

 1

		mowing
	Site code	Site name	Soil	Coordinates	pH Grazing	Precipitation (mm*a -1 )	Temperature (°C) Mean [Jan/Jul]	Days T<0
	Me	Mérindol	Calcareous Red Mediterranean	43.756456 N, 5.199526 E 43.750425 N, 5.210495 E	8.0 8.0	No* Low	583	14.5 [6.0/23.7]	49
	Ca	Caumont	Calcareous Red Mediterranean	43.898827 N, 4.941070 E 43.898319 N, 4.932395 E	8.2 6.9	No No	676	14.6 [5.9/24.2]	33
	SR	Saint-Rémy-de-Provence	Calcareous Red Mediterranean	43.768047 N, 4.831898 E 43.798952 N, 4.841659 E	7.8 6.8	No High	625	15.1 [6.6/24.3]	30
	SM	Saint-Martin-de Crau	Calcareous Red Mediterranean	43.705354 N, 4.797561 E 43.567465 N, 4.834645 E	8.1 6.8	No High	517	15.3 [7.4/24.4]	24
	Ni	Nîmes	Calcareous Red Mediterranean	43.784700 N, 4.259389 E 43.722868 N, 4.339264 E	8.0 7.8	No No*	701	14.9 [6.7/24.1]	34
	Mon Montpellier	Calcareous Red Mediterranean	43.645416 N, 3.691638 E 43.607580 N, 3.449269 E	8.9 6.7	No* No*	870	14.1[6.4/23.2]	51

Soil characteristics, grazing intensity, annual precipitation, mean annual air temperature (including January and July mean in brackets), and the number of frost days (T<0) at collection sites of the twelve populations. Sites are ordered from east to west. Climate data are based on daily averages from the nearest meteorological stations (1980-2010).*irregular Figure 1: Position of six sampling sites each comprising two populations from different soil types.

  The plug trays were placed in a growth chamber (temperature 15/20 °C, 12/12h, night/day) and watered regularly. Coleoptile emergence used as a proxy of germination was recorded every 2-3 days for seven weeks. In March 2016, 24 plugs perIn May 2016, stones of 10 -20 cm length and 7 to 15 cm width were placed in a circle around half of the seedlings of the common garden experiment to test their effect on plant growth. The stones were taken from the red Mediterranean soil of the Caumont population (Ca, Table1, Fig. were sown to peat plugs and placed in a greenhouse. In February 2015, two seedlings of each population were planted to each of 12 blocks (24 seedlings in total). Due to low germination, seedling number was only one per block for the Me population from calcareous soil (12 seedlings in total). Plants were moderately watered during the first year but not in the second year.

	1). Measurements were the same as in the population differentiation study under common
	conditions detailed above.
	A subset of four population pairs (red Mediterranean and calcareous soils x Me, Ca, SR, SM
	sites) was grown one year earlier in a similar common garden experiment to simulate grazing

population (corresponding to 24 patches or seed families) were randomly selected and transplanted to a previously ploughed experimental site in Avignon-Montfavet. Thus 48% of the initially collected patches were represented in the field. Seedling number was reduced to one (average size) per plug prior to transplantation. Two plugs per population were planted to random positions within each of 12 blocks (3 * 3m²). During the first four months, seedlings were moderately watered. Plots were hand-weeded to limit competition. Plant height, diameter, tiller number, leaf number and the length of the longest leaf were measured once a month. Young B. retusum plants allocate resources to tillering and leaf growth. Later, tillers elongate and older leaves die. Therefore, we calculated cumulative leaf length as an aboveground biomass estimate for young seedlings (leaf length * number of leaves) and cumulative tiller length for older plants (plant height * number of tillers). Adaptation to differences in soil conditions, management and soil moisture by clipping established plants. Clipping of second year plants prevents high mortality compared to clipping of young seedlings. The design was similar to the differentiation study. In November 2014, seeds

Table 2 :

 2 Differentiation in Brachypodium retusum populations from different sites and soil types, and differential population response to stone cover treatment. F-values and significance levels from two-way ANOVA before stone treatment (growth traits: May) and three-way ANOVA

	afterwards (July) including the stone effect. . P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.
			df	Germination Leaf number Leaf length	Plant height	Tiller number	Cumulative leaf length	Cumulative tiller length	Plant diameter
	Before	Site	5,338	31.41 ***	1.82 NS	11.81 ***	6.62 ***	4.87 ***	8.12 ***	6.98 ***
	treatment	Soil	1,338	36.83 ***	0.00 NS	5.30 *	0.00 NS	0.02 NS	1.05 NS	0.00 NS
	(May)	Soil × Site	5,338	3.08 **	0.64 NS	6.60 **	2.06 .	1.76 NS	2.00 .	2.13 .
		Site	5,315		1.73 NS	3.99 **	7.09 ***	6.93 ***	4.89 ***	8.48 ***	4.01 **
		Soil	1,315		0.00 NS	5.07 *	0.37 NS	0.34 NS	1.32 NS	0.38 NS	1.61 NS
	After	Stones	1,315		1.58 NS	1.09 NS	4.24 *	5.48 *	0.03 NS	5.67 *	4.16 *
	treatment	Soil × Site	5,315		0.97 NS	3.02 *	2.18 *	1.33 NS	2.04 .	1.76 NS	2.52 *
	(July)	Stones × Site	5,315		2.02 .	0.54 NS	1.56 NS	2.87 *	0.18 NS	2.24 *	0.38 NS
		Stones × Soil	1,315		0.10 NS	1.90 NS	0.59 NS	0.02 NS	1.14 NS	0.05 NS	0.44 NS
		Stones × Soil × Site	5,315		0.49 NS	0.18 NS	2.26 *	0.21 NS	1.18 NS	0.79 NS	0.26 NS

Table 3 :

 3 Differential response in a subset of populations to simulated grazing. F-values and significance levels from three-way ANOVA on site, soil (within site) and clipping effects. Vegetative traits measured in spring(May), inflorescence number in June of the second growing season. . P

	< 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.
		df	Leaf number Leaf length	Plant height	Tiller number	Cumulative leaf length	Cumulative tiller length	Plant diameter	Inflorescence number
	Site	3,132	1.20 NS	1.13 NS	0.27 NS	0.63 NS	1.47 NS	0.64 NS	1.30 NS	2.48 .
	Soil	1,132	0.02 NS	9.43 **	3.16 .	0.05 NS	4.13 *	2.87 .	1.54 NS	3.91 *
	Clipping	1,132	12.31 ***	3.19 .	220.98 ***	10.48 **	11.46 ***	211.72 ***	7.23 **	58.39 ***
	Soil × Site	3,132	0.67 NS	2.12 NS	0.81 NS	0.16 NS	2.05 NS	1.18 NS	0.67 NS	1.98 NS
	Clipping × Site	3,132	0.66 NS	0.49 NS	0.81 NS	0.02 NS	0.99 NS	1.49 NS	0.94 NS	0.23 NS
	Clipping × Soil	1,132	0.08 NS	0.09 NS	3.93 *	2.17 NS	0.12 NS	3.07 .	0.48 NS	3.37 .
	Clipping × Soil × Site	3,132	1.12 NS	0.61 NS	1.65 NS	0.76 NS	0.43 NS	1.08 NS	0.40 NS	0.29 NS

  Stone cover increased B. retusum growth and the differential response of populations suggests adaptation to this environmental factor. However, we could not relate this response to the home environment of tested populations. Further experiments are required to identify potential drivers of adaptation to stone cover (density at the surface or in different soil layers). The response to clipping was also clearly different among populations and the differential response could be linked to the major soil type suggesting that different grazing intensity in the past has selected for different ecotypes. B. retusum showed a high germination under dry soil conditions but there Appendix S2: Differentiation in Brachypodium retusum populations from different sites and soil types. F-values and significance levels from two-

	way ANOVA for measurement dates not presented in Table 2. . P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.
	Growth		df	Leaf number Leaf length	Plant height	Tiller number	Cumulative leaf length	Cumulative tiller length	Plant diameter
		Site	5,338	1.17 NS	18.20 ***	12.44 ***	0.43 NS	7.02 ***	5.37 ***
	March	Soil Soil × Site	1,338 5,338	3.39 . 0.93 NS	0.13 NS 2.78 *	0.01 NS 3.70 **	2.60 NS 0.36 NS	1.12 NS 0.97 NS	0.66 NS 2.15 *
		Site	5,338	4.99 ***	16.95 ***	12.18 ***	5.58 ***	9.89 ***	10.36 ***
	April	Soil Soil × Site	1,338 5,338	6.67 * 1.50 NS	1.06 NS 7.59 ***	1.23 NS 2.27 *	0.09 NS 1.60 NS	4.49 * 2.41 *	0.15 NS 1.92 .
		Site	5,338	1.56 NS	15.03 ***	5.69 ***	5.40 ***	5.58 ***	3.39 **	9.24 ***
	October	Soil Soil × Site	1,338 5,338	0.40 NS 1.83 NS	4.98 * 10.90 ***	0.04 NS 1.10 NS	0.08 NS 2.81 *	0.53 NS 2.73 *	0.80 NS 1.38 NS	1.21 NS 5.31 ***

was only weak evidence for a differential response of tested populations. The study demonstrated that population differentiation may well explain different colonization patterns observed in different Mediterranean regions

[START_REF] Caturla | Early post-fire regeneration dynamics of Brachypodium retusum Pers. (Beauv.) in old fields of the Valencia region (eastern Spain)[END_REF][START_REF] De Luis | Fire and torrential rainfall: effects on the perennial grass Brachypodium retusum[END_REF] Coiffait- Gombault et al., 2012)

. Since observed differences may largely influence seedling establishment in Mediterranean dry grasslands, plant origin needs to be carefully considered in ecological restoration involving planting or sowing of B. retusum.

Table 1 :

 1 ANOVA F-values and significance level for effects of fire and grazing on Brachypodium retusum in the two experiments: cover and weighted cover adjusted for total vegetation cover (both May), post-fire regeneration (cover difference between October 2016 and May 2017), inflorescence number, seed number per inflorescence (both July) and germination rate. F×G= fire x grazing interaction. . P < 0.1, * P < 0.05, ** P

	< 0.01, *** P < 0.001, NS not significant. Factor effect: ↓:decrease; ↑: increase.
			df	Cover (%)	Weighted cover (%)	Regeneration (%)	Inflorescences (number.4m -2 )	Seeds (number.inflo -1 )	Germination (%)
	A. Combined fire and grazing experiment
	2016	Fire	1.10 469.29 *** ↓	365.43 *** ↓		18.99 *** ↓	42.44 *** ↓	4.06 * ↑
		Grazing 1.10	0.39 NS	1.43 NS		0.18 NS	0.56 NS	0.01 NS
		F×G	1.10	0.13 NS	0.13 NS		0.09 NS	1.59 NS	0.03 NS
	2017	Fire	2.20	12.35 *** ↓	2.21 NS	143.04 *** ↑	11.48 *** ↑	5.86 ** ↑	1.25 NS
		Grazing 1.10	1.62 NS	0.58 NS	1.12 NS	0.01 NS	0.24 NS	0.35 NS
		F×G	2.20	3.64 *	3.62 *	0.65 NS	0.90S	0.18 NS	0.31 NS
	B. Fire experiment in long-term exclosures
	2016	Fire	1.10 311.00 *** ↓	115.60 *** ↓		1.10 NS	1.08 NS	0.87 NS
	2017	Fire	2.20	0.66 NS	1.85 NS	72.94 *** ↑	3.76 . ↑	2.27 NS	-

Table 2 :

 2 ANOVA F-values, significance levels for effects of fire and grazing on species richness (4m -2 ), evenness and Bray-Curtiss dissimilarity in the two experiments. F×G= fire x

			df	Species-Richness	Evenness	Bray-Curtis
	A. Combined fire and grazing experiment
	2016	Fire	1.10	4.97 * ↓	12.26 ** ↑	24.14 *** ↑
		Grazing	1.10	0.45 NS	0.01 NS	0.45 NS
		F×G	1.10	0.01 NS	3.12 NS	0.16 NS
	2017	Fire	2.20	4.18 * ↑	14.00 *** ↑	3.32 * ↑
		Grazing	2.20	0.03 NS	0.00 NS	0.04 NS
		F×G	2.20	0.42 NS	0.31 NS	0.40 NS
	B. Fire experiment in long-term exclosures
	2016	Fire	1.10	3.05 NS	4.82 . ↑	32.06 ** ↑
	2017	Fire	2.10	5.03 * ↑	3.95 * ↑	1.45 NS

grazing interaction. . P < 0•1, * P < 0•05, ** P < 0•01, *** P < 0•001, NS not significant. Factor effect: ↓: decrease; ↑: increase.

Table 1 :

 1 Effects of initial watering and grazing on B. retusum germination and growth (July 2017 and May 2018) for transplanted seedlings and sown seedlings. T×W= type x watering interaction, T×G= type x grazing interaction, W×G= watering × grazing interaction, T×W×G= type × watering × grazing interaction. ANOVA F-values, significance levels: . P < 0•1, * P < 0•05, ** P < 0•01, *** P < 0•001, NS not significant. Factor effect: ↓ (decrease) ↑ (increase).

	Date	Factors	df	Germination (semis)	Leaf number Leaf length Plant height	Tiller number	Cumulative leaf length	Cumulative tiller length	Plant diameter
	2017	Type	1	-	4.650 .	10.650 **	33.326 *** 34.278 ***	7.417 *	39.259 *** 126.101 ***
		Watering 1	1.106 NS 0.607 NS	0.020 NS	1.498 NS	4.649 * ↑	1.345 NS	0.681 NS	7.098 ** ↑
		Grazing	1	10.540 * ↓ 0.313 NS	3.821 . ↓	10.557 ** ↓ 16.291 ** ↑	0.573 NS	0.465 NS	13.222 ** ↑
		T×W	1	-	0.257 NS	0.005 NS	0.065 NS	2.381 NS	0.051 NS	0.959 NS	0.246 NS
		T×G	1	-	0.977 NS	1.574 NS	1.278 NS	0.023 NS	1.141 NS	0.484 NS	2.181 NS
		W×G	1	0.478 NS 0.006 NS	0.542 NS	0.713 NS	0.207 NS	0.588 NS	0.032 NS	1.062 NS
		T×W×G	1	-	5.523 *	3.804 .	11.354 **	7.116 *	5.733 *	10.157 **	0.153 NS
	2018	Type	1	-	1.605 NS	12.836 *	24.480 ***	7.387 *	7.183 *	18.079 **	20.008 ***
		Watering 1	-	0.430 NS	0.584 NS	0.002 NS	0.289 NS	1.293 NS	0.273 NS	0.887 NS
		Grazing	1	-	2.577 NS	19.594 *** ↓ 28.501 *** ↓ 1.506 NS	11.100 ** ↓ 12.621 *** ↓	1.482 NS
		T×W	1	-	0.175 NS	0.813 NS	0.063 NS	0.002 NS	0.687 NS	0.002 NS	0.303 NS
		T×G	1	-	1.157 NS	0.298 NS	0.041 NS	1.034 NS	1.233 NS	0.696 NS	0.789 NS
		W×G	1	-	0.076 NS	0.864 NS	1.068 NS	1.103 NS	0.207 NS	1.611 NS	0.014 NS
		T×W×G	1	-	2.241 NS	0.537 NS	0.144 NS	0.180 NS	1.308 NS	0.042 NS	0.291 NS

Table S1 :

 S1 Effects of initial watering and grazing on B. retusum germination and growth for transplanted seedlings and sown seedlings. T×W= seedling type x watering interaction, T×G= seedling type x grazing interaction, W×G= watering × grazing interaction, T×W×G= seedling type ×

	Date watering × grazing interaction. ANOVA F-values, significance levels: . P < 0•1, * P < 0•05, ** P < 0•01, *** P < 0•001, NS not significant. Factor Factors df Germination (semis) Tiller Cumulative Cumulative Plant Leaf number Leaf length Plant height number leaf length tiller length diameter Dec2017 Type -8.247 * 1.937 NS 18.202 ** 22.778 *** 12.335 ** 54.114 *** 45.803 *** effect: ↓ (decrease) ↑ (increase). Watering -0.979 NS 0.057 NS 0.159 NS 5.512 * ↑ 0.816 NS 4.573 * ↑ 4.655 * ↑
		Grazing		-	0.001 NS	0.191 NS	1.748 NS	1.523 NS	0.003 NS	0.062 NS	0.871 NS
	Date	Factors T×W T×G	df	Germination (semis) --	0.038 NS Leaf number Leaf length Plant height 0.115 NS 1.425 NS 2.771 NS 0.698 NS 0.404 NS	Tiller 0.023 NS number 0.498 NS	Cumulative 0.676 NS leaf length 2.116 NS	Cumulative 1.255 NS tiller length 0.001 NS	0.746 NS Plant diameter 0.282 NS
	May2017 Type W×G	1	--	101.467 *** 35.337 *** 38.501 *** 86.244 *** 63.465 *** 66.731 *** 134.109 *** 1.997 NS 0.676 NS 2.302 NS 4.305 * 0.311 NS 1.977 NS 2.728 .
	Watering 1 T×W×G Grazing 1 May2018 Type T×W 1 Watering T×G 1 Grazing	1.106 NS -10.540 * ↓ 41.998 *** ↓ 3.784 . ↓ 2.871 . ↑ 0.075 NS 1.850 NS 0.086 NS -1.605 NS 12.836 * -2.532 NS 0.205 NS -0.430 NS 0.584 NS -4.397 * 0.841 NS -2.577 NS 19.594 *** ↓ 28.501 *** ↓ 1.506 NS 0.990 NS 3.188 . ↑ 0.194 NS 0.612 NS 4.204 . ↓ 0.201 NS 24.480 *** 7.387 * 1.203 NS 0.279 NS 0.002 NS 0.289 NS 0.424 NS 0.001 NS	0.715 NS 0.221 NS 7.183 * 10.629 ** ↓ 1.151 NS 0.037 NS 3.548 * ↑ 18.079 ** 1.252 NS 0.784 NS 1.293 NS 0.273 NS 0.635 NS 0.179 NS 11.100 ** ↓ 12.621 *** ↓ 1.482 NS 2.368 NS 0.271 NS 0.281 NS 20.008 *** 0.887 NS 0.357 NS 0.012 NS
		W×G T×W	1	0.478 NS -	0.743 NS 0.175 NS	0.337 NS 0.813 NS	0.063 NS 0.063 NS	0.648 NS 0.002 NS	0.084 NS 0.687 NS	0.314 NS 0.002 NS	0.082 NS 0.303 NS
		T×W×G T×G	1	--	1.568 NS 1.157 NS	2.707 NS 0.298 NS	0.248 NS 0.041 NS	7.236 * 1.034 NS	2.920 NS 1.233 NS	2.316 NS 0.696 NS	10.628 ** 0.789 NS
	July17	Type W×G	1	--	4.650 . 0.076 NS	10.650 ** 0.864 NS	33.326 *** 34.278 *** 7.417 * 1.068 NS 1.103 NS 0.207 NS	39.259 *** 126.101 *** 1.611 NS 0.014 NS
		Watering 1 T×W×G	--	0.607 NS 2.241 NS	0.020 NS 0.537 NS	1.498 NS 0.144 NS	4.649 * ↑ 0.180 NS	1.345 NS 1.308 NS	0.681 NS 0.042 NS	7.098 ** ↑ 0.291 NS
		Grazing	1	-	0.313 NS	3.821 . ↓	10.557 ** ↓ 16.291 ** ↑ 0.573 NS	0.465 NS	13.222 ** ↑
		T×W	1	-	0.257 NS	0.005 NS	0.065 NS	2.381 NS	0.051 NS	0.959 NS	0.246 NS
		T×G	1	-	0.977 NS	1.574 NS	1.278 NS	0.023 NS	1.141 NS	0.484 NS	2.181 NS
		W×G	1	-	0.006 NS	0.542 NS	0.713 NS	0.207 NS	0.588 NS	0.032 NS	1.062 NS
		T×W×G	1	-	5.523 *	3.804 .	11.354 **	7.116 *	5.733 *	10.157 **	0.153 NS

Table S2 :

 S2 Effects of initial watering, grazing, seedling type and observation date on B. retusum survival. ANOVA F-values, significance levels: . P < 0•1, * P < 0•05, ** P < 0•01, *** P < 0•001, NS not significant.

	Factors	df	Survival rate
	Type	1	7.889 **
	Watering	1	0.850 NS
	Grazing	1	3.151 .
	Date	4	65.450 ***
	Type × watering	1	0.366 NS
	Type × grazing	1	1.313 NS
	Watering × grazing	1	31.211 ***
	Type × date	4	1.733 NS
	Watering × date	4	0.279 NS
	Grazing × date	4	1.072 NS
	Type × watering × grazing	1	1.368 NS
	Type × watering × date	4	0.679 NS
	Type × grazing × date	4	1.315 NS
	Watering × grazing × date	4	4.275 **
	Type × watering × grazing × date	4	0.600 NS

[START_REF] Saatkamp | Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland[END_REF]. The position of water treatments was randomized within half-blocks

ACKNOWLEDGEMENTS

We thank Daniel Pavon, Elise Buisson, Hervé Ramone, Kevin Maurin and Cándido Gálvez Ramírez (Semillas Silvestres) for collecting seeds. Vivien Carrere and Lara Amorsi helped in setting up the common garden experiment. We are grateful to Anne Blondeau Da Silva for advice in using R Pstat package. This work was supported by the French Federal State PACA (PhD grant) and the University of Avignon (Excellence program).

Chapter I

ACKNOWLEDGEMENTS

We thank Stéphane Ruy for water potential measurements and Vivien Carrere, Lara Amorsi, Hervé Ramone, Sébastien Dailly, Julien Formento, Hugo Lezarme, Kevin Maurin, Cannelle Moinardeau, Julie Chenot, Anne Aurière, Lorenzo Ottaviani, Jean-François Alignan, Olivier Blight and Elise Buisson for field assistance. We are grateful to the "Conservatoire des Espaces Naturels" (CEN-PACA) for the permit to collect seeds in the Nature Reserve "La Crau". This work was supported by the "Région PACA" (PhD grant) and the University of Avignon (Excellence program). We also thank two anonymous reviewers for their helpful and constructive comments. Chapter II ACKNOWLEDGEMENTS We thank Sébastien Dailly, Julien Formento, Hugo Lezarme, Cannelle Moinardeau, Julie Chenot, Jean-François Alignan, Olivier Blight, Daniel Pavon and Elise Buisson for field assistance. Perrine Lagarde and Aline Le Menn helped in running germination tests. We also thank Fanny Sauguet for identifying field plots and the sheep farmers Mr. Tavan and Ms Trouillard, the Federal State Nature Conservation Authority (CEN-PACA) and Crau Nature Reserve for the permit to set up the experiment. The study was funded by the French Federal State PACA (PhD grant) and the University of Avignon. Chapter III ACKNOWLEDGEMENTS We thank Olivier Blight, Lucie-Valentine Dupont, Perrine Lagarde and Lorenzo Ottaviani for field assistance. We also thank Fanny Sauguet for identifying field plots and the sheep farmers Mr and Ms Groulet, the Federal State Nature Conservation Authority (CEN-PACA) and Crau Nature Reserve for the permission to set up the experiment. The study was funded by the French Federal State PACA (PhD grant).

Chapter IV

Merci à Yunier

Chapter II

Adaptive differentiation among populations of the Mediterranean dry grassland species Brachypodium retusum -the role of soil conditions, grazing and humidity.

Abstract

 Background and Aims

Genetic differentiation in plant species may result from adaptation to environmental conditions but also from stochastic processes. The drivers selecting for local adaptation and the contribution of adaptation to genetic differentiation are often unknown. Restoration and succession studies have revealed different colonization patterns for Brachypodium retusum, a common Mediterranean grass. In order to understand these patterns, we tested population differentiation and adaptation to different environmental factors.

 Methods

Structured sampling of twelve populations from six sites and two soil types within site was used to analyze the spatial and environmental structure of population differentiation. Sampling sites differ in grazing intensity and climate. We tested germination and growth in a common garden.

In subsets, we analyzed the differential response to stone cover, grazing and soil moisture.

 Key Results

We found significant differences among populations. Site explained population differentiation better than soil suggesting a dominant influence of climate and/or genetic drift. Stone cover had a positive influence on seedling establishment and populations showed a differential response.

However, this response was not related to environmental differences between collection sites.

Regrowth after clipping was higher in populations from the more intensively grazed Red Mediterranean soils suggesting an adaptation to grazing. Final germination was generally high even under drought but germination response to differences in soil moisture was similar across populations.

 Conclusions

Adaptive population differentiation in germination and early growth may have contributed to different colonization patterns. Thus, the provenance of B. retusum needs to be carefully considered in ecological restoration. What is the effect of fire and grazing on vegetative growth and sexual reproduction of Brachypodium retusum, the dominant species of Mediterranean Thero-Brachypodietea grasslands ("steppes") and on species composition of the associated plant community?

 Location

"La Crau", South-Eastern France.

 Methods

We set up a split-plot experiment testing the effects of fire season (winter, summer) and grazing on plant cover, inflorescence number, seed set and germination of B. retusum in traditionally managed grasslands. We further analyzed plant species composition and diversity in vegetation relevés. The same fire treatments were tested in a second experiment in long-term exclosures abandoned 17 years ago.

 Key Results

B. retusum showed a significant rapid post-fire recovery but increase in plant cover was not faster than in other species of the plant community. Fire increased B. retusum inflorescence production and seed set per inflorescence and this effect was stronger in the summer fire treatment. At community level, fire significantly increased species richness, evenness and Bray-Curtiss dissimilarity in the second post-fire season and again, the summer fire effect was stronger. Grazing exclusion for two seasons had only a small effect on B. retusum and plant community. The effect of both fire treatments on B. retusum was similar in long-term exclosures. In these exclosures, fire resulted in a community shift towards traditionally managed steppes.

 Conclusions

The concomitant positive effects of experimental burning on B. retusum reproduction and on plant diversity of Mediterranean steppe vegetation suggest that the system is adapted to fire as an important driver of community dynamics. Annual species surviving as seeds are as successful in post-fire recovery as perennial resprouters. Prescribed burning may be an alternative strategy to restore community structure in abandoned steppes showing a decline in typical grassland species after several years of grazing abandonment.

Keywords: exclosures; Mediterranean grassland; plant community; plant-environment interactions; population dynamics; prescribed burning; Thero-Brachypodietea.

treatments was randomized within half-blocks and that of grazing treatment was randomized within blocks.

A thermal weeder was used to burn plots to simulate winter fire and summer wildfire. Thermal weeding was achieved using a portable device with a gas cylinder and a ramp comprising four nozzles for flame weeding (Appendix S4). Due to fast fire spread during summer, we removed the device once the vegetation was burning. We stopped the fire manually when flames arrived at the plot border. To prevent grazing, the control half-blocks were fenced with stretched, nonelectrified nets (1 m high). The other half-blocks allowed free sheep grazing during the grazing period from April to June. The mean grazing intensity was of 646 sheep*ha -1 *season -1 at Négreiron site and 328 sheep* ha -1 *season -1 at Collongue site.

In July 2016, the aboveground vegetation was harvested in two plots of 50*50 cm in each grazed and ungrazed half-block to measure biomass removal by sheep. Samples were dried at 80°C to a constant biomass and weighed afterwards. Grazing reduced aboveground biomass from 70 ± 11g/m² in ungrazed plots to 50 ± 9g/m² at the end of the grazing period.

Effect of fire in long-term exclosures

The same fire treatments were established in long-term grazing exclosures. The aboveground vegetation was harvested as described above. Standing biomass (110 g/m² ± 16) was twice as high as in traditionally grazed steppe area. The design included three plots per block comprising the three fire treatments and six replicate blocks (18 plots in total). Corresponding to the first experiment, three blocks were set up at the Négreiron site fenced in August 2001 (3 ha) and three blocks at the Collongue site fenced in June 1999 (2.9 ha). The distance between blocks and to exclosure fences was at least 50 m.

Data collection

B. retusum survey

The cover percentage of the species was estimated at six dates (May, July, October 2016 andFebruary, May, July 2017) including two growing seasons. At the same time, total vegetation cover was estimated. Cover was measured as the vertical projection of all aboveground organs.

Visual cover estimation is as reliable as the more objective pin-point or point-intercept method and allows an analysis of larger areas [START_REF] Damgaard | Estimating mean plant cover from different types of cover data: a coherent statistical framework[END_REF] Meteo-France).

Appendix S2: Vascular plant species found at the study sites.

Species

Aegilops geniculata Roth

Aegilops triuncialis L.

Aira cupaniana Guss.

Ajuga iva (L.) Schreb.

Allium sphaerocephalon L.

Andryala integrifolia L.

Anisantha madritensis (L.) Nevski, Anisantha rubens (L.) Nevski Arenaria serpyllifolia L.

Asperula cynanchica L.

Asphodelus ayardii Jahand. & Maire

Avena barbata Pott ex Link Avena sterilis L.

Tragopogon porrifolius L.

Trifolium campestre Schreb.

Trifolium cherleri L.

Trifolium glomeratum L.

Trifolium scabrum L.

Trifolium stellatum L.

Trifolium subterraneum L.

Trifolium suffocatum L.

Trifolium tomentosum L.

Tyrimnus leucographus (L.) Cass.

Urospermum dalechampii (L.) Scop. ex F.W.Schmidt

Urospermum picroides (L.) Scop. ex F.W.Schmidt

Valerianella discoidea (L.) Loisel.

Verbascum sinuatum L.

Veronica arvensis L.

Vulpia ciliata Dumort.

Vulpia myuros (L.) C.C.Gmel. Chapter

Transition to Chapter IV

In Chapter III, we showed that fire had a positive effect on vegetative regeneration and reproduction of B. retusum even though its recovery is lower than community average.

Moreover, it has a positive effect on species richness and diversity of the associated community.

Short-term grazing exclosure (2 years) had no effect on B. retusum but long-term abandonment (17 years) reduced relative B. retusum cover and resulted in a community shift towards ruderal perennials.

In Chapter IV, we tested in situ germination and seedling establishment to evaluate colonisation capacity in the field. In Mediterranean dry grasslands, the limiting factors may be water availability and grazing although grazing is required to maintain the system with its dominant plant species and a higher soil moisture favour other species not adapted to summer drought.

We hypothesize that initial watering favours germination and seedling recruitment whereas early grazing has a negative effect.

Contribution of the thesis -Knowledge on environmental factors limiting germination and early establishment:

water availability and grazing.

-Information on general in situ colonisation capacity -Recommendations for conservation management and plant re-introduction.

Chapter IV Which factors limit early establishment of Brachypodium retusum a dominant species of Mediterranean grasslands? Chapter IV

Abstract

In Mediterranean dry grasslands, water availability and grazing regimes are known to be limiting factors for plant species establishment. However, their join effect on species establishment have received little attention especially on Brachypodium retusum, the dominant perennial grass species of Mediterranean dry grasslands which populations are particularly impacted by land use changes.

In our study, we aimed to analyse the effects of grazing and watering on seedling recruitment and early establishment of B. retusum in a former cultivated field located in "La Crau", a vast steppe vegetation area in Southern France, for improving the re-establishment of this species in future ecological restoration projects.

A split-plot experiment was set up in order to test the effects of grazing and initial watering on in situ seeds germination. Grazing and initial watering effects have also been tested on seedling survival, seedling height, leaf length, tiller number, leaf number and seedling diameter for (i) transplanted seedlings and (ii) sown seedlings.

Grazing negatively affected germination rates whereas watering effect was not significant. At the end of the survey period, survival was 49 % for transplanted seedlings and 36 % for sown seedlings after two grazing seasons. One original significant interaction occurred between grazing and watering. Initial watering had a positive effect in grazed plots compensating for recurrent grazing "perturbations". Grazing also significantly reduced plant height, leaf number and length but these effects disappeared six months after the end of the grazing period.

Nevertheless, grazing also significantly increased tillering while watering had an expected significant positive effect on plant growth. These results are then discussed for restoring B.

retusum populations in former arable fields. Sowing in ungrazed fenced areas instead of watering and transplanting seedlings in grazed areas could be then advised and would allow to reduce the economic cost and environmental impacts of such operations.

Keywords: Dry grassland; Plant-environment interactions; Poaceae; Population dynamics; Thero-Brachypodietea

RESULTS

Field germination ranged from 7.1 % (unwatered and grazed plots) to 12.9 % (watered and ungrazed plots). Grazing had a significant negative impact on germination (Table 1, Fig. 2) as B. retusum seeds from ungrazed plots germinated significantly better than those from grazed plots. The effect of watering and the grazing × watering interaction were not significant. Genetic diversity is affected by gene flow, drift and selection but also strongly depends on breeding system [START_REF] Hamrick | Effects of life history traits on genetic diversity in plant species[END_REF]. In Chapter I, a high genetic diversity was found and only one population showed a small number of private alleles. B. retusum is capable of sexual (cross-pollination) and vegetative reproduction (through ramet-forming rhizomes). However, the relative contribution of sexual and vegetative reproduction is not known. In Chapter I, we demonstrated that in spite of vegetative reproduction, the species maintain a high diversity suggesting that gene flow and sexual reproduction are high in the Western Mediterranean.

SUPPORTING INFORMATION

General discussion

The ecological consequences of genetic diversity are numerous as it affects all higher organisation levels such as individuals, populations and even communities, with, in some cases, a similar magnitude to the effects of species diversity [START_REF] Hughes | Ecological consequences of genetic diversity[END_REF]. Genetic diversity may affect several ecological processes such as primary productivity, population recovery from disturbance, interspecific competition, community structure, and fluxes of energy and nutrients.

Genetic diversity and variation enables species and populations to adapt to environmental changes and allows organisms to establish in different environments [START_REF] Reed | Correlation between Fitness and Genetic Diversity[END_REF][START_REF] Frankham | Genetics and extinction[END_REF]).

Adaptive differentiation is common in plants and often occurs conjointly with drift. A main current question in ecology and evolution is how local is adaptation [START_REF] Mckay | How local is local?"-a review of practical and conceptual issues in the genetics of restoration[END_REF][START_REF] Sutherland | Identification of 100 fundamental ecological questions[END_REF]. In Chapters I and II, we found a regional pattern of adaptive differentiation but also evidence for drift. Genetic drift can contribute to population differentiation and reduce the response to selection pressures [START_REF] Wright | Evolution in Mendelian Populations[END_REF][START_REF] Loveless | Ecological Determinants of Genetic Structure in Plant Populations[END_REF]. It was found to be major force causing population differentiation in quantitative traits (e.g. [START_REF] Lande | Natural Selection and Random Genetic Drift in Phenotypic Evolution[END_REF][START_REF] Linhart | Evolutionary Significance of Local Genetic Differentiation in Plants[END_REF]. Moreover, geographic distance between populations may also contribute to the restricted gene flow [START_REF] Slatkin | Gene flow and the geographic structure of natural populations[END_REF]) and thus to differentiation. Sexton et al. (2014) found that concomitant isolation-by-distance and isolationby-environment is the major pattern found in plant species. However, in Chapter I, isolationby-environment remained significant even after controlling for isolation-by-distance confirming local adaptation.

The magnitude of local adaptation depends on several factors such as the environmental heterogeneity, the strength of selection pressure, among-population connectivity and genetic variation within populations [START_REF] Leimu | A Meta-Analysis of Local Adaptation in Plants[END_REF][START_REF] Bolnick | The magnitude of local adaptation under genotype-dependent dispersal[END_REF]. Local adaptation is considered to be stronger in populations that occur in heterogeneous environments, that experience high selection pressure, and that show restricted gene flow and sufficient genetic variation [START_REF] Pigliucci | Differentiation for flowering time and phenotypic integration in Arabidopsis thaliana in response to season length and vernalization[END_REF][START_REF] Conner | A primer of ecological genetics[END_REF]. Strong directional selection is

Appendices

Appendix I: Brief description of thematic fields used in the thesis and brief glossary.

Population biology

A population is a group of individuals belonging to the same species (interbreeding individuals) and living in the same geographic area [START_REF] Krebs | The experimental analysis of distribution and abundance[END_REF]. Appendix II: In situ and common garden seed set.

Observations in the field suggested a low in situ seed production. Seed production and seed set were therefore measured in situ and in common garden. In order to measure population differentiation in the common garden studies of the Chapters I and II, in one inflorescence per sampled patch, the number of seeds and flowers was counted to calculate the seed set.

Remaining inflorescences were shaken in tubes with glass beads using a vortex mixer to separate seeds from glumes. Transparency under transmitted overhead projector light was used as a criterion to remove empty seeds without embryo. In situ seed set could thus be measured.

This experiment was also used to compare the seed set in a common garden with the seed set at the collection sites (see above).

Separate models were run for seed set at collection (parental generation, influenced by maternal environment) and common garden sites (F1). The parental seed set model included site, soil and their interaction. Soil and site effects combine seed set differences resulting from genetic differentiation but also from phenotypic plasticity according to different environmental conditions. The F1 seed set model additionally included block as all other common garden study models. The main objective of this approach was to test whether the general seed set level, but also differences between populations, are similar at original collection sites and under standard common garden conditions. As for the germination tests, seed set percentage was arcsin (sqrt(x/100)) transformed prior to analysis.

A Tuckey HSD posthoc test was calculated to analyse differences between treatment levels more in detail if the treatment main effect or interactions were significant. Posthoc tests were preferred over a priori linear contrasts because we had no clear a priori hypothesis on specific adaptation to tested environmental factors. All statistical analyses were run in R (R, version 3.3.1, R Development Core Team ( 2013)).

Seed set data were obtained from a subset of populations planted in the common garden and compared with seed set of parental plants at the original collection sites. Mean seed set was lower than 10% in parental plants in situ, but between 20 and 25% in the common garden (Figure A). Seed set in parental plants differed between sites (F5, 663: 56•346 ***), but the site effect depended on soil type (site x soil, F5, 663: 3•28 **) whereas the soil of origin had no overall effect (F1, 663: 0•95). Seed set was higher on Red Mediterranean soils than on calcareous soils at three sites and higher on Calcareous soils at one site (Saint-Rémy-de-Provence). In the common garden, these differences in seed set between original sites and soils disappeared.

Extended abstract

The ecological restoration of Mediterranean Thero-Brachypodietea grasslands ("steppes")

based on spontaneous succession following soil disturbance is a very slow process. Even after several decades of restoration, the species composition is often different from that of the reference community. In particular, key species hardly establish. Brachypodium retusum is a dominant widespread core species of such dry Mediterranean grasslands managed by grazing.

However, approaches to restore such grasslands and to re-establish B. retusum have failed until now in the greatest French Mediterranean steppe area of the Crau plain. Previous studies showed that the recreation of initial environmental conditions is not sufficient to restore the plant community with its dominant species. Even the transfer of B. retusum rich hay has not been successful to re-establish populations suggesting a low seedling recruitment. The absence of the dominant species has a strong effect on community structure and functioning.

Furthermore, the species plays an important role in sheep nutrition and its re-establishment on degraded grassland systems would thus represent an agro-economic benefit.

Surprisingly, former studies in other Mediterranean systems have not confirmed the slow recolonisation. The species rapidly re-occur after wildfires and is generally frequent in habitats of moderate disturbance. This may be explained by the strong resprouting capacity from belowground organs representing a competitive advantage over annual species. However, a high re-colonisation was even found after intensive soil disturbance by ploughing.

The PhD thesis aims to identify the causes for the low establishment of Brachypodium retusum in Mediterranean steppes and in particular in the Crau area. Two main hypotheses are tested (i) the genetic differentiation explains differences in colonisation capacity, (ii) colonisation depends on environmental conditions such as humidity, grazing or fire that may limit or favour seedling recruitment. This project allows us to better understand the ecology of this key species in order to improve the efficiency of restoration measures.

Specifically, this PhD thesis aims to answer to the following major questions:

(1) What are the spatial and environmental scales of genetic differentiation? Is differentiation adaptive? What are the major drivers of differentiation and adaptation? What are the implications for provenance choice in ecological restoration?

(2) Which ecological factors limit establishment? In particular, do fire, grazing, initial watering and their interactions improve or reduce the establishment of B. retusum acting on seed production, germination, survival and growth?

Extended abstract

The thesis comprises a general introduction, four research chapters and a general discussion.

Each research chapter is written as a stand-alone paper. One paper is currently published, one is in revision (minor), one is submitted and the last one in preparation for a submission by the end of October 2018. In Chapter I, the mechanisms driving population differentiation and adaptation were analysed by comparing pairwise θST and PST. Seeds or leaves from seventeen populations were collected in France (14 populations), Spain (2 populations) and Italy (1 population) for a total of 322 individuals genotyped with 323 AFLP markers. A subset of thirteen French populations was grown for two years in a common garden experiment to measure vegetative growth and reproduction. Based on AFLP marker analysis, diversity indices and their relations to climate and soil conditions were calculated. Population differentiation in neutral AFLP markers was estimated using pairwise θST. First axis PCA scores of quantitative traits were fitted to determine

Extended abstract

PST values (adaptive differentiation), and PST were compared to θST. Global dataset θST (0.102, P < 0.001) indicates significant differentiation between populations. PST (0.171; P < 0.001) was higher than θST (0.072; P < 0.001) suggesting that phenotypic population differentiation is driven by directional selection leading to adaptive differentiation. This adaptive differentiation at a regional scale (Southern France) advocates a careful choice of plant origin in ecological restoration.

The aim of Chapter II was to focus on adaptive population differentiation by manipulating environmental factors in common garden and growth chamber experiments. Different subsamples of populations tested in the genetic analyses (Chapter 1) were used to analyse the spatial and environmental structure of population differentiation. These subsamples comprised twelve populations from six sites and two soil types (Red Mediterranean, calcareous) within site. Sampling sites differ in grazing intensity and climate. In these populations of different site and soil origin, we analysed the differential response to stone cover, grazing and soil moisture.

We found significant differences among populations. Site explained population differentiation better than soil suggesting a dominant influence of climate and/or genetic drift. Stone cover had a positive influence on seedling establishment and populations showed a differential response.

However, this response was not related to environmental differences between collection sites.

Regrowth after clipping was higher in populations from the more intensively grazed Red

Mediterranean soils suggesting an adaptation to grazing. Final germination was generally high even under drought but germination response to differences in soil moisture was similar across populations. Adaptive population differentiation in germination and early growth may have contributed to different colonisation patterns. The results confirm that the provenance of B.

retusum needs to be carefully considered in ecological restoration.

Seed production and seedling establishment represent a strong filter for re-colonisation of plant species. In Mediterranean dry grasslands, three main factors influence population dynamics.

Sheep grazing is one of the key management factors driving plant species composition and diversity. Wildfires and fire management were also important drivers in the past but their role in maintaining the ecosystem is nowadays less clear because fire prevention and abandonment of traditional prescribed burning have largely reduced their impact in the last decades. Drought stress may be a third factor limiting seedling recruitment.

The last two parts of the thesis analysed environmental factors that may limit establishment:

the effect of grazing and fire on vegetative regeneration as well as on the sexual reproduction of adult B. retusum (Chapter III) and the impact of watering and grazing on the growth of transplanted and sown seedlings of local origin (Chapter IV).

The aim of Chapter III was to understand the effect of fire and grazing on vegetative growth and sexual reproduction of adult Brachypodium retusum since seed production in potential source populations may largely influence seed availability and recruitment at adjacent restoration sites. These factors were also tested on species composition of the associated plant community in Mediterranean steppe of the Crau (South-Eastern France) because fire cannot be suggested as a restoration tools for increase B. retusum colonisation without considering its effects on the whole plant community.

We set up a split-plot experiment testing the effects of fire season (late winter, summer) and grazing on plant cover, inflorescence number, seed set and germination of B. retusum in traditionally managed grasslands. We further analysed plant species composition and diversity in vegetation relevés. The same fire treatments were tested in a second experiment in long-term exclosures abandoned 17 years ago using the same protocol of measurements.

B. retusum showed a significant rapid post-fire recovery but increase in plant cover was not faster than in other species of the plant community. Nevertheless, summer fire increased inflorescence production of the species and seed set per inflorescence. At community level, fire significantly increased species richness, evenness and Bray-Curtiss dissimilarity and again, the effect of summer fire was stronger. Grazing exclusion for two seasons had only a small effect on B. retusum and plant community. The effect of the two fire treatments on B. retusum was similar in long-term exclosures. In these exclosures, fire resulted in a shift in plant species composition towards the traditionally managed steppe.

The concomitant positive effects of experimental burning on B. retusum reproduction and on plant diversity of Mediterranean steppe vegetation suggest that the system is adapted to fire as an important driver of community dynamics. Seedling recruitment of annual species is as successful in post-fire vegetative recovery as resprouting of perennials such as B. retusum.

Prescribed burning may be an alternative strategy to maintain community structure in abandoned Mediterranean grasslands. However, plant community and dominant species tolerate the absence of grazing in the short term (two years) showing no significant changes in tested response variables.

In Chapter IV we analysed the effects of grazing and watering on seedling recruitment and early establishment of Brachypodium retusum. Grazing is known to favour the species but the effect on seedling recruitment may still be negative. Inversely, soil moisture increases

Extended abstract

competition and favours ruderal species in the long run but initial watering may still be beneficial to establish young seedlings.

We set up a split-plot experiment testing the effects of grazing and initial watering on seedling height, leaf length, tiller number, leaf number and seedling diameter for (i) transplanted seedlings and (ii) sown seedlings on ex-arable fields of the Crau.

Survival after one year was 40 % for transplanted seedlings and 27 % for sown seedlings.

Grazing negatively affected field germination whereas the watering effect was not significant.

Grazing reduced plant height but the effect disappeared six months after the grazing period.

Watering had a significantly positive effect on early plant growth that disappeared in the second growing season. Main effects of both factors on survival were not significant but a significant interaction occurred: initial watering had a positive effect only in grazed plots compensating for recurrent "grazing stress". In ecological restoration, grazing exclosure in the beginning may reduce negative drought effects on survival. If not possible, initial watering is then an alternative way to limit seedling mortality.

In conclusion, the adaptive differentiation between populations demonstrated in my studies may have contributed to regional differences in terms of colonising capacity. The combination of drought and grazing stress limits seedling recruitment and survival during the hot and dry Mediterranean summer. Fire generally increases seed production of potential source populations and may thus be an original tool for restoring degraded areas through indirectly favouring the arrival of seeds on restoration sites.