Fabien Anthelme

Marie-Lise Benot

Kathrin Kiehl

François Mesléard

Vicky Temperton

Truman Young D'

Un Gran Agradecimiento A Luis

Daniel Llambi

Thierry Duthoit

Tania De Almeida

Cannelle Moinardeau

Quentin Lambert

Marie Perrin

Christopher Burot

Julie Braschi

Julie Lescure

Virginie Montelle

Aline Le Men

Dominique Messaoudi Merci Margaux D'ambly

Léo Rocher

Antoine Beltrame

Christel Vidaller

Tiago Toma

Manon Hess

Je Tiens

Yoann Suty

Jeanne Pénaud

Sophie Delastre

Damien Mottier

Loïc Larue

Hélène Durbecq-Bassoul

Gérard Durbecq

Soeurs Thomas

Mathias Durbecq

Cécile Labeyrie

Emilie Souzeau

Kael Durbecq

Elio Linaly

Durbecq-Labeyrie

Aure Que J'

Durbecq-Bassoul

Keywords: assemblage des communautés végétales, écologie de la restauration, écosystème de référence, effets de priorité, dégradation du sol, pelouse de montagne, prairie de montagne, succession végétale, restauration écologique, transfert de propagule ecological restoration, mountain grassland, plant community assembly, plant succession, priority effects, propagule transfer, reference ecosystem, restoration ecology, soil disturbance Bromopsis erecta, brush harvesting, community ecology, ecological engineering, grassland restoration, grazing, harrowing, montane grasslands, Rhinanthus alectorolophus, seedbed preparation, seedling establishment, survival Semi-natural montane grasslands, Seedling recruitment, Nurse plant cover, Erosion control, Plant establishment, Facilitation ecological restoration, restoration ecology, mountain grassland, soil disturbance, plant community assembly, plant succession, priority effects

II

Les techniques de restauration appliquées ont indiqué que la réduction de la compétition par la préparation du sol avant l'ajout de graines issues de la brosseuse avait un effet positif sur le recrutement des espèces transférées, et peut donc être clairement recommandée dans mon système d'étude. De plus, j'ai constaté que le faible effet négatif du pâturage ne justifiait pas toujours les coûts des exclos. Le paillis de foin a favorisé le recrutement des semis d'espèces cibles, mais le transfert de propagules sans couche de paillis devait être compensé par un couvert végétal temporaire pour être efficient, ce qui suggère que la restauration des prairies de montagne avec des sols peu profonds et caillouteux bénéficie clairement d'un effet facilitateur de couvert végétal mort (foin) ou vivant (blé). Enfin, le chapitre 4 a fourni des preuves que l'assemblage de la communauté végétale était influencé par l'ordre d'arrivée des espèces, mais a aussi mis en évidence de fortes variations de réponse entre les espèces, suggérant des effets de priorités spécifiques à chaque espèce. La force des effets de priorité varie en fonction des caractéristiques de la niche qui diffèrent entre les espèces et peuvent influencer (négativement ou positivement) l'établissement des espèces arrivant ultérieurement.

I

Résumé

La dégradation écologique à grande échelle des prairies et pelouses augmente dans le monde entier, principalement en raison des changements d'utilisation des terres et de la destruction des habitats. La construction d'une ligne électrique à haute tension dans la zone d'étude, la vallée de la Haute-Durance dans les Hautes-Alpes, a entraîné une dégradation des sols et des communautés végétales le long de voies d'accès transitoires et de plates-formes de construction. Dans les écosystèmes perturbés de la zone d'étude, la restauration est limitée par un manque de propagules disponibles et un faible recrutement des graines sur sols peu profonds et caillouteux caractérisés par une sécheresse estivale marquée. Mon étude se concentre sur la restauration de prairies sèches à mésophiles riches en espèces couvrant une grande partie de la zone de construction, situées entre 1000 et 1400 mètres d'altitude. La plupart de ces prairies sont des habitats prioritaires Natura 2000 tels que N6210 'Prairies sèches semi-naturelles de Festuco-Brometalia et faciès de garrigue sur substrats calcaires'(directive UE habitat 92/43 / CEE).

Ma thèse vise à mieux comprendre les facteurs limitant la restauration des prairies de montagne après une perturbation du sol, et à identifier des techniques de restauration pour compenser la dégradation. Le chapitre 1 se concentre sur l'identification d'une référence appropriée qui est une étape cruciale pour la restauration écologique. Afin de comprendre les mécanismes favorisant l'établissement des espèces cibles, cinq pelouses semi-naturelles dégradées ont été utilisées pour mettre en place trois expériences différentes, en utilisant une approche basée sur les filtres d'assemblage des communautés pour analyser les résultats de la restauration. Dans la première expérimentation (Chapitre 2), j'ai examiné si la préparation du sol était nécessaire pour fournir des conditions de sol plus appropriées et réduire la compétition avec la végétation préexistante, et pour voir si le pâturage extensif traditionnel devrait être exclu lors des premières étapes de restauration. Dans la deuxième expérimentation (Chapitre 3), je me suis concentrée sur différentes techniques de transfert de propagules de plus en plus utilisées dans les prairies de montagne (propagules récoltées à la brosseuse et transfert de foin), et j'ai analysé l'effet potentiellement facilitateur d'un couvert végétal mort (foin) et vivant (blé) sur le recrutement des propagules transférées et sur l'érosion du sol. Enfin, comme l'assemblage des communautés est également fortement influencé par la dispersion, qui elle-même peut conduire à des effets de priorité, j'ai testé le semis séquentiel de différentes combinaisons d'espèces végétales (dominantes vs. subordonnées) dans une troisième expérimentation et j'ai analysé comment il affecte l'assemblage des communautés (Chapitre 4).

Les résultats ont montré que l'analyse des interactions plante-environnement fournit un outil important pour identifier les communautés de référence ou les sites sources pour la récolte de propagules, d'autant plus si ces sources ne sont pas disponibles à proximité des habitats dégradés.

My thesis aims to understand better the factors limiting the restoration of mountain grasslands after soil disturbance, and to identify successful restoration techniques to compensate the degradation. Chapter 1 is focused on the identification of appropriate reference communities, which is a crucial step in ecological restoration. In order to understand the mechanisms favouring target species establishment, five degraded seminatural grasslands were used to set up two different experiments, using a filter-based community assembly approach of community dynamics to analyse the restoration outcome. In the first one (Chapter 2), I examined whether soil preparation is required to improve soil conditions and to reduce competition of pre-existing vegetation, and whether traditional extensive grazing should be excluded in initial stages of grassland restoration. In the second experiment (Chapter 3), I focused on different techniques of propagule transfer increasingly used in mountain grasslands (brush-harvested propagules and hay transfer), and I analysed the potentially facilitative effect of dead (hay) and living (wheat) plant cover on the recruitment of transferred brush-harvested propagules, and on soil erosion. Finally, as community assembly is also highly influenced by dispersal that may lead to priority effects, I tested sequential sowing of different combinations of plant species (dominants vs. subordinates) (Chapter 4) and I analysed how they affect community assembly.

The results demonstrated that the analysis of plant-environment interactions provides a useful tool to identify reference communities or donor sites for seed transfer, particularly if donor sites are not available adjacent to degraded habitats. The restoration techniques applied indicated that the reduction of competition by soil preparation before seed addition of brush material had a positive effect on the transferred species recruitment and can thus clearly be recommended in my study system. Furthermore, I found that the weak negative effect of grazing may not always justify fencing costs.

Table index

XIII

Table index

General introduction ………………………………………………………………………………………………..… 1 Table I.1. Filter-based restoration actions carried out during the thesis ……………………..…..… 15 Chapter 1. Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition ……………………………….…….. 17 Table 1.1. Similarities between restoration sites (1R, 2R, 3R, 11R, 20R) and the non-degraded sites (from site 4 to 24, without R) of the study zone (similarity decreasing from left to right) using the Euclidean distance for environmental variables shown in Fig. 3 ………………….….. 28 Supplement S1.1. Estimation of annual direct incident solar radiation (ASR) using the method of McCune and Keon (2002) ……………………………………………………………………………………...….. 32 Table S1.1. Correlations between environmental variables……………………………………………… 33 . Table S1.2. Relationships between grassland plant composition (as represented in Fig. 2) and six environmental gradients using "envfit" with N = 999 permutations (R package "vegan"). The significant variables are in bold …………………...…………………………………………………..…..… 33 Table S1.3. Complete names of plant species represented in the RDA biplot (Fig. 3) …….…… 34 Chapter 2. Seedling recruitment in mountain grassland restoration: effects of soil preparation and grazing ………………………………………………………………...…………………….…… 39 Table 2.1. Effect of harrowing and grazing on number of seedlings per m², species richness and the survival of two focal species. Results on seedling number and richness are presented separately for the whole plant community and the subsample of transferred species. H x G = grazing-by-harrowing interaction. Results of GLMM with Chi-Square (χ2) and significance levels: . P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant …………………………… 47 Table S2.1. Number of seedlings of transferred species from brush material measured in the greenhouse and in the field. Nomenclature follows [START_REF] Tison | Flore de la France méditerranéenne continentale[END_REF] Competitive, stress and resistance strategy according to [START_REF] Grime | Benefits of plant diversity to ecosystems: immediate, filter and founder effects[END_REF], Erschbamer (2007), and [START_REF] Pierce | Allocating CSR plant functional types: the use of leaf economics and size traits to classify woody and herbaceous vascular plants[END_REF]. Number of seeds sown / m² adjusted according to seed producer expertise on field germination (Phytosem, Gap, France). Frequency of occurrence and average cover according to [START_REF] Durbecq | Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition[END_REF] ……………………………………………….……….… 89 Table S4.1. Analysis of sown species cover, their priority and secondary indices. Results of Wilcoxon-test (ꭓ²) Integrated framework of ecological restoration and community assembly filters after a disturbance. The natural variability of the community before disturbance is characterised by the range of stable states. A range of reference communities encompass the range of stable states to consider the potential restored community trajectories under both deterministic and stochastic processes. At the end of the disturbance, non-linear community trajectories undergo the pressure of the three main assembly filters (different filter sizes indicate their hypothesized relative strength over time and double arrow indicates biotic interactions and environmental feedbacks). The recovery debt represents the gap between the community under restoration and the references [START_REF] Moreno-Mateos | Anthropogenic ecosystem disturbance and the recovery debt[END_REF][START_REF] Strobl | Integrated assessment of ecosystem recovery using a multifunctionality approach[END_REF]. Modified from [START_REF] Hobbs | Towards a Conceptual Framework for Restoration Ecology[END_REF], including elements of [START_REF] Chang | Integrating succession and community assembly perspectives[END_REF] The four major steps of the proposed method to select references. ① Delimit an extend study area around restoration sites; ② Identify environmental factors structuring plant communities (data collection and driver analysis); ③ Evaluate environmental distances between restoration and potential reference sites (dissimilarity coefficient); ④ Select the most similar references to restoration sites (dissimilarity coefficient) ……………. 22 Fig. 1.2. Location of the study area with restoration sites (1R, 2R, 3R, 11R and 20R, in red) and reference sites (numbered from north to south, without R, in blue) along the construction of a new electricity line ……………….…………………………………………………….….… 24 Fig. 1.3. RDA biplot using Hellinger-transformed plant species cover (species names in blue) and reference sites (green) constrained by environmental variables (red arrows). Environmental variables: ASR = Annual Solar Radiation; P2O5 = plant available phosphorous; soil moisture; soil pH; elevation and K2O = plant available potassium. Full species names: Table S1.3 ……………………………………………………………………………………………………..………………. 27 . Fig. 1.4. NMDS based on environmental variables showing a) the three most similar nondegraded sites (without R) for each restoration site (with R) and b) the non-degraded site that present the lowest average environmental distance with all the restoration sites. Different restoration sites are represented by different colours. The grey numbers represent the environmental distance (dissimilarity coefficient). See Table 1 for more details …....…. 29 Fig. 2.2. Number of seedlings per m² of (a) all species, and of (b) transferred species only, in the four restoration treatments. The direction of main effects is indicated using "<<" (P<0.01), "<" (P<0.05), "(<)" (P<0.1; marginally significant) ………………………………….…….… 48 Fig. 2.3. (a) Species richness of seedlings of all species and of (b) transferred species in the four restoration treatments. The direction of main effects is indicated "<" (P<0.05), "(<)" (P<0.1; marginally significant) and "=" (not significantly different) ………………….………….… 48 The direction of main effects is indicated using "<" (P<0.05) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05) ……………………………………..……………………………….…….…………………………..…………… 50 Fig. S2.1. Seedling emergence of transferred species from brush material measured in the greenhouse and in the field. For the field data, species occurring in control plots of a given site were excluded from measurements of this respective site. Number of seedlings are detailed in the table above ……………………………………………………………………………………….….. 56 The direction of main effects is indicated using "<<" (P<0.01), "<" (P<0.05), "(<)" (P<0.1) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05) ……………………………………..………………..…….… 57 Chap. 3. Mountain grassland restoration using hay and brush material transfer combined with temporary wheat cover ………………………………………………………..…….….… 61 [START_REF] Goldberg | Competitive Effect and Response: Hierarchies and Correlated Traits in the Early Stages of Competition[END_REF]. Dominants are displayed in red and subordinates in purple. Competitive response (proportion of focal species cover or biomass when grown with other species compared to monoculture) and effect (i.e. proportion of cover or biomass of other species when they are grown with the focal species compared to monoculture) are displayed according to the hypotheses drawn for each species group (dominant or subordinate. Hypothesis n°1: Subordinates show a low competitive response and therefore establish less well in pre-established communities, but resist if sown at first (b, purple line). Hypothesis n°2: Subordinates show a low competitive effect and therefore, do not or hardly affect other species regardless of arrival date (c, purple line). Hypothesis n°3: Dominant species show a highly competitive response and are therefore not affected by other species, even if sown later (b, red line). Hypothesis n°4: Dominant species show a strong competitive effect and therefore strongly affect other species when arriving first, but to a lesser degree when arriving later (c, red line) ………………………………………………...………………………………………....… 87 Fig. 4.2. Experimental design with a. the 40 plots including the ten replicates of the four Ecological restoration and restoration ecology are closely related [START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF]. The Society for Ecological Restoration (SER), a worldwide organisation founded in 1987 to guide restoration science and policy, defines restoration ecology as the scientific process that develops theories guiding ecological restoration, and ecological restoration as "the process of assisting the recovery of an ecosystem that has been degraded, damaged, or destroyed" (hereafter collectively referred to as 'disturbance'; Gann et al. 2019). Disturbance is an event that disrupts ecosystem, community, or population structure, thus changing the physical environment and its resources [START_REF] Pickett | The ecology of natural disturbance and patch dynamics[END_REF]. Such ecological disturbance may result in a partial or total loss of plant biomass [START_REF] Grime | Plant strategies and vegetation processes[END_REF][START_REF] Sousa | The Role of Disturbance in Natural Communities[END_REF][START_REF] Rykiel | Towards a definition of ecological disturbance[END_REF][START_REF] White | Disturbance, succession and community assembly in terrestrial plant communities[END_REF]. Therefore, the aim of most restoration ecologists is to recover the ecosystem with its original structure, composition, functions, and dynamics, by assisting habitat repair and recolonisation of local animal and plant species that were present before the disturbance.

Although ecosystems are continuously facing natural disturbances [START_REF] White | Disturbance, succession and community assembly in terrestrial plant communities[END_REF], human activities are nowadays the most important source of disturbance (UN Environment 2019). Since the beginning of the industrial revolution, humanity has been responsible for global environmental changes transforming ecosystems at an unprecedented rate causing significant damage to systems on which living beings depend [START_REF] Hobbs | Restoration Ecology: Repairing the Earth's Ecosystems in the New Millennium[END_REF][START_REF] Steffen | Global Change and the Earth System: A Planet Under Pressure[END_REF]. Human disturbances are responsible for massive natural habitat losses, for the extension of urban and agricultural areas (Donald 2004;[START_REF] Pansu | Impacts of anthropogenic activities on biodiversity : a spatial and temporal approach by analysis of environmental DNA[END_REF] and for an unprecedented global species extinction, often described as the irreversible sixth extinction crisis [START_REF] Ceballos | The sixth extinction crisis: Loss of animal populations and species[END_REF][START_REF] Chapin | Consequences of changing biodiversity[END_REF][START_REF] Lenzen | International trade drives biodiversity threats in developing nations[END_REF]. Land use transformation facilitated biological invasions and overexploitation of natural resources, pollution and climate change are continuous key processes contributing to biodiversity loss [START_REF] Vitousek | Human Domination of Earth's Ecosystems[END_REF]. The growing need to 'repair ecosystems'in the face of global change is now taken into account in political decisions. The United Nations have even declared the years 2021-2030 as the "Decade of Ecosystem Restoration". Hence, there is an increasing need to improve knowledge on how to restore degraded habitats? Since the end of 20 th century, many standards and practical guides have been published including the SER International Primer on Ecological Restoration (SER 2004), Ecological Restoration for Protected Areas: Principles, Guidelines and Best Practices [START_REF] Day | Guidelines for applying the IUCN protected area management categories to marine protected areas[END_REF], the National Standards for the Practice of Ecological Restoration in Australia (McDonald et al. 2016), and the International principles and standards for the practice of ecological restoration (Gann et al. 2019). These guides highlight the key stages of ecological restoration projects. However, the application of these principles must be adapted to the local context, such as habitat type and specific disturbance effects. Ecological restoration is therefore a context-based approach and restoration ecologists need to be familiar with general ecological theory that is the baseline of all restoration projects [START_REF] Gornish | Using strip seeding to test how restoration design affects randomness of community assembly[END_REF][START_REF] Lindenmayer | Improving Restoration Programs Through Greater Connection With Ecological Theory and Better Monitoring[END_REF]Palmer et al. 2006;[START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Temperton | Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF][START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF].

I.2. A synthesis of theory and practice

Today, it is commonly accepted by restoration ecologists that ecological theory and ecological restoration are closely connected. Since the beginning of restoration ecology as a discipline, many authors have emphasized this interdependence of theory and practice, which has spurred a particular enthusiasm resulting in several book publications [START_REF] Bradshaw | Restoration: an acid test for ecology[END_REF][START_REF] Hobbs | Novel ecosystems: implications for conservation and restoration[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Temperton | Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF]Walker et al. 2007). However, ecological theory is still not well integrated in restoration practice and recently, several authors advocated a stronger consideration of theory [START_REF] Audino | Drivers of community assembly in tropical forest restoration sites: role of local environment, landscape, and space[END_REF][START_REF] Corline | Understanding community assembly rules in managed floodplain food webs[END_REF][START_REF] Halassy | Trait-based approach confirms the importance of propagule limitation and assembly rules in old-field restoration[END_REF][START_REF] Lindenmayer | Improving Restoration Programs Through Greater Connection With Ecological Theory and Better Monitoring[END_REF]. The objectives of restoration ecology and ecological sciences are different since the former intends to guide the repair of ecosystems that have been degraded, while the latter aims to ultimately understand and explain how organisms interact with their environment. However, the restoration of degraded ecosystems requires a good understanding of ecosystem structure, mechanism, dynamics and natural variability [START_REF] Menninger | Restoring ecological communities: from theory to practice[END_REF][START_REF] Suding | The dynamic nature of ecological systems: Multiple states and restoration trajectories[END_REF]. In fact, the theoretical models of succession and assembly of communities are at the heart of restoration practice [START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF]. Ecological theory provides a framework that helps identifying the underlying mechanisms and ecological processes behind the observed patterns, and guidelines may be designed to improve predictions for the outcome of restoration actions [START_REF] Temperton | Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Gornish | Using strip seeding to test how restoration design affects randomness of community assembly[END_REF][START_REF] Lindenmayer | Improving Restoration Programs Through Greater Connection With Ecological Theory and Better Monitoring[END_REF]). Thus, many authors agree that restoration projects must be 'sciencebased restoration projects'by systematically integrating scientific methods and theoretical predictions as guiding concepts [START_REF] Menninger | Restoring ecological communities: from theory to practice[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Palmer | Ecological Theory and Community Restoration Ecology[END_REF][START_REF] Temperton | Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF][START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF].

Furthermore, the relationship between theory and practice is mutually beneficial (Falk et al. 2013;[START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Suding | The dynamic nature of ecological systems: Multiple states and restoration trajectories[END_REF]. Ecological theory and scientific knowledge also benefit from applied restoration research since experiments offer the chance to test and examine the theories [START_REF] Bradshaw | Restoration: an acid test for ecology[END_REF][START_REF] Palmer | Ecological Theory and Community Restoration Ecology[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Young | Community Succession and Assembly Comparing, Contrasting and Combining Paradigms in the Context of Ecological Restoration[END_REF]. Restoration projects provide in situ 'playgrounds'to analyse biological processes complementary to ex situ experiments, by manipulating community responses under different environmental conditions [START_REF] Menninger | Restoring ecological communities: from theory to practice[END_REF][START_REF] Young | Community Succession and Assembly Comparing, Contrasting and Combining Paradigms in the Context of Ecological Restoration[END_REF]. Testing theories under controlled and repeatable conditions of restoration projects provide opportunities to analyse successional trajectories of restored communities which contributes to an improvement of ecological theory [START_REF] Menninger | Restoring ecological communities: from theory to practice[END_REF][START_REF] Hobbs | Restoration Ecology: Repairing the Earth's Ecosystems in the New Millennium[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF]Laughlin 2014;Falk et al. 2013).

I.3. Restoration ecology and plant community assembly models

Among the fundamental theories and concepts of restoration ecology (lists of concepts are detailed in: Lindenmayer 2020; [START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Perring | Advances in restoration ecology: rising to the challenges of the coming decades[END_REF][START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF], my thesis focuses on early plant community assembly and succession, and the associated filter-based models [START_REF] Chang | Integrating succession and community assembly perspectives[END_REF][START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Hulvey | Using filter-based community assembly models to improve restoration outcomes[END_REF][START_REF] Temperton | Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF]). Plant succession and assembly models aim at identifying the processes driving the establishment of species from local species pools. The identification of key processes allows predictions of plant community trajectories in restoration projects [START_REF] Young | Community Succession and Assembly Comparing, Contrasting and Combining Paradigms in the Context of Ecological Restoration[END_REF]. A hierarchical framework with three main filters has been suggested to classify environmental constraints of restoration: i) the dispersal filter -the first barrier limiting species dispersal (Öster et al. 2009), (ii) the abiotic filter -the environmental conditions that favour or limit recruitment [START_REF] Hobbs | Ecological Filters, Thresholds, and Gradients inn Resistance to Ecosystem Reassembly[END_REF], and iii) the biotic filter -the interactions between species (e.g. competition, facilitation; [START_REF] Menninger | Restoring ecological communities: from theory to practice[END_REF]. The filters are interdependent, and feedbacks between environmental and biotic filters continuously modify the ecosystem, drive niche modification and therefore, the processes of community assembly [START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF][START_REF] Koffel | Facilitation-vs. competition-driven succession: the key role of resource-ratio[END_REF]). This filter-based 'integrated community concept'framework was synthetized by [START_REF] Lortie | Rethinking plant community theory[END_REF] and is nowadays largely applied in ecological restoration [START_REF] Durbecq | Seedling recruitment in mountain grassland restoration: Effects of soil preparation and grazing[END_REF][START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Hulvey | Using filter-based community assembly models to improve restoration outcomes[END_REF][START_REF] Török | Beyond the species pool: modification of species dispersal, establishment, and assembly by habitat restoration[END_REF].

In my thesis, I particularly focus on the processes affecting community assembly in early successional stages after soil disturbance. The dispersal filter was overcome by adding propagules combined with different techniques to manipulate both the biotic and abiotic filters, in order to enhance restoration success. Niche modification, niche pre-emption [START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF][START_REF] Vannette | Historical contingency in species interactions: towards nichebased predictions[END_REF], competition and facilitation between plant species [START_REF] Callaway | Competition and Facilitation: A Synthetic Approach to Interactions in Plant Communities[END_REF][START_REF] Koffel | Facilitation-vs. competition-driven succession: the key role of resource-ratio[END_REF]) are the main theoretical concepts that are discussed. Despite increasing knowledge on successional dynamics, associated filters and community assembly processes [START_REF] Chang | Integrating succession and community assembly perspectives[END_REF][START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Halassy | Trait-based approach confirms the importance of propagule limitation and assembly rules in old-field restoration[END_REF][START_REF] Hulvey | Using filter-based community assembly models to improve restoration outcomes[END_REF], the occurrence of stochastic processes may compromise predictions and may result in alternative stable states [START_REF] Suding | Alternative states and positive feedbacks in restoration ecology[END_REF][START_REF] Suding | The dynamic nature of ecological systems: Multiple states and restoration trajectories[END_REF][START_REF] Temperton | The Search for Ecological Assembly Rules and Its Relevance to Restoration Ecology. Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF][START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF]. The potential existence of alternative stable states needs to be considered in the identification of the pre-disturbance community that may also have oscillated within a range of states (Fig. I.1). Similarly, such alternative states may characterise the endpoints of succession and restoration outcome [START_REF] Suding | Alternative states and positive feedbacks in restoration ecology[END_REF][START_REF] Suding | The dynamic nature of ecological systems: Multiple states and restoration trajectories[END_REF][START_REF] Temperton | The Search for Ecological Assembly Rules and Its Relevance to Restoration Ecology. Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF][START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF]. Therefore, the reference community may not be unique, and choosing a range of reference communities that encompass the range of community states before the disturbance may be more representative of the natural variability of community responses to environmental conditions (Gann et al. 2019;[START_REF] Ruiz-Jaen | Restoration Success: How Is It Being Measured?[END_REF][START_REF] Suganuma | Indicators of restoration success in riparian tropical forests using multiple reference ecosystems[END_REF]. The recovery debt represents the gap between the community under restoration and the references [START_REF] Moreno-Mateos | Anthropogenic ecosystem disturbance and the recovery debt[END_REF][START_REF] Strobl | Integrated assessment of ecosystem recovery using a multifunctionality approach[END_REF]. Modified from [START_REF] Hobbs | Towards a Conceptual Framework for Restoration Ecology[END_REF], including elements of [START_REF] Chang | Integrating succession and community assembly perspectives[END_REF].

II. The study system

II.1. Habitat type -Semi-natural mountain grasslands

Grasslands are open habitats, with few shrubs or trees, dominated by grasses and forbs. They represent between 25% and 40% (including shrublands and tundra) of Earth's terrestrial biomes [START_REF] Blair | Grassland Ecology[END_REF][START_REF] Goodale | Priority effects are affected by precipitation variability and are stronger in exotic than native grassland species[END_REF]. In Europe, natural grasslands are limited to continental steppes or extreme habitats such as high elevation, unstable chalk scree and cliff grasslands. All other grasslands are 'semi-natural'depending on extensive grazing and/or cutting [START_REF] Kuneš | The origin of grasslands in the temperate forest zone of east-central Europe: long-term legacy of climate and human impact[END_REF] or 'intensive'including sowing of forage grasses and fertilization [START_REF] Hejcman | Origin and history of grasslands in Central Europe -a review[END_REF]. European semi-natural grasslands developed with livestock farming during the Neolithic Age [START_REF] Gibson | Grasses and Grassland Ecology[END_REF][START_REF] Pärtel | Biodiversity in temperate European grasslands: origin and conservation[END_REF]. Since this period (3000 BC -AD 1000), human occupancy was closely related to livestock keeping and grazing, which explains the absence of shrub encroachment and the open character of these occupied areas [START_REF] Kuneš | The origin of grasslands in the temperate forest zone of east-central Europe: long-term legacy of climate and human impact[END_REF].

Semi-natural grasslands are often species-rich with specialized species not occurring in other habitats [START_REF] Dengler | Biodiversity of Palaearctic grasslands: a synthesis[END_REF][START_REF] Habel | European grassland ecosystems: threatened hotspots of biodiversity[END_REF][START_REF] Pärtel | Biodiversity in temperate European grasslands: origin and conservation[END_REF][START_REF] Wallisdevries | Challenges for the conservation of calcareous grasslands in northwestern Europe: integrating the requirements of flora and fauna[END_REF][START_REF] Wilson | Plant species richness: the world records[END_REF]. They provide numerous ecosystem services such as regulation of carbon storage and nutrient cycling, reduction of water run-off and soil erosion, nutrition of grazing livestock and open habitats for plants and animals (e.g. pollinators) [START_REF] Amiaud | Grassland multifunctionality in providing ecosystem services[END_REF][START_REF] Byrne | The effect of management practices on bumblebee densities in hedgerow and grassland habitats[END_REF][START_REF] Jean-Louis | Status and assets of permanent grassland in France and in Europe[END_REF][START_REF] Yan | Quantitative analysis of the contributions of climatic and human factors to grassland productivity in northern China[END_REF]. Due to land use intensification and land abandonment, semi-natural grasslands, and their related biodiversity and ecosystem services, are severely endangered and show a dramatic decline (-12% land cover from 1975 to 1998 in the EU) [START_REF] Amiaud | Grassland multifunctionality in providing ecosystem services[END_REF][START_REF] Stoate | Ecological impacts of early 21st century agricultural change in Europe -A review[END_REF][START_REF] Török | Beyond the species pool: modification of species dispersal, establishment, and assembly by habitat restoration[END_REF]Valkó et al. 2016). Additionally, direct habitat destruction is still contributing to semi-natural grassland losses (Poschlod & WallisDeVries 2002;[START_REF] Veen | Grasslands in Europe: Of High Nature Value[END_REF]). However, since 1979, the conservation of species-rich grasslands has become a major concern in Europe: their conservation value was recognized by environmental policy, and an inventory of these habitats was requested to preserve their endangered vegetation and to develop adequate management [START_REF] Wolkinger | Dry grasslands of Europe[END_REF]EEA 2004). Following the creation of the Habitats Directive, many sites comprising semi-natural grasslands were integrated in the Natura 2000 network, in order to prevent habitat degradation, to improve the guidelines and practices of ecological restoration, and to increase grassland species diversity [START_REF] Stoate | Ecological impacts of early 21st century agricultural change in Europe -A review[END_REF][START_REF] Wallisdevries | Challenges for the conservation of calcareous grasslands in northwestern Europe: integrating the requirements of flora and fauna[END_REF][START_REF] Wilsey | Restoration in the face of changing climate: importance of persistence, priority effects, and species diversity[END_REF].

II.2. Localisation -The 'Haute-Durance'valley in the French Alps

The upper Durance valley ('Haute-Durance') is located in the southern France Alps, in the Hautes-Alpes (05) department, Provence-Alpes-Côte d´Azur region (Fig. 2). The construction of a high-voltage transmission line led to a local destruction of semi-natural grasslands along a 100 km strip from L'Argentière-la-Bessée (44°78'78''N, 6°59''41'E) via Embrun (44°60''04'N; 6°54''24'E) to La-Bâtie-Neuve (44°57'93''N, 6°20''77'E),. These seminatural grasslands are located between 1,000 and 1,300 meters above sea level (asl), at the upper montane altitudinal belt. The climate is temperate to sub-Mediterranean depending on elevation and exposition. The Durance valley is characterized by 209 hours of sunshine per month (based on data from 1973 to 2010, meteorological station of Embrun), and 740 mm of annual precipitation with considerable intra-valley variation. The rainfall is thus lower than in other regions of the Alps as the valley is protected by the Pelvoux massif in the west and the Mont Viso in the east. At the Embrun meteorological station (800 m asl), average July and January temperatures are 20 °C, and 2°C, respectively, with an annual mean of 10.7 °C. Due to the higher altitude, temperatures of the study sites are 1.5 to 3 °C lower. At the study sites, snow and frost occur from October to April with a usually continuous snow cover in January and February. The geology of the region is complex. The bedrock is predominantly calcareous, including black marlstone, dolomite, gypsum and siliceous glacial deposits occur at the surface.

The 'Haute-Durance'grasslands are traditionally managed and maintained open by grazing by cattle or sheep and to a lesser degree by mowing. Mesobromion plant communities are dominating, with transitions to Arrhenateretum at more humid sites, and to Xerobromion at dryer sites. The main environmental factors driving plant communities are elevation, slope, aspect, and dominant bedrock. The 'Haute-Durance'grasslands belong to the Natura 2000 priority habitat type "Semi-natural dry grasslands of Festuco-Brometalia and scrubland facies on calcareous substrates" (N6210, EU directive habitat 92/43/EEC; [START_REF] Calaciura | -Brometalia) (*important orchid sites)[END_REF]. Grasslands of this habitat type are often nutrient-poor and particularly species-rich, with up to 80 plant species/m2 [START_REF] Wallisdevries | Challenges for the conservation of calcareous grasslands in northwestern Europe: integrating the requirements of flora and fauna[END_REF] including rare or endangered plant and animal species such as Astragalus alopecurus Pall. and the butterfly Maculinea arion L.. The two main threats to these grasslands are i) decreasing livestock densities and subsequent land abandonment that led to shrub encroachment [START_REF] Ostermann | The need for management of nature conservation sites designated under Natura 2000[END_REF], and ii) soil destruction by land use transformation.

II.3. Restoration context -Environmental degradation and ecological compensation

The renovation of the high-voltage transmission lines of the 'Haute-Durance'valley including the construction of transitory access tracks and platforms involved different measures to compensate for the ecological impact. This thesis project is part of the ecological compensation action 'Experimental restoration of degraded open habitats', set up by the environmental consultancy ECO-MED and two laboratories IMBE (Mediterranean Institute of Biodiversity and Ecology -Avignon University, CNRS, IRD) and LESSEM (Mountain Ecosystems and Societies Lab -INRAE Grenoble) in the framework of a four-party agreement with the construction company RTE.

The compensation takes place according to the sequence "avoid, reduce, and compensate" (called 'séquence ERC'in France). Article R. 122-14 of the Environment Code defines the compensation measures as follows:

The purpose of the compensatory measures is to provide compensation for the direct and indirect negative effects of the project that cannot be avoided or sufficiently reduced. They are primarily implemented on the damaged site in order to recover and then guarantee its longterm ecological functionality. The whole sequence should preserve and, if possible, improve the environmental quality of the concerned habitat.

If a project leads to the degradation of environmental quality and if significant residual negative effects persist despite avoidance and reduction measures, the contracting authority needs to set up sustainable and effective compensation measures.

In our study, the compensation action includes (1) an experimental approach comparing the effectiveness of restoration techniques and aiming at a better understanding of plantplant and plant-environment interactions during the restoration process (this thesis), and (2) a large-scale direct propagule transfer using brush harvesting to restore all degraded open habitats of the study area (10 ha). The construction work ended between May and July 2018 and the experiments of part (1) were set up on five construction sites in autumn 2018 (Fig.

III. Thesis aims III.1. Grassland restoration: the identification of reference communities, limiting factors and potential solutions

Identifying the ecosystem that has been degraded and represents the state of the undisturbed system is the first step of any ecological restoration project. The definition of this hypothetical state is therefore a starting point that provides a framework to guide the planning and the implementation of the project towards a reference system to reach (James [START_REF] Aronson | Ecological restoration and ecological engineering: Complementary or indivisible?[END_REF][START_REF] Giardina | Science Driven Restoration: A Candle in a Demon Haunted World-Response to Cabin[END_REF][START_REF] Hobbs | Towards a Conceptual Framework for Restoration Ecology[END_REF]. The reference concept emerged in the 1990s to identify the desirable state for restoration [START_REF] Aronson | Restoration and Rehabilitation of Degraded Ecosystems in Arid and Semi-Arid Lands. I. A View from the South[END_REF]. The reference has been particularly used in forest and aquatic restoration projects but was less well developed in terrestrial restoration processes until the end of the last century. Only since the 21 st century, the concept has been adopted by the scientific community initiated by the SER Primer ( 2004), that highlights the importance of references to define restoration goals and to evaluate the success of the project. However, the identification of references is still not sufficiently taken into account in restoration projects [START_REF] Wortley | Evaluating Ecological Restoration Success: A Review of the Literature: Trends and Gaps in Empirical Evaluations[END_REF], and the specific selection of references often lacks clearly defined criteria. Furthermore, as the reference may already be degraded prior to the disturbance, and/or the information on the historical community might not be available, identifying references based on robust environmental indicators of the restoration site may be a straightforward solution. It also allows taking into account alternative stable states, as well as the natural variability and dynamics of the study system (Gann et al. 2019;[START_REF] Ruiz-Jaen | Restoration Success: How Is It Being Measured?[END_REF][START_REF] Suganuma | Indicators of restoration success in riparian tropical forests using multiple reference ecosystems[END_REF].

In my study area, plant communities are shaped by traditional mowing and grazing, and as in several grassland restoration projects, restoration ecologists must negotiate with farmers to fence the areas under restoration and protect sown seedlings from herbivory and trampling. As extensive agricultural management shaped semi-natural mountain grasslands by mowing and grazing for millennia [START_REF] Gibson | Grasses and Grassland Ecology[END_REF][START_REF] Kuneš | The origin of grasslands in the temperate forest zone of east-central Europe: long-term legacy of climate and human impact[END_REF], the maintenance, or even reintroduction of traditional extensive management is essential [START_REF] Hejcman | Origin and history of grasslands in Central Europe -a review[END_REF]. Many plant species and ecosystem services of semi-natural grasslands are totally dependent on the maintenance of these practices (Petit et al. 2004;[START_REF] Schermer | Institutional impacts on the resilience of mountain grasslands: an analysis based on three European case studies[END_REF], which deserves further consideration for both efficient conservation and restoration. Intensive grazing may result in the loss of biodiversity while extensive grazing is essential to preserve these ecosystems (Petit et al. 2004;[START_REF] Hejcman | Origin and history of grasslands in Central Europe -a review[END_REF]Janicka and Pawluśkiewicz 2018). The viability of seeds in the soil contributed to the preservation of semi-natural grasslands but also seed dispersal, in particular by grazing and mowing (Janisová 2018). However, longterm extensive grazing has favoured clonal reproduction whilst decreasing sexual reproduction of grasslands species [START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Piqueray | Revue bibliographique sur la restauration des pelouses calcicoles en Europe : contraintes rencontrées et solutions proposées[END_REF][START_REF] Römermann | Influence of former cultivation on the unique Mediterranean steppe of France and consequences for conservation management[END_REF].

'Passive'or 'Natural grassland restoration'(sensu Atkinson and Bonser 2020) may be hampered by the poor density of grassland species in the soil seed bank [START_REF] Buisson | Species transfer via topsoil translocation: lessons from two large Mediterranean restoration projects[END_REF][START_REF] Grman | Dispersal and establishment filters influence the assembly of restored prairie plant communities[END_REF]Thompson & Grime 1979;[START_REF] Turnbull | Are plant populations seed-limited? A review of seed sowing experiments[END_REF], by their low dispersal capacity and by the fragmentation of grassland habitats limiting the seed rain [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] Muller | Effect of topsoil removal and plant material transfer on vegetation development in created Mediterranean meso-xeric grasslands. N Hölzel (ed)[END_REF][START_REF] Münzbergová | Seed, dispersal, microsite, habitat and recruitment limitation: identification of terms and concepts in studies of limitations[END_REF]. Therefore, overcoming dispersion limitation is an important issue and seed addition is often a successful method to restore species-rich grasslands [START_REF] Öster | Dispersal and establishment limitation reduces the potential for successful restoration of semi-natural grassland communities on former arable fields[END_REF][START_REF] Török | Fast restoration of grassland vegetation by a combination of seed mixture sowing and low-diversity hay transfer[END_REF] or, at least, to accelerate restoration [START_REF] Halassy | Trait-based approach confirms the importance of propagule limitation and assembly rules in old-field restoration[END_REF][START_REF] Kövendi-Jakó | Three years of vegetation development worth 30 years of secondary succession in urban-industrial grassland restoration[END_REF]. The use of local and native seeds is recommended to assure adaption to local environmental conditions and to maintain genetic structure [START_REF] Barrel | Native seeds for the ecological restoration in mountain zone -Production and use of preservation mixtures[END_REF][START_REF] Bucharova | Mix and match: regional admixture provenancing strikes a balance among different seed-sourcing strategies for ecological restoration[END_REF]Vander Mijnsbrugge et al. 2010). However, native species of local origin are still not widely available, and the seed mixtures used for revegetation in Europe are often dominated by species of non-local origin. Consequently, transferring local seeds from nearby non-degraded communities is increasingly used in grassland restoration [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]Scotton et al. 2012). A multitude of techniques are currently being used and studied, such as dry hay transfer vs. sowing of seed mixtures [START_REF] Auestad | Nature will have its way: local vegetation trumps restoration treatments in semi-natural grassland[END_REF][START_REF] Kövendi-Jakó | Three years of vegetation development worth 30 years of secondary succession in urban-industrial grassland restoration[END_REF], brushharvesting material sowing vs. green hay transfer (Albert et al. 2019) and seed sowing combined with various additional treatments [START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF][START_REF] Pawluśkiewicz | Effects of Different Introduction Methods on Plant Species Establishment Success in Wet Grassland Restoration[END_REF].

Seed addition experiments demonstrated that limited recruitment due to unfavourable site conditions is the next major constraint influencing plant establishment [START_REF] Bissels | Effects of different mowing regimes on seedling recruitment in alluvial grasslands[END_REF][START_REF] Pywell | Restoration of species-rich grassland on arable land: assessing the limiting processes using a multi-site experiment[END_REF][START_REF] Tilman | Community Invasibility, Recruitment Limitation, and Grassland Biodiversity[END_REF][START_REF] Turnbull | Are plant populations seed-limited? A review of seed sowing experiments[END_REF]. Although soil disturbance may negatively affect soil structure and components [START_REF] Di | Nitrate leaching in temperate agroecosystems: sources, factors and mitigating strategies[END_REF][START_REF] Holland | The environmental consequences of adopting conservation tillage in Europe: reviewing the evidence[END_REF], soil preparation, such as harrowing or topsoil removal, may promote the recruitment of plant species [START_REF] Hölzel | Restoration of a species-rich flood meadow by topsoil removal and diaspore transfer with plant material[END_REF]Klaus et al. 2017;[START_REF] Long | Seed and microsite limitations mediate stochastic recruitment in a low-diversity prairie restoration[END_REF][START_REF] Myers | Seed arrival, ecological filters, and plant species richness: a meta-analysis[END_REF]) by limiting the competition for resources by an already established vegetation (Bischoff et al. 2018;[START_REF] Kupferschmid | Dispersal and microsite limitation in an abandoned calcareous grassland of the southern prealps[END_REF][START_REF] Poschlod | Diaspore and gap availability are limiting species richness in wet meadows[END_REF][START_REF] Stevenson | Seed bed treatment effects on vegetation and seedling establishment in a New Zealand pasture one year after seeding with native woody species[END_REF]. Soil preparation may also improve abiotic conditions and increase seed trapping and adhesion [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF][START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF][START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF].

Seedling recruitment and early plant community assembly are influenced by abiotic factors as well as by species strategy [START_REF] Grime | Plant strategies and vegetation processes[END_REF] influencing biotic interactions (i.e. plantplant and plant-soil interactions) (Bever 2003;[START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF][START_REF] García-Girón | Biotic interactions hold the key to understanding metacommunity organisation[END_REF]. Biotic interactions can be either negative (e.g. competition, predation, amensalism, parasitism) [START_REF] Callaway | Competition and Facilitation: A Synthetic Approach to Interactions in Plant Communities[END_REF][START_REF] Grime | Competition and Diversity in Herbaceous Vegetation (reply)[END_REF] or positive (e.g. facilitation, commensalism, mutualism, symbiosis) (M. [START_REF] Bertness | The Role of Positive Interactions in Communities: Lessons from Intertidal Habitats[END_REF][START_REF] Brooker | Facilitation in plant communities: the past, the present, and the future[END_REF][START_REF] Bruno | Inclusion of facilitation into ecological theory[END_REF][START_REF] Choler | Facilitation and Competition on Gradients in Alpine Plant Communities[END_REF]. All types of interactions are important in ecological restoration, because plant-soil feedback and spontaneous colonisation continuously reshape the habitat since early successional stages. Therefore, the use of biotic drivers, such as ecosystem engineers (Jones et al. 1996) or nurse species, may favour ecosystem restoration (cf. also keystone or nexus species, [START_REF] Temperton | The Search for Ecological Assembly Rules and Its Relevance to Restoration Ecology. Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF]. For example, in active restoration by seed addition, the use of mulch may facilitate seedling recruitment by protecting seedlings against high solar radiation and drought [START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF][START_REF] Mollard | Impacts of mulch on prairie seedling establishment: Facilitative to inhibitory effects[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF]. Sowing of annual ('nurse') plant species together with target species may compensate for the absence of a protecting mulch layer. Such annuals provide temporary living cover that may facilitate seedling recruitment and establishment [START_REF] Padilla | The role of nurse plants in the restoration of degraded environments[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF]. Furthermore, plant cover may reduce soil erosion [START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF]Gu et al. 2020;[START_REF] Liu | The effects of vegetation on runoff and soil loss: Multidimensional structure analysis and scale characteristics[END_REF][START_REF] Peratoner | Organic seed propagation of alpine species and their use in ecological restoration of ski runs in mountain regions[END_REF], an important criterion for the success of restoration measures. In particular in mountain grasslands, soil stabilisation and erosion control are important because steep slopes and low vegetation cover favour soil erosion [START_REF] Huc | SEM'LESALPES -Des semences d'origine locale pour la restauration de milieux ouverts en montagne alpine[END_REF][START_REF] Löbmann | Towards a better understanding of shallow erosion resistance of subalpine grasslands[END_REF][START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]). However, a living cover may also increase root and light competition [START_REF] Donath | Influence of competition by sown grass, disturbance and litter on recruitment of rare flood-meadow species[END_REF]. Biotic and abiotic filters need to be taken into account in restoration actions, since both filters continuously interact with each other through feedback loops (Fig. I.1).

After degradation, plant community assembly may be modified by natural succession or by management actions leading to different trajectories [START_REF] Clewell | Ecological Restoration, Second Edition: Principles, Values, and Structure of an Emerging Profession[END_REF]). These different trajectories may result in alternative states that are more or less similar to the reference [START_REF] Suding | Alternative states and positive feedbacks in restoration ecology[END_REF][START_REF] Temperton | The Search for Ecological Assembly Rules and Its Relevance to Restoration Ecology. Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF]. In ecological restoration, it is important to determine relevant management strategies to drive succession towards the desired states. Stochastic dispersal events, biotic and abiotic interactions influencing community assembly induce priority effects. Priority effects occur when one species arrives first and affects its neighbourhood and/or its environment and the establishment of later species [START_REF] Temperton | The Search for Ecological Assembly Rules and Its Relevance to Restoration Ecology. Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF][START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF][START_REF] Weidlich | Priority effects caused by plant order of arrival affect below-ground productivity[END_REF]. They have been identified as a core concept in restoration ecology since they influence early community succession [START_REF] Van Der Putten | Plant species diversity as a driver of early succession in abandoned fields: a multi-site approach[END_REF][START_REF] Vaughn | Short-term priority over exotic annuals increases the initial density and longer-term cover of native perennial grasses[END_REF] and can have mid-to long-term consequences on plant community assembly [START_REF] Fry | Plant, soil and microbial controls on grassland diversity restoration: a long-term, multi-site mesocosm experiment[END_REF][START_REF] Fukami | Assembly History Interacts with Ecosystem Size to Influence Species Diversity[END_REF][START_REF] Garcia-Giron | Historical contingency via priority effects counteracts environmental change on metacommunity dynamics across decades[END_REF][START_REF] Mergeay | Priority effects and species sorting in a long paleoecological of repeated community assembly through time[END_REF][START_REF] Švamberková | The legacy of initial sowing after 20 years of exarable land colonisation[END_REF][START_REF] Werner | Persistent asymmetrical priority effects in a California grassland restoration experiment[END_REF]. Therefore, modifying the sequence of the arrival of plant species, and thus modifying priority effects is one possible way to influence the structuring of communities [START_REF] Hillerislambers | Rethinking Community Assembly through the Lens of Coexistence Theory[END_REF][START_REF] Vannette | Historical contingency in species interactions: towards nichebased predictions[END_REF][START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF]. The first arriving species may modify the micro-local environmental conditions and consequently the establishment of later arriving species by inhibiting (Cole 1983) or facilitating [START_REF] Bertness | Competition and Facilitation in Marsh Plants[END_REF]) their seedling recruitment (Donath et al. 2007;[START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF]. Many other mechanisms may also drive priority effects, such as niche pre-emption or niche modification [START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF][START_REF] Helsen | A test of priority effect persistence in semi-natural grasslands through the removal of plant functional groups during community assembly[END_REF], plant-soil feedbacks and soil legacies (Bever 2003;[START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF][START_REF] Grman | Within-Year Soil Legacies Contribute to Strong Priority Effects of Exotics on Native California Grassland Communities[END_REF][START_REF] Putten Wh Van Der | Plant-soil feedbacks: the past, the present and future challenges[END_REF] or even allelopathic effects [START_REF] Levine | Mechanisms References 123 underlying the impacts of exotic plant invasions[END_REF]. Finally, priority effects do not only influence the early community assembly but also shape the potential future community trajectories, including their composition and associated functions [START_REF] Grman | Within-Year Soil Legacies Contribute to Strong Priority Effects of Exotics on Native California Grassland Communities[END_REF][START_REF] Stuble | Priority Treatment Leaves Grassland Restoration Vulnerable to Invasion[END_REF][START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF][START_REF] Werner | Persistent asymmetrical priority effects in a California grassland restoration experiment[END_REF].

III.2. Research questions

The thesis project aims to better understand the factors limiting the restoration of mountain grasslands after soil disturbance, and to identify successful restoration techniques compensating the degradation. I addressed the following scientific questions:

In restoration ecology, the reference is a key concept that is well defined in theory. In practice, however, the specific selection of references often lacks clear methodology. Thus, my major questions are: In order to understand the mechanisms favouring target species seedling establishment, I asked: (3.a) Which method of plant material transfer is most successful in establishing species transferred from the donor community? (3.b) Does the sowing of a nurse plant (wheat) facilitate seedling establishment from brush material? (3.c) Do transfer techniques and wheat sowing increase the similarity between restored and reference communities? (3.d) Does propagule addition limit soil erosion? Chapter 3 And finally, I manipulated priority effects to restore plant community composition, asking: (4.a) Does the order of species arrival manipulated by sequential sowing influence community establishment? (4.b) More specifically, does sowing dominant species first results in competitive exclusion of subordinate species? And (4.c) Does sowing subordinates first provide a sufficient establishment of both subordinates and dominants? Chapter 4

III.3. Thesis chapters

Chapter 1 focused on the identification of an appropriate reference community, which is a crucial step in ecological restoration as it allows defining restoration targets and evaluating restoration success (Gann et al. 2019;[START_REF] Giardina | Science Driven Restoration: A Candle in a Demon Haunted World-Response to Cabin[END_REF][START_REF] Hobbs | Towards a Conceptual Framework for Restoration Ecology[END_REF]. The community prior to its degradation is considered as the reference (Fig. 3; [START_REF] Aronson | Restauration et réhabilitation des écosystèmes dégradés en zones arides et semi-arides : le vocabulaire et les concepts[END_REF]SER 2004). However, information on the reference may not be available and communities may have already been degraded before the latest anthopogenic disturbance. As in our project, this situation is very common in European grassland restoration [START_REF] Baasch | Enhancing success in grassland restoration by adding regionally propagated target species[END_REF][START_REF] Bischoff | Dispersal and establishment of floodplain grassland species as limiting factors in restoration[END_REF]. Furthermore, the use of several positive references is increasingly recommended to include alternative stable states and natural variability of the study system (Gann et al. 2019;[START_REF] Ruiz-Jaen | Restoration Success: How Is It Being Measured?[END_REF][START_REF] Suganuma | Indicators of restoration success in riparian tropical forests using multiple reference ecosystems[END_REF]. However, selecting references in practice often lacks clear criteria. In this chapter, I suggest a framework based on ecological theory, and more precisely on relationships between vegetation and environmental drivers, to facilitate the identification of a range of reference communities. In the next three chapters of my thesis, I suggest a filter-based community assembly approach of community dynamics by simultaneously manipulating dispersal, abiotic and biotic filters to analyse restoration outcomes [START_REF] Chang | Integrating succession and community assembly perspectives[END_REF][START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Hulvey | Using filter-based community assembly models to improve restoration outcomes[END_REF][START_REF] Temperton | The Search for Ecological Assembly Rules and Its Relevance to Restoration Ecology. Assembly Rules and Restoration Ecology: Bridging the Gap Between Theory and Practice[END_REF]. In order to answer questions (2) and (3), five degraded semi-natural grasslands were used to set up two different experiments (Fig. 1).

In the first experiment, I manipulated both abiotic and biotic assembly filters by examining whether soil preparation is required to provide more suitable soil conditions and to reduce competition of pre-existing vegetation, and whether traditional extensive grazing should be excluded in initial stages of grassland restoration to protect seedlings. This would justify soil tillage and fencing costs in grassland restoration projects (chapter 2). In the second experiment, I focused on different techniques of propagule transfer increasingly used in mountain grasslands to understand the mechanisms favouring seedling establishment of transferred species. I analysed both biotic and abiotic filters by testing the effects of brushharvested propagules and hay transfer, as well as the potentially facilitative effect of dead and living plant cover on the recruitment of transferred brush-harvested propagules (biotic filter), and on soil erosion (abiotic filter; chapter 3). Finally, as community assembly is also highly influenced by dispersal that may lead to priority effects, I altered the biotic filter by sowing different combinations of plant species (dominant vs. subordinate) in a third experiment and I analysed how sequential sowing affects community assembly (chapter 4).

Abstract

In restoration ecology, the reference ecosystem represents a key concept, which is well defined from a theoretical point of view. In practice, however, selecting reference systems, such as reference plant communities, often lacks clear methodology. In order to facilitate this selection, we provide a framework based on ecological theory and more precisely on relationships between vegetation and environmental factors to identify reference plant communities. The four major steps are: 1) the delimitation of a geographical zone in which habitat types similar to restoration sites occur; 2) the identification of environmental factors structuring non-degraded plant communities within this geographical zone; 3) the comparison of the environmental factors between non-degraded and degraded sites; 4) the selection of the non-degraded sites most similar to restoration sites in terms of environmental factors to use them as references. We concept-proved our approach by identifying reference communities using environmental factor combinations for five mountain grassland sites degraded by the construction of a high-voltage line. In a multivariate analysis of eighteen non-degraded sites, we identified six major environmental factors explaining plant species compositions. A second multivariate analysis including degraded sites provided environmental distances of the eighteen non-degraded to each of the degraded sites. The results demonstrated that the environmentally most similar sites were not necessarily the geographically closest ones. In conclusion, the analysis of regional plant-environment interactions provides an important tool to identify reference communities or donor sites for seed transfer if not available adjacent to degraded habitats.

Keywords: abiotic factors, grassland restoration, plant succession, degradation, target reference

Introduction

The definition of reference systems is a crucial step in ecological restoration as it allows defining restoration targets and evaluating restoration success [START_REF] Hobbs | Towards a Conceptual Framework for Restoration Ecology[END_REF][START_REF] Giardina | Science Driven Restoration: A Candle in a Demon Haunted World-Response to Cabin[END_REF]McDonald et al. 2016). The reference paradigm represents a cornerstone concept, which is well defined from a theoretical point of view [START_REF] Aronson | Restoration and Rehabilitation of Degraded Ecosystems in Arid and Semi-Arid Lands. I. A View from the South[END_REF][START_REF] Aronson | Ecological restoration and ecological engineering: Complementary or indivisible?[END_REF]SER Primer 2004). However, in practice, the specific selection of reference ecosystems or communities often lacks clear scientific criteria, or this step is not sufficiently taken into account in restoration planning. A review of 301 articles focusing on terrestrial ecological restoration projects showed that less than 75% of the studies used a reference, 62% used "negative reference(s)" representing the pre-restoration degraded state, and only 60% used "positive target reference(s)" defined by the pre-degradation system, with only 22% presenting both. Both negative and positive references may be used together to evaluate restoration success: i) a negative one to evaluate the success of an active restoration compared with the degraded system, and ii) a positive one to assess success by comparing the restored and the reference systems [START_REF] Benayas | Enhancement of Biodiversity and Ecosystem Services by Ecological Restoration: A Meta-Analysis[END_REF][START_REF] Wortley | Evaluating Ecological Restoration Success: A Review of the Literature: Trends and Gaps in Empirical Evaluations[END_REF].

In many studies, the ecosystem prior to its degradation is considered as a (positive) reference [START_REF] Aronson | Restauration et réhabilitation des écosystèmes dégradés en zones arides et semi-arides : le vocabulaire et les concepts[END_REF]SER Primer 2004). However, ecosystems may already be degraded prior to the latest disturbance that is considered as starting point of restoration, and/or the information on the state of the ecosystem before degradation might not be available. This is a very common situation in European grassland restoration where ecosystems suffer from land abandonment or intensification [START_REF] Bischoff | Dispersal and establishment of floodplain grassland species as limiting factors in restoration[END_REF][START_REF] Baasch | Enhancing success in grassland restoration by adding regionally propagated target species[END_REF]).

An important objective of restoration is to accelerate or jumpstart succession by facilitating late-successional species [START_REF] Palmer | Ecological Theory and Community Restoration Ecology[END_REF]. Late-successional stages or even endpoints of succession are determined by abiotic conditions although historical contingency may influence final community composition [START_REF] Fukami | Species divergence and trait convergence in experimental plant communities[END_REF][START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF]. While it is useful to consider endpoints (attractors) of succession, earlier successional stages and potential variation of endpoints also need to be taken into account [START_REF] Prach | Does succession run towards potential natural vegetation? An analysis across seres[END_REF]. The succession towards natural vegetation representing such endpoints is often prevented by human land management. A constant and/or predictable disturbance regime, such as grazing by livestock of semi-natural grasslands, may result in stable intermediate states of plant succession (or "alternate steady states" or metastable states; [START_REF] Clewell | Ecological Restoration, Second Edition: Principles, Values, and Structure of an Emerging Profession[END_REF]). These intermediate states can thus be considered as quasi-stable representing an equilibrium with environmental conditions and land management [START_REF] Young | Community Succession and Assembly Comparing, Contrasting and Combining Paradigms in the Context of Ecological Restoration[END_REF][START_REF] Bouzillé | Gestion des habitats naturels et biodiversité. Concept, méthodes et démarches[END_REF].

The use of several positive references may be a solution to take into account such intermediate states and is increasingly common in ecological restoration [START_REF] Ruiz-Jaen | Restoration Success: How Is It Being Measured?[END_REF][START_REF] Suganuma | Indicators of restoration success in riparian tropical forests using multiple reference ecosystems[END_REF]McDonald et al. 2016). The definition of various reference systems resulting from different land management types would provide a range of restored ecosystem trajectories (Le Floc'h & Aronson 1995;[START_REF] Cortina | Ecosystem structure, function, and restoration success: Are they related?[END_REF].. Furthermore, considering the potential variation in successional endpoints allows integrating ecosystem dynamics and natural variability [START_REF] Asbjornsen | Defining Reference Information for Restoring Ecologically Rare Tallgrass Oak Savannas in the Midwestern United States[END_REF][START_REF] Sluis | Multiscale metrics differentiate among tallgrass prairie restorations and remnant ecosystems along a restorative continuum[END_REF][START_REF] Erskine | Using reference sites to guide ecological engineering and restoration of an internationally significant uranium mine in the Northern Territory, Australia[END_REF]. In cases of high spatial heterogeneity such as varying slope and aspect, small-scale differences may compromise the preference of adjacent plant communities and populations usually recommended for active ecological restoration [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF], Vander Mijnsbrugge et al 2010;[START_REF] Scotton | Practical handbook for seed harvest and ecological restoration of species-rich grasslands. Practical handbook for seed harvest and ecological restoration of species-rich grasslands[END_REF]. Additionally, restoration sites may sometimes be surrounded by other more or less degraded ecosystems. In such cases, references need to be found at higher distances requiring a clear concept and methodology to identify the most appropriate sites.

In our study, we provide a framework to define positive reference communities by using environmental factors that drive plant species composition. This method is particularly useful in the following cases: i) information on plant species composition prior to latest degradation is not available; ii) the sites were already (at least partially) degraded before the latest disturbance for which restoration is required and several environmental factors structuring plant communities remain within the natural range after a particular ecosystem degradation; iii) if due to economic or legal constraints, only one propagule donor site can be selected for several restoration sites.

We first present a framework to select reference communities by using environmental factors that determine plant species composition. To concept-prove our framework, we applied the approach to a mountain grassland project involving the restoration of temporary access tracks required to build a new high-voltage electricity line. Habitat conditions at the sites change at relatively small scales thus complicating the search for appropriate reference communities. Such extensively grazed and/or mowed grassland ecosystems represent a particular case of succession in which regular management truncates succession by preventing the development of woody vegetation [START_REF] Young | Community Succession and Assembly Comparing, Contrasting and Combining Paradigms in the Context of Ecological Restoration[END_REF]. Most European grasslands are thus considered as such alternative quasi-stable states since they are maintained by regular grazing and mowing. Their species composition is not only dependent on climate and soil conditions but also on management type [START_REF] Isselstein | Agronomic aspects of biodiversity targeted management of temperate grasslands in Europe -A review[END_REF][START_REF] Härdtle | Vegetation responses to environmental conditions in floodplain grasslands: Prerequisites for preserving plant species diversity[END_REF]). However, management regimes are similar in our study region allowing an analysis of environmental factors driving plant community composition. Due to the scope of the construction work, the different non-degraded grasslands selected as potential references should thus make it possible to obtain a wide range of quasi-stable states representative of the different restoration sites.

Framework to identify reference communities by combinations of environmental factors

We suggest a 4-step methodological framework to select references by analyzing the species composition of non-degraded systems and measuring the environmental factors structuring them (Fig. 1.1): 1) delimit a geographical zone in which habitat types similar to restoration sites occur; 2) identify environmental factors structuring non-degraded plant communities within this geographical zone and measure them in non-degraded and degraded sites; 3) evaluate environmental distances between degraded sites and non-degraded sites; 4) identify the non-degraded sites most similar to restoration sites in terms of environmental factors. First, a rough estimation of habitat type based on existing habitat or biogeographical maps is required to delimit the geographical zone in which the reference communities may be found [START_REF] Alard | Biodiversity in changing landscapes: from species or patch assemblages to system organisation[END_REF]. Existing pedoclimatic or historical vegetation data may increase the precision of habitat type information. Secondly, within the delimited geographical zone, major environmental factors that typically drive species composition in the study area are measured on non-degraded sites. Factors that are significantly modified by the habitat degradation cannot be taken into account. At the same time, the relationship between these environmental factors and plant species composition is analysed. The number of analysed sites needs to be representative of the variation of the geographical zone and sufficient to identify the best subset of environmental factors using multivariate statistics. Once identified, the major environmental drivers of non-degraded sites need to be measured on sites to be restored. The non-degraded sites presenting the most similar values compared with restoration sites can therefore be used as reference sites. This method allows identifying the best corresponding plant communities for each restoration site within the study zone. For each restoration site, one or more reference sites that correspond best in terms of structuring environmental factors and thus of potential plant community composition will be chosen.

Proof of concept

We tested our approach to identify reference communities using combinations of environmental factors in mountain grasslands degraded by the construction of a highvoltage electricity line. The construction work included the creation of access tracks and working platforms on 13 ha of mountain grasslands. The grasslands were separated in hundreds of patches separated by woodlands. Due to small-scale changes in abiotic conditions and degradation of adjacent sites by land abandonment, arable use or afforestation, the closest sites were often inappropriate references. The identification of reference communities was thus a priority action to define restoration targets but also to find appropriate grassland sources for hay transfer used as a restoration technique [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]. Following the four suggested steps we asked: (1) What are the environmental factors explaining best the species composition of non-degraded mountain grassland sites? (2) What are the non-degraded sites most similar to the degraded sites in terms of these environmental factor combinations? (3) Are the environmentally most similar sites also the geographically closest ones?

Material and methods

Restoration sites and delimitation of geographical zone

The study area is the upper Durance valley ("Haute-Durance") in the Southern French Alps from L'Argentière-la-Bessée to La-Batie-Neuve (44°78'78''N, 6°59''41'E; 44°57'93''N, 6°20''77'E, Fig. 2). The study sites are located 100 to 600 m above the valley floor on slopes at an elevation of 1000 to 1400 m (upper montane altitudinal belt). The climate is temperate to sub-Mediterranean depending on elevation and exposition. The Pelvoux and Mont Viso mountains provide a rain shadow, reducing precipitation to 740 mm per year, with a mean annual temperature of 10.7°C (based on data from 1991 to 2010, meteorological station of Embrun). The bedrock is predominantly calcareous, but many sites are covered by quaternary glacial deposits. The degraded areas were characterized by dry to mesophilic grasslands. Most of these grasslands are Natura 2000 priority habitats: "Semi-natural dry grasslands of Festuco-Brometalia and scrubland facies on calcareous substrates" (N6210, EU directive habitat 92/43/EEC). The construction work affected soil structure and soil organic matter composition by stripping soil, compacting and decompacting. A total of 18 species-rich non-degraded grassland sites as well as five restoration sites were selected as study sites in the Haute-Durance valley (Fig. 1.2). Selection criteria were traditional management by grazing (sheep, cattle) and mowing, geographical representation of the study area and representative microclimatic conditions (semi-dry to mesophilic) in avoiding driest and most humid sites. We further excluded restoration sites with particularly strong soil modification by gravel addition.

Data collection

To link species composition to environmental conditions, vegetation cover of all vascular plant species was evaluated between 21 June and 5 July 2018 in 3 randomly placed quadrats (2m x 2m) within each of the 18 non-degraded grassland sites resulting in a total of 54 quadrats. We measured abiotic factors that are usually not or little influenced by soil disturbance involved in access track or working platform creation. These factors included soil variables such as phosphorus, potassium and carbonate content, pH value and moisture, as well as geomorphological variables influencing climatic conditions such as slope, aspect and elevation. Three soil samples per site were thus taken to a depth of 20 cm (diameter 5 cm). They were dried and sieved using a 2 mm mesh size. Plant available phosphorus (P2O5) was determined using the Olsen Sterling method (1954), exchangeable potassium ions (K2O) was determined according to Thomas (1982) and [START_REF] Ciesielski | A comparison between three methods for the determination of cation exchange capacity and exchangeable cations in soils[END_REF]. Soil moisture was measured in the field using Theta Probe ML3 sensors. The soil pH was measured in a 1:5 water solution. The carbonate content was estimated using a 3-level scale of effervescence with 1 M HCl (non-effervescent; slightly effervescent; strongly effervescent). In mountain ecosystems, plant species composition and richness are strongly influenced by slope and aspect driving solar radiation [START_REF] Pykälä | Plant species richness and persistence of rare plants in abandoned semi-natural grasslands in northern Europe[END_REF]. We estimated the annual direct incident solar radiation (ASR) using the method of McCune & Keon (2002). The ASR takes into account that southwest facing slopes show a higher radiation than southeast-facing slopes in folding the aspect (Supplement S1.1). This folded aspect is combined with slope and latitude to calculate the ASR.

Statistical analysis

Factor selection: test of variance inflation to avoid collinearity and correlation matrix

In order to reduce collinearity within the measured environmental variables we tested Variance Inflation Factors (VIF; [START_REF] Zuur | A protocol for data exploration to avoid common statistical problems[END_REF]. Highly correlated variables (VIF>2) were sequentially removed (R package "vegan", [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2[END_REF]. We used the pairwise correlation analysis (Pearson correlation coefficient) to crosscheck VIF removal procedure. Since slope and aspect are included in the ASR, they were not considered. Carbonate content (correlated to pH) was removed according to VIF analysis whereas elevation, ASR, soil moisture, soil pH, P2O5 and K2O were kept and used in subsequent analyses in order to test the response of plant species composition to environmental factors of non-degraded sites.

Analysis of factors driving species composition

We analysed the plant species composition of the non-degraded sites using redundancy analysis (RDA) to model the relationship between species composition and environmental variables (R package "vegan", [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2[END_REF]. Species cover was first Hellinger-transformed since this transformation provides a better resolution in linear ordination techniques than chi-square distance [START_REF] Legendre | Ecologically meaningful transformations for ordination of species data[END_REF]. We removed rare species (occurring in less than eight plots) from the analysis in order to improve the detection of relationships between community composition and abiotic factors. Removing rare species reduces bias due to stochastic sampling effects since rare species occurrence is often a poor predictor of environmental conditions [START_REF] Mccune | Analysis of ecological communities, MjM Software Design[END_REF]. The significance of the six selected variables (ASR, elevation, soil moisture, soil pH, P2O5, K2O) was tested using the "envfit" function with N = 999 permutations (R package "vegan"; [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2[END_REF]) to determine relationships with plant community composition.

Environmental distances between non-degraded and restoration sites

The environmental distance (= dissimilarity) between each restoration and nondegraded sites was assessed by calculating the Euclidean distance using the "vegdist" function (R package "vegan"; [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2[END_REF]. Non-metric multidimensional scaling (NMDS) based on Euclidean distances of environmental factors was used to illustrate the most similar reference for each restoration site (R code: https://github.com/RenaudJau/ChooseRef). In order to compare the distribution of plant communities according to environmental factors values and to geographical zone, a second NMDS was performed on plant species composition of non-degraded sites. Sites were grouped by each environmental factor (high, medium, low factor values) and major geographical zones (south; central; north) in different ordination plots. All analyses were performed using R 3.5.3 (R Core Team 2019).

Results

Environmental drivers of plant species composition

One-hundred and eighty plant species belonging to 40 families were identified across the 18 study grasslands. The RDA model relating plant species composition and environmental variables at non-degraded sites explained 44.8% of the total variance. The first two constrained axes accounted for 20.2% and 11.4% of total variance and were mainly determined by P2O5, soil pH, soil moisture and solar radiation (Fig. 1.3). The environmental variables retained by the VIF analysis did not show strong correlations confirming that applying it efficiently removed correlated environmental variables (Table S1.1). Four of the six selected variables (ASR: p = 0.004, P2O5: p = 0.025, soil moisture: p = 0.023, pH: p = 0.026) had a significant influence on plant species composition whereas the influence of elevation (p = 0.201) and K2O (p = 0.830) was not significant (Table S1.2). Sites located on the right side of Fig. 3 were associated with low ASR, relatively low (neutral) pH and high soil moisture, as well as high P2O5. They were well represented by mesophilic species, such as Dactylis glomerata, Galium verum, Poa pratensis, Plantago lanceolata and Lathyrus pratensis.

In contrast, plants associated with higher ASR and pH included Bromopsis erecta, Brachypodium rupestre, Pilosella officinarum, Lotus corniculatus and Teucrium chamaedrys. Sites with higher abundance of Astragalus danicus, Pillosella officinarum, Lotus corniculatus and Teucrium chamaedrys were negatively correlated with elevation and soil moisture, whereas sites with higher abundance of Salvia pratensis and Dactylis glomerata were positively correlated with both environmental variables. S1.3.

Identification of reference sites by comparing similarity in environmental factors

The five restoration sites revealed different combinations of environmental factors resulting in different environmental distances with non-degraded sites (Fig. 1.4). Restoration sites 1R and 3R showed a low environmental distance to the non-degraded site 21 indicating high similarity in measured abiotic factors. However, the second environmentally closest nondegraded sites were different (1R: site 22, 3R: site 18). The three sites closest to 2R in terms of environmental variables were 6, followed by 4 and 5. The most similar sites to 11R were 12, 10 and 16, and the most similar to 20R were 9, 17 and 14 (Fig. 1.4.a, Table 1.1). If technical and/or logistic constraints require the choice of one reference community as a donor site for plant material for all five degraded sites, the non-degraded site 9 would represent the best reference (Table 1.1), which has the lowest mean distance to five restored sites (Fig.

1.4.b).

Table 1.1. Similarities between restoration sites (1R, 2R, 3R, 11R, 20R) and the non-degraded sites (from site 4 to 24, without R) of the study zone (similarity decreasing from left to right) using the Euclidean distance for environmental variables shown in ).

Chap. 1. Identification of references in restoration ecology

The geographical zonation of plant species composition hardly corresponded to environmental factor values. NMDS according to geographical position separated the southernmost zone from the northernmost zone whereas the central zone was not well separated from the latter one (Fig. S1.1). The Northern zone was characterized by low levels of P2O5 and soil moisture, low solar radiation (ASR) and neutral pH. The Central zone showed highest soil moisture and P2O5, medium ASR and the most acidic pH. The Southern zone was represented by high solar radiation and a basic pH. However, there were many exceptions to these general trends and environmental zonation was very different from geographical zonation in P2O5 and soil moisture. The lack of correspondence between geographical and environmental zonation explains that the environmentally most similar non-degraded site was not necessarily the geographically closest one.

Discussion

Our results indicate that among the seven tested environmental factors, ASR, soil moisture, P2O5 and soil pH best explain the distribution of plant communities in our 18 species-rich mountain grasslands and can thus be used as indicators for reference communities in this particular case.

The simultaneous analysis of environmental factors such as pedoclimatic variables, and vegetation, allows identifying major ecological processes that govern the studied system, since they are considered as major drivers of plant community composition [START_REF] Box | Macroclimate and plant forms: an introduction to predictive modelling in phytogeography[END_REF]. Elevation known has an important driver of mountain grassland composition did not significantly influence plant species composition since limited to a relatively small elevational gradient of 400 m. ASR included slope and aspect that are known to determine plant species composition in mountain grasslands [START_REF] Srinivasan | Vegetation-environment relationships in a South Asian tropical montane grassland ecosystem: restoration implications[END_REF]. As many other plant communities, grasslands are controlled by edaphic factors, such as water content, organic matter and nutrient concentration [START_REF] Sebastiá | Role of topography and soils in grassland structuring at the landscape and community scales[END_REF][START_REF] Klimek | Plant species richness and composition in managed grasslands: The relative importance of field management and environmental factors[END_REF]). Several studies have emphasized the relation between pH and phosphorus. Phosphorus availability decreases with increase in pH because at alkaline pH, phosphate ions react with calcium or magnesium [START_REF] Cerozi B Da | The effect of pH on phosphorus availability and speciation in an aquaponics nutrient solution[END_REF][START_REF] Li | Abiotic and biotic controls on dynamics of labile phosphorus fractions in calcareous soils under agricultural cultivation[END_REF]. This is consistent with our results that show a negative correlation between soil pH and exchangeable phosphorus (P2O5).

The lack of methods for reference community choice has often been cited as a shortcoming in restoration ecology [START_REF] Halle | Advances in restoration ecology: insights from aquatic and terrestrial ecosystems[END_REF]) although some recent studies have developed a more explicit methodology. [START_REF] Suganuma | Indicators of restoration success in riparian tropical forests using multiple reference ecosystems[END_REF] selected nine references divided into three categories of riparian forest according to their ecological integrity (3 references in old growth forest, 3 in degraded forest and 3 in secondary forest). [START_REF] Erskine | Using reference sites to guide ecological engineering and restoration of an internationally significant uranium mine in the Northern Territory, Australia[END_REF] combined vegetation surveys and mapping data to select five reference sites for the ecological restoration of uranium mines, while [START_REF] Mcmanamay | Identifying nonreference sites to guide stream restoration and long-term monitoring[END_REF] suggest a spatial framework in six steps to identify reference systems for stream restoration. All these studies used multiple references that were selected according to relationships between environmental factors and vegetation patterns. They included floristic surveys (contemporary data) and the search of historical data. These studies suggest extending the surveys beyond the close vicinity of restoration sites and using statistical tools to identify and to rank structuring variables in order to select appropriate reference communities. In our framework, we filled this gap in providing a method to identify reference plant communities based on statistical analyses of specific relationships between environmental factors and plant community composition.

Since soil conditions are among the most important factors driving plant species composition, a careful variable choice is needed to exclude factors strongly influenced by previous ecosystem degradation. Soil disturbance, such as tillage or soil removal, may lead to a drastic reduction in organic matter and total nitrogen storage, and can strongly affect their distribution in the soil profile [START_REF] Hölzel | Restoration of a species-rich flood meadow by topsoil removal and diaspore transfer with plant material[END_REF][START_REF] Dolan | Soil organic carbon and nitrogen in a Minnesota soil as related to tillage, residue and nitrogen management[END_REF][START_REF] Du | Tillage and Residue Removal Effects on Soil Carbon and Nitrogen Storage in the North China Plain[END_REF]. Intermediate arable use, as the most common degradation of European grasslands, increases soil nutrient content, in particular plant available potassium and phosphorous [START_REF] Bischoff | Succession of floodplain grasslands following reduction in land use intensity: the importance of environmental conditions, management and dispersal[END_REF]). In such a case of eutrophication, the depletion of fertilizer residues is crucial to obtain abiotic conditions that are appropriate for the establishment of plant species adapted to the pre-degradation state [START_REF] Tallowin | Residual effects of phosphorous fertilisation on the restoration of floristic diversity to wet hay 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]). Thus, our method of reference community identification is difficult to apply to ecological restoration of fertilized soils. However, if habitat degradation is limited to mechanical soil disturbance, many soil variables typically driving plant species composition remain unchanged. In our approach, we avoided the use of the soil parameters cited above (total and mineral nitrogen, organic matter) that are known to be influenced by mechanical soil disturbance. Thus, we are convinced that our selected abiotic factors are appropriate for the search of best matching reference sites. Such data collected in situ (contemporary data) may be complemented by historical data (wildfire records, land use changes) that may still affect plant species composition [START_REF] Asbjornsen | Defining Reference Information for Restoring Ecologically Rare Tallgrass Oak Savannas in the Midwestern United States[END_REF][START_REF] Urgenson | Social perspectives on the use of reference conditions in restoration of fire-adapted forest landscapes[END_REF]. Our method is applicable to ecological restoration beyond grasslands if the specific environmental factors structuring the plant communities are correctly identified. For example, in ecological restoration of lagoon ecosystems corresponding to ancient salt marshes (EU habitat code 1150), environmental factors structuring plant communities are water level, duration of flooding and degree of salinity that may also be used to identify reference communities [START_REF] Bouzillé | Gestion des habitats naturels et biodiversité. Concept, méthodes et démarches[END_REF].

In restoration practice, adjacent sites of very similar environmental conditions are not always available. Either the degradation includes the whole habitat with completely different conditions outside the degraded zone (e.g. degraded grasslands within woodlands), adjacent sites are not accessible (or not available as donor sites for seed transfer) or abiotic conditions change at very small scales. In such cases, our approach allows the identification of geographically more distant reference or donor sites that match best with environmental conditions of restoration sites. In this case, the selection of multiple potential references is therefore necessary to select the one(s) that will be closest to the restored sites in terms of environmental variables driving plant community composition. The concept of multiple references is a helpful tool in restoration practice allowing a more flexible choice depending on availability (identification of donor sites) or on different priorities for environmental factor match. Additionally, even if the restoration sites are close, the best potential reference sites are not necessarily the same, depending on the environmental factors that can vary on a relatively small scale. Finally, in grassland restoration practice, the identification of multiple references allows to combine different donor sites if non-degraded sites do not comprise the entire species pool (area not sufficient, environmental conditions do not fully correspond). Although not directly tested in our proof-of-concept, the method also allows the integration of land management variation resulting in different quasi-stable states of plant succession.

To conclude, our approach to use the close relationships between environmental conditions and plant community composition is a straightforward method to identify reference communities when environmental structuring factors are not affected by degradation. It can be easily applied to restoration practice but requires a good knowledge of plant ecology, in particular on plant-environment interactions, environmental filters and local species pools [START_REF] Lortie | Rethinking plant community theory[END_REF][START_REF] Cristofoli | Restauration écologique: contexte, contraintes et indicateurs de suivi[END_REF]. A better understanding of plant-environment interactions and of potential effects of degradations on physical and chemical soil conditions would be required to improve the method.

Supporting Information

Supplement S1.1. Estimation of annual direct incident solar radiation (ASR) using the method of McCune and Keon (2002): a) "To approximate heat load, the equation can be shifted from a maximum on south slopes to a maximum on southwest slopes and a minimum on northeast slopes. This is accomplished by 'folding'the aspect about the NE -SW line, such that NE becomes zero degrees and SW becomes 180°": Chapter 2 focuses on drivers limiting seedling recruitment in mountain grassland restoration.

Folded aspect = | 180 -|aspect -225| | b) f (latitude,
Seeds were harvested using a brush harvester in one of the 18 references determined in chapter 1 and transferred to five degraded sites. I set up a combined soil preparation and grazing experiment to evaluate the effect of both factors on seedling recruitment.

Chapter Two

Seedling recruitment in mountain grassland restoration: effects of soil preparation and grazing

This chapter was published in 2021:

Durbecq A, d'Ambly M, Buisson E, Jaunatre R, Cluchier A, Bischoff A (2021) Seedling recruitment in mountain grassland restoration: Effects of soil preparation and grazing. Applied Vegetation Science. 24, e12564. https://onlinelibrary.wiley.com/doi/abs/10.1111/avsc.12564

Introduction

Seedling recruitment is an important process driving the composition and diversity of plant communities [START_REF] Grubb | The Maintenance of Species-Richness in Plant Communities: The Importance of the Regeneration Niche[END_REF][START_REF] Zeiter | Recruitment Limitation Constrains Local Species Richness and Productivity in Dry Grassland[END_REF]. Understanding the factors constraining seedling recruitment is thus crucial to predict and improve restoration success. Dispersal [START_REF] Münzbergová | Seed, dispersal, microsite, habitat and recruitment limitation: identification of terms and concepts in studies of limitations[END_REF][START_REF] Frances | Importance of Seed and Microsite Limitation: Native Wildflower Establishment in Non-native Pasture[END_REF]) and microsite limitation [START_REF] Myers | Seed arrival, ecological filters, and plant species richness: a meta-analysis[END_REF][START_REF] Long | Seed and microsite limitations mediate stochastic recruitment in a low-diversity prairie restoration[END_REF]) are known to be such constraining factors in re-colonisation processes. In grassland restoration, the small soil seed bank of typical late-successional species may result in insufficient seedling recruitment [START_REF] Grman | Dispersal and establishment filters influence the assembly of restored prairie plant communities[END_REF][START_REF] Buisson | Species transfer via topsoil translocation: lessons from two large Mediterranean restoration projects[END_REF]. Poor seed dispersal, as well as the lack of connectivity between source populations due to landscape fragmentation, often limit the seed rain [START_REF] Münzbergová | Seed, dispersal, microsite, habitat and recruitment limitation: identification of terms and concepts in studies of limitations[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][START_REF] Muller | Effect of topsoil removal and plant material transfer on vegetation development in created Mediterranean meso-xeric grasslands. N Hölzel (ed)[END_REF]) and successful restoration may require seed addition to overcome seed limitation [START_REF] Öster | Dispersal and establishment limitation reduces the potential for successful restoration of semi-natural grassland communities on former arable fields[END_REF][START_REF] Török | Fast restoration of grassland vegetation by a combination of seed mixture sowing and low-diversity hay transfer[END_REF]Valkó et al. 2016). Thus, seed addition methods such as sowing local seed mixtures, hay transfer or soil transfer, are increasingly used in grassland restoration [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]Scotton et al. 2012;[START_REF] Kiss | Establishment gaps in species-poor grasslands: artificial biodiversity hotspots to support the colonization of target species[END_REF].

Seed-addition experiments demonstrated that limited recruitment is the next major constraint influencing plant establishment [START_REF] Tilman | Community Invasibility, Recruitment Limitation, and Grassland Biodiversity[END_REF][START_REF] Turnbull | Are plant populations seed-limited? A review of seed sowing experiments[END_REF][START_REF] Pywell | Restoration of species-rich grassland on arable land: assessing the limiting processes using a multi-site experiment[END_REF]. Unfavourable microsite conditions may hamper germination and reduce seedling survival often resulting in microsite limitation [START_REF] Bissels | Effects of different mowing regimes on seedling recruitment in alluvial grasslands[END_REF]. Soil disturbance, such as topsoil removal or soil tillage, increases recruitment opportunities [START_REF] Hölzel | Restoration of a species-rich flood meadow by topsoil removal and diaspore transfer with plant material[END_REF][START_REF] Myers | Seed arrival, ecological filters, and plant species richness: a meta-analysis[END_REF][START_REF] Long | Seed and microsite limitations mediate stochastic recruitment in a low-diversity prairie restoration[END_REF]Klaus et al. 2017). Soil preparation (also called "seedbed preparation"; Shaw et al. 2020) may promote the recruitment of plant species by (i) limiting the competition of an already established vegetation, and by (ii) improving abiotic conditions of seedling recruitment [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]. A low pre-existing vegetation cover may have a facilitating effect on recruitment by protecting seedlings from high solar radiation and drought stress [START_REF] Callaway | Competition and Facilitation: A Synthetic Approach to Interactions in Plant Communities[END_REF]. However, if pre-existing vegetation is dense, the competitive effect on introduced species may prevail because already established species limit available space and resources [START_REF] Kupferschmid | Dispersal and microsite limitation in an abandoned calcareous grassland of the southern prealps[END_REF][START_REF] Poschlod | Diaspore and gap availability are limiting species richness in wet meadows[END_REF]. Preparing the soil may favour seedling recruitment of new species by opening the vegetation and destroying the root systems of competitive perennial species (Bischoff et al. 2018). Soil preparation further creates micro-reliefs improving seed and water retention as well as seed adhesion [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF]. However, soil disturbance increases nitrogen mineralisation increasing plant available nitrogen in the soil (eutrophication) and may negatively affect the soil fauna and soil structure potentially favouring erosion [START_REF] Di | Nitrate leaching in temperate agroecosystems: sources, factors and mitigating strategies[END_REF][START_REF] Holland | The environmental consequences of adopting conservation tillage in Europe: reviewing the evidence[END_REF]).

Human land management by grazing and mowing is a key factor regulating the functioning and structure of semi-natural grasslands [START_REF] Hejcman | Origin and history of grasslands in Central Europe -a review[END_REF]. Several studies confirmed the essential role of grazing in maintaining plant species richness of grassland ecosystems [START_REF] Dupré | Differences in species richness and life-history traits between grazed and abandoned grasslands in southern Sweden[END_REF][START_REF] Pykälä | Effects of restoration with cattle grazing on plant species composition and richness of semi-natural 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]. However, in the initial stages of grassland restoration, grazing may hamper seedling recruitment since seedlings are particularly vulnerable to trampling and biomass removal by herbivory [START_REF] Bakker | Herbivores as mediators of their environment: the impact of large and small species on vegetation dynamics[END_REF][START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]. Trampling may prevent species from germinating by crushing and damaging seeds or coleoptiles [START_REF] Rother | Demographic bottlenecks in tropical plant regeneration: A comparative analysis of causal influences[END_REF]). Moreover, trampling on slopes may lead to soil destabilization [START_REF] Tasser | Effects of land use in alpine grasslands on the probability of landslides[END_REF], increases soil erosion [START_REF] Farrell | Seeding alters plant community trajectory: Impacts of seeding, grazing and trampling on semi-arid re-vegetation[END_REF] and soil compaction [START_REF] Allington | Long-Term Livestock Exclusion in an Arid Grassland Alters Vegetation and Soil[END_REF]. However, in mountain ecosystems, in which the particular pedo-climatic conditions (soil erosion, shallow and stony soils) reduce seedling recruitment, micro-reliefs created by hoof prints may increase seed trapping [START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF]) and improve seed adhesion to the soil [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF]. Accordingly, [START_REF] Eichberg | Sheep trampling on surface-lying seeds improves seedling recruitment in open sand ecosystems[END_REF] found an increased seedling recruitment in a trampling simulation experiment on sandy soils also suffering from low stability and high run-off. Biomass removal by herbivory damages seedlings and often reduces initial seedling survival [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]Vidaller et al. 2019b). However, [START_REF] Farrell | Seeding alters plant community trajectory: Impacts of seeding, grazing and trampling on semi-arid re-vegetation[END_REF] only found a combined negative effect of trampling and herbivory whereas herbivory alone did not affect seedling establishment. Furthermore, [START_REF] Kladivová | Interacting effects of grazing and habitat conditions on seedling recruitment and establishment[END_REF] even revealed a positive effect of grazing on both seedling recruitment and establishment but the grazing effect depended on habitat conditions at the specific microsite.

In our study, we aim to test the effect of soil preparation and grazing on seedling recruitment (combining germination and early survival) in a seed-addition experiment conducted in semi-natural mountain grasslands. Using brush material transferred from a species-rich donor grassland to restore degraded sites, we addressed the following questions: (1) Does soil preparation by harrowing before sowing improve seedling recruitment and survival of the transferred species? (2) What is the effect of early grazing on seedling recruitment and survival of the transferred species?

Material and methods

Study area

The study area is the upper Durance valley ("Haute-Durance") in the Southern French Alps. The construction of a high-voltage transmission line led to a local destruction of species-rich grassland communities within a 1 km wide and 100 km long strip from L'Argentière-la-Bessée to La-Batie-Neuve (44°78'78''N, 6°59''41'E; 44°57'93''N, 6°20''77'E). The experimental sites representing a subsample of these grasslands are located between 1100 and 1300 meters above the sea level (upper montane altitudinal belt). The climate is temperate to sub-Mediterranean depending on slope, exposition and elevation. At the closest meteorological station (Embrun), the annual precipitation is about 740 mm and the mean annual temperature 10.7°C. Considering the lower elevation of the station (880 m) the temperature is roughly 2°C lower resulting in an annual mean of 8.7°C at our experimental sites. The bedrock is predominantly calcareous with some quaternary glacial deposits. Nondegraded grasslands of the study area belong to Natura 2000 priority habitats: "Seminatural dry grasslands of Festuco-Brometalia and scrubland facies on calcareous substrates" (N6210*, EU directive habitat 92/43/EEC; Calaciura and Spinelli 2008) that are traditionally managed either by mowing, cattle and sheep grazing. On transitory access tracks and construction platforms, the vegetation was removed, and the soil compacted to facilitate the transport of heavy equipment. To recreate former soil conditions after electricity line construction the soil was de-compacted by deep tillage between May and July 2018.

Donor site and seed harvest

The donor site to collect brush material is located within the study area at Freissinières at an elevation of 1100 m (44°73'61''N; 6°56'72''E). The soil and climatic conditions of the donor site corresponded to the average of the five experimental sites. The seed material was harvested in July 2018 using a brush harvester mounted on a quad and equipped with an integrated vacuum system. On a surface of 4000 m², we obtained 2.6 kg of brush material. The brush material comprised seeds (60% of total mass) and vegetative parts (40%). Seed counts in ten subsamples of 0.5 g revealed an average density of 887 seeds/g of brush material. A single harvest was considered as sufficient since the short summer season in the study area has resulted in a concentration of the seed production period for most species.

Experimental set-up and design

The experiment was set up in October 2018 using a full factorial split-plot design replicated on five sites (blocks) to test the effect of previous soil preparation, grazing and its interaction on seedling recruitment (see [START_REF] Durbecq | Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition[END_REF], chapter 1, for the position of experimental sites). Each of the five sites comprised eight plots of 16 m² each (4 m × 4 m) resulting in a total of 40 plots. Four plots representing a half-block were exposed to extensive grazing and the remaining four were fenced preventing grazing (whole-plot factor, Fig. 2.1). The sowing and soil preparation treatments (split-plot factors) were randomly assigned within half-blocks. The distance between plots was 1 m except for the Embrun site where this distance was reduced to 50 cm due to limited space. The full design also comprised unsown control plots randomly assigned to half-blocks. The unsown plots were used to evaluate which plant species of the donor grassland already occurred at the restoration sites before the brush material was transferred (soil seed bank, seed rain). Average vegetation cover of these unsown controls was 28% in the first year compared with 90% at the donor site. Species occurring in the control plots of a given site were considered as "spontaneously emerging" for this site. Only plant species of the donor site not occurring in the control plots were considered as "transferred" (Table andFig The method may underestimate the density of transferred species since species occurring in unsown control plots may still have been transferred to the sown plots.

Sites already covered with spontaneous vegetation were mown prior to experimental set-up. Harrowing was selected as a soil preparation method using a rotary harrow adjusted to a working depth of 8 cm. In October 2018, just after harrowing, the brushed material was spread at a density of 104 g/plot corresponding to a seed density of 3.75 g/m² and approximatively 5765 seeds/m². The chosen seed density was based on recommendations of a previous research project in the French Alps [START_REF] Koch | Les semences locales dans la restauration écologique en montagne. Production et utilisation de mélanges pour la préservation[END_REF]. In agreement with the traditional local grazing regime, the unfenced experimental plots were grazed twice a year: in late October just after experimental set up, and in early June. Two sites were grazed by cattle, three sites by sheep, corresponding to the average proportion of both grazing types in the study region. The sites were neither fertilised nor irrigated.

Data collection

Vegetation relevés at the donor site and germination tests in the greenhouse

Prior to seed harvest, the cover of all occurring vascular plant species was estimated in five representative 3 m × 3 m plots as the vertical projection cover of above-ground vegetation in late June 2018. We used [START_REF] Tison | Flore de la France méditerranéenne continentale[END_REF] for plant species identification and as reference for plant species names. At the harvest in July 2018, the average phenological stage of all species was recorded in order to evaluate seed availability. Eighty percent of the species recorded in June were found to have seeds in July.

In order to obtain information on the germination potential of seeds contained in the brush material and to evaluate germination in the field compared with germination potential, we set up a germination test in November 2018. Subsamples of the brush material were transferred to five trays (0.03 m²) filled with standard peat substrate and placed in an unheated greenhouse. Each tray received 0.54 g of brush material corresponding to 18 g/m² and a density of approximately 18 000 seeds per m². The trays were regularly watered, and germination was recorded until April 2019. Greenhouse germination of the harvested brush material was recorded once a week for four months and seedlings were removed after identification.

Seedling survey and vegetation relevés at restoration sites

To quantify seedling recruitment of both transferred species and spontaneously emerging species, plant numbers were counted in three quadrats of 40 cm × 40 cm placed along the diagonal of each plot. Since we were not able to distinguish seedlings and plants re-sprouting from below-ground organs, the seedling counts may include young ramets of clonally growing plants. In order to identify spontaneously emerging species, we conducted at the same time vegetation relevés on the unsown control plots (9 m²).

Seedling survival of focal species

Seedlings of two typical species of the donor site, Bromopsis erecta (Huds.) Fourr. (former name: Bromus erectus) and Rhinanthus alectorolophus (Scop.) Pollich were monitored over 11 weeks in order to assess seedling survival. B. erecta is the characteristic species of semi-dry grasslands (Mesobromion type sensu Ellenberg 1996) occupying a large range in Europe. It is further one of the typical species of the habitat type (N6210*, EU directive habitat 92/43/EEC; [START_REF] Calaciura | -Brometalia) (*important orchid sites)[END_REF]. B. erecta is a perennial grass growing on calcareous and often nutrient-poor soils. R. alectorolophus is a summer annual, hemi-parasitic species growing in semi-dry to mesophilic calcareous grasslands. As B. erecta, it is among the characteristic species of the N6210*-habitat type. R. alectorolophus has a wide range of hosts that includes grasses and legumes [START_REF] Sandner | Multiple choice: hemiparasite performance in multi-species mixtures[END_REF].

The species is used as a tool in restoration of species-rich grasslands as it reduces competition by grasses and may thus have a positive effect on plant diversity (Bullock and Pywell 2005;[START_REF] Heer | Hemiparasite-density effects on grassland plant diversity, composition and biomass[END_REF]. In all sown plots (four plots per site), ten randomly chosen seedlings of both species were tagged in May 2019 using coloured rings and poles (Fig. S2.2 S2.2). A total of 400 seedlings were tagged over the five sites. The position of each individual was mapped, and each seedling was numbered in order to facilitate localization and recognition. Seedling survival and reproduction of these two focal species were recorded in late July 2019.

Data analysis

We analysed (i) seedling numbers and species richness of the total plant community ("all species" = spontaneously emerging species + transferred species), and of transferred species only, as well as (ii) the survival of the two focal species, Bromopsis erecta and Rhinanthus alectorolophus.

Generalized linear mixed models were applied to assess the effect of soil preparation (harrowing: yes/no) and of grazing (yes/no) on seedling recruitment and the survival of B. erecta and R. alectorolophus. The full model included the two factors and the grazing-by-soil preparation interaction as fixed effects and site as a random effect. In order to include the split-plot design of the experiment, a grazing-by-site interaction (random) was fitted to the model to test the whole-plot factor (grazing). Harrowing (split-plot factor) and the site-byharrowing interaction were tested against the model residuals. Abundance data were rightskewed but models with Poisson error distribution and log-link function showed overdispersion. Thus, negative binomial error distribution and log-link function were finally used to test total abundance and abundance of transferred species. Species richness was tested using Gaussian error distribution and identity link. Both models additionally included quadrat within plots as a random factor. Survival was analysed at plot level and did not include quadrat. A binomial error distribution was fitted using a logit-link function.

Non-Metric Multidimensional Scaling (NMDS) based on Bray-Curtis similarity was applied to compare plant species composition in different treatments (Borcard et al. 2011). A permutation multivariate analysis of variance (PERMANOVA, Anderson 2001) with 9999 permutations was used to analyse whether the community composition was significantly different between the grazing and soil preparation treatments (R package "vegan", [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2[END_REF]. All analyses were performed using R 3.5.3 (R Core Team 2019).

Results

Seedling emergence

A total of 21 plant species germinated in the greenhouse corresponding to 37.5% of the total species number observed at the donor site. At the restoration sites, 29 donor site species (51.8%) were found. Taking into account the difference in the amount of seed material used in the greenhouse test and at the restoration sites, the final germination was 40 times higher in the greenhouse (7.31 seeds/g) than in the field (0.17 seeds/g; Table and Fig. S2.1). In particular, Plantago media L. Festuca marginata (Hack.) K.Richt. and Schedonorus pratensis (Huds.) P.Beauv. showed a much higher greenhouse germination whereas the difference between greenhouse and field was much smaller in B. erecta (4 times higher in the greenhouse). R. alectorolophus did not germinate in the greenhouse.

Seedling recruitment

Soil preparation by harrowing reduced overall seedling recruitment including that of spontaneously emerging species. Grazing had a marginally significant negative effect on seedling recruitment (Table 2.1, Fig. 2.2a). The opposite effect of harrowing was observed for transferred plant species. Twice as many seedlings of transferred species were found in the harrowed than in the non-harrowed plots (Fig. 2.2b). Grazing resulted in a 50% reduction of the seedling density of transferred species, but due to the high between-site variation the grazing effect was only marginally significant. The grazing-by-soil preparation interaction was not significant for both response variables.

Table 2.1. Effect of harrowing and grazing on number of seedlings per m², species richness and the survival of two focal species. Results on seedling number and richness are presented separately for the whole plant community and the subsample of transferred species. H x G = grazing-by-harrowing interaction. Results of GLMM with Chi-Square (χ2) and significance levels: . P < 0. of (a) all species, and of (b) transferred species only, in the four restoration treatments. The direction of main effects is indicated using "<<" (P<0.01), "<" (P<0.05), "(<)" (P<0.1; marginally significant).

Plant species richness

The treatment effects on overall species richness were not (grazing) or marginally (increase when harrowed) significant (Table 2.1, Fig. 2.3a). The species richness of transferred species was 30% higher in the harrowed compared to the non-harrowed plots (Fig. 2.3b). Similarly to the abundance of transferred species, the richness of transferred species was reduced by grazing but the effect was only marginally significant. No significant grazing-by-soil preparation interaction was observed. Fig. 2.3. (a) Species richness of seedlings of all species and of (b) transferred species in the four restoration treatments. The direction of main effects is indicated "<" (P<0.05), "(<)" (P<0.1; marginally significant) and "=" (not significantly different).

Plant species composition

The species composition was not clearly separated between treatments resulting in a large overlap of NMDS polygons (Fig. 2.4). Neither harrowing (P = 0.198), nor grazing (P = 0.392) nor the grazing-by-soil preparation interaction (P = 0.461) were significant. The plots without soil preparation nor grazing showed higher variation in species composition resulting in a larger polygon. Independent of treatment, semi-dry grassland species such as B. erecta, Saponaria ocymoides L. and Eryngium campestre L. occurred in the upper part of the ordination plot, and ruderal species such as Capsella bursa-pastoris (L.) Medik. Atriplex patula L. Anisantha sterilis (L.) Nevski (former name: Bromus sterilis), Veronica polita Fr. and Polygonum aviculare L. in the lower right part.

Seedling survival of two focal species

Seedling survival of B. erecta was high in all treatments ( 75%, Fig. 2.5a). The main effects of grazing and soil preparation were not significant, but a significant grazing-by-soil preparation interaction indicated that the grazing effect depended on soil preparation (Table 2.1). Grazing reduced seedling survival only in non-harrowed plots. With few exceptions, B. erecta did not reach the reproductive stage in the first year. The survival rate of R. alectorolophus was low (<45%) compared to that of B. erecta (Fig. 2.5b). Similar to B. erecta, no significant main effect was found for R. alectorolophus but a significant grazingby-soil preparation interaction explained by a negative grazing effect in non-harrowed plots (Fig. 2.5b). Most surviving R. alectorolophus individuals of the un-grazed, non-harrowed plots were flowering in July, with a significant negative effect of grazing (χ2 = 7.348, df = 1, P = 0.007; Fig. S2.3). Few individuals reached the reproductive stage in the three other treatment types. Similarly to survival, the significant grazing-by-soil preparation interaction (χ2 = 4.100, df = 1, P = 0.043) was explained by a negative grazing effect in non-harrowed plots. The direction of main effects is indicated using "<" (P<0.05) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05).

Discussion

The low field emergence compared with greenhouse germination showed that seedling recruitment may be a bottleneck in ecological restoration of grasslands (Öster et al. 2009). In our study on mountain grasslands, soil preparation largely increased the recruitment of transferred species, whereas competition by spontaneously emerging ruderal species was reduced. Grazing had only a weak negative effect on seedling density. While neither harrowing nor grazing had an effect on focal species survival when applied alone, a significant interaction effect indicated that the grazing effect depended on soil preparation or vice versa. The negative grazing effect was generally stronger in non-harrowed plots. Despite significant grazing and harrowing effects on seedling recruitment and early survival, none of the treatments influenced the first-year plant community composition.

Several studies have demonstrated that soil preparation prior to seed addition increases germination success and emergence of added seeds because it limits competition of preexisting vegetation [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF]Bischoff et al. 2018[START_REF] Harvolk-Schöning | Floodplain meadow restoration revisited: Long-term success of large scale application of diaspore transfer with plant material in restoration practice[END_REF]). However, pre-existing vegetation or a mulch layer may also facilitate seedling recruitment (Donath et al. 2007;Scotton et al. 2012). In particular in dry and open habitats, the absence of vegetation cover increases drought stress reducing germination and seedling survival [START_REF] Callaway | Competition and Facilitation: A Synthetic Approach to Interactions in Plant Communities[END_REF][START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF]. Our restoration sites in the Upper Durance valley clearly benefitted from previous harrowing indicating that the reduction of competition and improved conditions for germination [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF] prevailed over the reduction of facilitation effects. Similarly, [START_REF] Schmiede | Enhancing plant biodiversity in species-poor grassland through plant material transfer -the impact of sward disturbance[END_REF] and [START_REF] Harvolk-Schöning | Floodplain meadow restoration revisited: Long-term success of large scale application of diaspore transfer with plant material in restoration practice[END_REF] found that the cover of target species was higher following soil disturbance and seed introduction in a floodplain grassland. To our knowledge, our study is the first showing a simultaneous negative effect of soil preparation on spontaneously emerging species and a positive effect on transferred species. This effect was observed shortly after construction work leading to vegetation degradation and may be even stronger at restoration sites with higher spontaneous vegetation cover (here only 28%). The reduced competition by already established species created favourable microsite conditions for the recruitment of transferred species. Soil tillage particularly reduced the abundance of ruderal, competitive species (sensu Grime 1988), such as Anisantha sterilis (L.) Nevski, Capsella bursa-pastoris (L.) Medik. Atriplex patula L. and Polygonum aviculare L. Without soil disturbance, such competitive early successional species hamper the establishment of the transferred latesuccessional species (Donath et al. 2007;[START_REF] Jaunatre | Topsoil removal improves various restoration treatments of a Mediterranean steppe (La Crau, southeast France)[END_REF]. Regardless of competition, harrowing has also been shown to create favourable micro-reliefs improving the adhesion of the transferred seeds to the soil and thus favouring recruitment and establishment [START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF]. [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF] showed that seed movements and seedling establishment clearly depended on soil surface structure with holes limiting seed removal and increasing germination. The quantity of removed seeds also depends on the number of transferred seeds and their morphology (size, weight, shape). A better understanding of interactions between soil preparation, seed adhesion to the soil and seed morphology may help to increase the recruitment of transferred species and thus improve the efficiency of restoration measures [START_REF] Jakobsson | A comparative study of seed number, seed size, seedling size and recruitment in grassland plants[END_REF].

Contrary to the soil treatment, the effect of grazing was not selective between transferred species representing the target species of restoration, and non-target species. Grazing had a small but generally negative effect on seedling recruitment. The negative grazing effect was smaller than in other studies of the same geographical region but under Mediterranean climate [START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]Vidaller et al. 2019b) in which the transferred species clearly established better when grazing was excluded. Grazing animals negatively affect seedling recruitment by trampling and later on by biomass removal. Trampling by livestock does not only damage seedlings but also strongly affects the soil by erosion [START_REF] Farrell | Seeding alters plant community trajectory: Impacts of seeding, grazing and trampling on semi-arid re-vegetation[END_REF] or destabilization [START_REF] Tasser | Effects of land use in alpine grasslands on the probability of landslides[END_REF], particularly in mountain grassland slopes. On the other hand, trampling may have a positive effect on seedling recruitment of transferred species, since pre-existing vegetation is damaged by bruising, crushing, plant displacement or burial in mud [START_REF] Bilotta | The Impacts of Grazing Animals on the Quality of Soils, Vegetation, and Surface Waters in Intensively Managed Grasslands[END_REF], thus limiting competition with nontarget species. Similar to soil preparation, trampling may also create holes reducing seed removal of transferred species [START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF]. Soil preparation and grazing may thus have a common positive effect: the creation of microsites favourable to recruitment by limiting competition, increasing seed adhesion and water retention [START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF][START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF]. The outcome of the trade-off between microsite creation and seedling damage depends on the productivity of the sites and the timing of grazing. Biomass removal by grazing or mowing is particularly required on highly productive sites where competitive grasses often hamper seedling recruitment and establishment [START_REF] Schmiede | Enhancing plant biodiversity in species-poor grassland through plant material transfer -the impact of sward disturbance[END_REF]Kladivova and Münzbergova 2016). Nutrient-poor sites rather need protection of seedlings against grazing than biomass removal since competition is much lower [START_REF] Kirmer | Sowing of low and high diversity seed mixtures in ecological restoration of surface mined-land[END_REF]Scotton et al. 2012;Vidaller et al. 2019aVidaller et al. 2019b)). The marginally significant negative grazing effect in our study may thus be explained by the intermediate productivity level of our restoration sites. Post-pasture sowing has been cited as a method to optimise timing, because it allows seedlings to develop before the next grazing, reducing trampling and herbivory at the most vulnerable stage [START_REF] Eichberg | Sheep trampling on surface-lying seeds improves seedling recruitment in open sand ecosystems[END_REF]. Our study sites were first grazed before seedling emergence, 2-3 weeks after brush material transfer, and the following grazing period occurred seven months later. Thus, the first grazing probably had a positive effect in reducing competition by pre-existing vegetation, but the second grazing period may have negatively affected young seedlings that predominantly emerged in spring, finally resulting in a slightly negative effect of grazing. The vegetation period usually starts after snow melt in March resulting in a low probability to attain maturity before the June grazing period. The timing of grazing needs to be taken into account to limit the damage on seedlings, particularly if grazing exclusion is not possible. Livestock type may influence grazing effects [START_REF] Tóth | Livestock Type is More Crucial Than Grazing Intensity: Traditional Cattle and Sheep Grazing in Short-Grass Steppes[END_REF]. In our study, we focused on an overall grazing effect including sheep and cattle, and the grazer species effect was pooled with experimental site. It may be interesting for future studies to distinguish effects of cattle and sheep grazing on seedling recruitment.

The analysis of seedling survival of two focal species confirmed these conclusions. The grazing effect was small and limited to plots without soil preparation. The vegetation of these non-harrowed plots was denser and thus more attractive for grazers explaining the higher grazing effect. Grazing livestock prefers dense vegetation with higher food resource over sparse vegetation resulting in a higher biomass removal [START_REF] Török | Traditional Cattle Grazing in a Mosaic Alkali Landscape: Effects on Grassland Biodiversity along a Moisture Gradient[END_REF][START_REF] Meuret | When Art and Science Meet: Integrating Knowledge of French Herders with Science of Foraging Behavior[END_REF]. Bromopsis erecta showed a generally high survival rate after three months suggesting that the species, characteristic of semi-dry soils and semi-natural sites, is very well adapted to the pedo-climatic conditions of our study zone and resistant to grazing [START_REF] Calaciura | -Brometalia) (*important orchid sites)[END_REF]. Rhinanthus alectorolophus showed, however, a much lower survival since the abundance of potential host plants was low during the first year of restoration. The species is a hemi-parasite and its performance depends on the availability and performance of host plants [START_REF] Matthies | Response of a root hemiparasite to elevated CO2 depends on host type and soil nutrients[END_REF][START_REF] Bullock | Habitat-specific dispersal: environmental effects on the mechanisms and patterns of seed movement in a grassland herb Rhinanthus minor[END_REF]. It cannot establish under low vegetation cover and its population may go locally extinct as it is an annual species whose year-to-year survival relies on the production of seeds and seedling recruitment [START_REF] Coulson | Colonization of grassland by sown species: dispersal versus microsite limitation in responses to management[END_REF][START_REF] Bullock | Habitat-specific dispersal: environmental effects on the mechanisms and patterns of seed movement in a grassland herb Rhinanthus minor[END_REF]. Whereas the grazing effect on the survival of R. alectorolphus was not significant, the effect on flower production was negative. The result suggests that the species may survive grazing, but grazing damage prevent species from flowering.

The differential effects of soil preparation and grazing on spontaneous and transferred species did not result in a change in first-year plant species composition. The abundance of spontaneously emerging species was between six and ten times higher than that of transferred species, explaining that the observed significant soil treatment effect on transferred species was not yet visible at community level. The soil of disturbed sites generally contains high numbers of seeds of annuals and/or ruderal species [START_REF] Bischoff | Dispersal and establishment of floodplain grassland species as limiting factors in restoration[END_REF]Donath et al. 2007). These species often dominate the vegetation of early successional stages before being replaced by typical grassland species that are usually perennials developing later in the succession [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]Valkó et al. 2016;[START_REF] Kiss | Establishment gaps in species-poor grasslands: artificial biodiversity hotspots to support the colonization of target species[END_REF]. The first year after the restoration of Eastern European grasslands, [START_REF] Lengyel | Grassland restoration to conserve landscape-level biodiversity: a synthesis of early results from a large-scale project[END_REF] observed that annual weeds were dominant, but the cover of those weeds decreased dramatically after the third year due to the growth of perennial grasses. The authors observed an increase in the cover of target species from the first to the fourth year of restoration. A similar low initial restoration treatment effect due to initial low establishment of transferred target species and a subsequent high long-term restoration success (eight years) is known for riparian mesophilous grasslands [START_REF] Auestad | Near-natural methods promote restoration of speciesrich grassland vegetation -revisiting a road verge trial after 9 years[END_REF]Bischoff et al. 2018). Monitoring the plant community over several years will be necessary in order to evaluate whether the observed initial differences in seedling recruitment and early survival significantly influence plant species composition and restoration success in the long run.

Conclusion

Most species of the donor site were found at the restoration sites indicating that the applied brush harvesting technique was appropriate and allowed testing soil preparation and grazing effects. Similarly to studies in mesophilous grasslands [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF], Bischoff et al. 2018), soil preparation had a clearly positive effect on the seedling recruitment. The reduction in overall seedling density dominated by spontaneously emerging species demonstrated that the reduction in competition was the principal mechanism explaining the positive effect of previous soil tillage by harrowing on the recruitment of transferred species [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF][START_REF] Schmiede | Enhancing plant biodiversity in species-poor grassland through plant material transfer -the impact of sward disturbance[END_REF]. We thus recommend soil preparation before seed-based ecological restoration even in semi-dry grasslands to improve seedling recruitment of transferred target species. Our study did not confirm a strong negative effect of grazing on seedling recruitment found by several other authors (Scotton et al. 2012;Vidaller et al. 2019b). The marginally significant negative effect of extensive grazing may be tolerated if farmers are reluctant in putting up fences as in our study area. Further research is needed on the timing of sowing relative to grazing periods, in order to benefit from positive grazing effects by reduction of competition while avoiding seedling damage.

Chap. 2. Seedling recruitment in mountain grassland restoration 51 in the first year. The direction of main effects is indicated using "<<" (P<0.01), "<" (P<0.05), "(<)" (P<0.1) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05).

Supporting information

(2.a) Does soil preparation by harrowing before sowing improve recruitment and survival of the transferred species?

In chapter 2, the soil preparation (tillage) applied before addition of brush-harvested propagules largely increased the recruitment of transferred species harvested in one of the reference communities, whereas the density of spontaneously emerging species was reduced. Therefore, this technique can clearly be recommended in my study system to enhance the recruitment of target species.

(2.b) What is the effect of early extensive grazing on seedling recruitment and survival of the transferred species?

The effect of livestock grazing on transferred species recruitment was weakly negative, suggesting that fencing costs are not always justified. However, as my study sites were first grazed before seedling emergence of transferred species, and as the following grazing period occurred seven months later, the first grazing probably had a positive effect, but the second grazing period may have negatively affected young seedlings. Thus, the timing of grazing probably needs to be considered to limit seedling damage, particularly if grazing exclusion is not possible.

Chapter 3 presents a test of different seed transfer techniques and nurse plant sowing in order to overcome low seed dispersal and to understand the mechanisms favouring target species recruitment and establishment. Hay transfer, brush-harvested propagule transfer, and brush-harvested propagule transfer combined with wheat sowing were applied. Each restored plant community was compared to the best references identified in the chapter 1.

Mountain grassland restoration using hay and brush material transfer combined with temporary wheat cover

Abstract

Mountain grassland restoration success may be hampered by limited seed dispersal and poor soil seed banks of many grassland species. These constraints can be overcome by actively introducing propagules from nearby non-degraded communities.

We tested different restoration techniques in order to understand the mechanisms favouring target species seedling recruitment and establishment. In five degraded mountain grasslands, we analysed (i) the effect of two techniques increasingly used in ecological restoration to overcome low seed dispersal: transfer of brush-harvested seed material and hay transfer, and (ii) the potentially facilitative effect of a temporary plant cover (common wheat) on the recruitment of transferred brush-harvested propagules.

We found that both propagule transfer techniques were successful in establishing plant species of the donor community with an increase of plant species richness, cover and abundance of transferred species. Hay transfer was more efficient in transferring species of the donor grassland than brush-harvested material transfer. Brush-harvested material transfer only increased abundance and cover of donor grassland species when sown together with wheat.

The results indicated that hay mulch favoured seedling recruitment of target species, and that propagule transfer without hay mulch needs to be compensated by additional temporary plant cover in order to create favourable conditions for seedling recruitment. A comparison with best reference communities for each restoration grassland confirmed that hay transfer and brush material transfer with wheat sowing were successful in driving plant community composition towards the desired reference state.

In conclusion, restoration of mountain grasslands with shallow and stony soils clearly benefits from a facilitative effect of dead (hay) or living (wheat) vegetation cover.

Introduction

Grassland ecosystems represent 25% of terrestrial biomes [START_REF] Blair | Grassland Ecology[END_REF][START_REF] Wilsey | Restoration in the face of changing climate: importance of persistence, priority effects, and species diversity[END_REF] and are essential habitats for the conservation of biodiversity and associated ecosystem services [START_REF] Stoate | Ecological impacts of early 21st century agricultural change in Europe -A review[END_REF][START_REF] Amiaud | Grassland multifunctionality in providing ecosystem services[END_REF]Valkó et al. 2016). Since the 1980ies, the conservation of species-rich semi-natural grasslands has become a major concern in Europe. Most European semi-natural grasslands were integrated in the habitat management Natura 2000 network in order to stop habitat degradation and to re-establish grassland species diversity using ecological restoration approaches [START_REF] Wilsey | Restoration in the face of changing climate: importance of persistence, priority effects, and species diversity[END_REF].

Successful grassland restoration may be primarily hampered by limited dispersal capacity and poor soil seed banks of many grassland species, and by a grazing management favouring clonal reproduction over sexual reproduction [START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Török | Beyond the species pool: modification of species dispersal, establishment, and assembly by habitat restoration[END_REF]). These constraints are well documented and can be overcome by actively introducing propagules [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]Scotton et al. 2012;[START_REF] Hölzel | EDITORIAL: Species introduction -a major topic in vegetation restoration[END_REF]. The use of native plant species of local origin is recommended to assure adaption to local environmental conditions and to maintain genetic diversity (Vander Mijnsbrugge et al. 2010;[START_REF] Bucharova | Mix and match: regional admixture provenancing strikes a balance among different seed-sourcing strategies for ecological restoration[END_REF]. Consequently, transferring local seeds from nearby non-degraded communities is increasingly used in ecological restoration [START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]. Diverse techniques are currently applied depending on specific environmental conditions at restoration and donor sites [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]Hedberg & Kotowski 2010). The increasing number of studies illustrates the scientific interest in comparing restoration effectiveness of various seed harvesting and transfer techniques [START_REF] Scotton | Efficiency of mechanical seed harvesting for grassland restoration[END_REF], such as hay transfer vs. sowing of seed mixtures [START_REF] Auestad | Nature will have its way: local vegetation trumps restoration treatments in semi-natural grassland[END_REF][START_REF] Kövendi-Jakó | Three years of vegetation development worth 30 years of secondary succession in urban-industrial grassland restoration[END_REF], brush material vs. green hay transfer (Albert et al. 2019), or sowing combined with various additional treatments [START_REF] Pawluśkiewicz | Effects of Different Introduction Methods on Plant Species Establishment Success in Wet Grassland Restoration[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF].

In our study, we compared the restoration effectiveness of two mechanical harvesting techniques increasingly used in mountain grassland restoration: the transfer of brushharvested seed material and the transfer of dry hay both collected from the same donor site located close to the degraded grasslands. The brush harvester strips and aspirates the plant material without cutting (Scotton et al. 2012). The harvested brush material comprises seeds but also vegetative parts of fruits and, to a lesser degree, other vegetative material. Low-growing and less abundant species may be under-represented in this material [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF][START_REF] Scotton | Seed Harvesting for Ecological Restoration: Efficiency of Haymaking and Seed-Stripping on Different Grassland Types in the Eastern Italian Alps[END_REF][START_REF] Wagner | Green hay transfer for grassland restoration: species capture and establishment[END_REF]), but brush harvesting allows concentrating seeds thus reducing humidity and facilitating storage. It also reduces the mulch layer that may hamper germination [START_REF] Mollard | Impacts of mulch on prairie seedling establishment: Facilitative to inhibitory effects[END_REF]. Dry hay transfer involves cutting of plant material close to the soil surface, drying it on the field before storage and spreading it on the restoration site. The higher vegetative biomass compared with brush material may hamper germination but protects seedlings against high solar radiation, drought and soil erosion [START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF][START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF].

As seedling recruitment and establishment are important constraints in semi-natural grassland restoration (Öster et al. 2009), we also tested whether simultaneous transfer of brush material and sowing of an annual plant species may compensate for the absence of a protecting mulch layer. Such annuals provide temporary living cover that may facilitate seedling recruitment and establishment [START_REF] Padilla | The role of nurse plants in the restoration of degraded environments[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF]. Similarly to hay mulch, a living cover may, however, increase root and light competition [START_REF] Donath | Influence of competition by sown grass, disturbance and litter on recruitment of rare flood-meadow species[END_REF]. We tested a mix of two lowland varieties of common wheat Triticum aestivum as a facilitative species. These varieties were expected to disappear within a couple of years since climatic conditions of our mountain study sites limit their seed production and self-sowing. Triticum cover may reduce soil erosion, an important criterion for the functionality of restoration measures. Such soil stabilisation and erosion control are particularly important in mountain grasslands on slopes facing a high risk of soil loss and propagule run-off [START_REF] Huc | SEM'LESALPES -Des semences d'origine locale pour la restauration de milieux ouverts en montagne alpine[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF].

In order to understand the underlying mechanisms of seedling recruitment and establishment, we analysed first-year seedling abundance and second-year cover, respectively. To determine which technique is the most appropriate in mountain grassland restoration, we addressed the following questions: (1) Which method of plant material transfer is most successful in establishing species transferred from the donor community? (2) Does sowing of wheat improve the seedling establishment of brush material? (3) Do transfer techniques and wheat sowing increase the similarity between restored and reference communities? (4) What is the influence of these treatments on soil erosion?

Material and methods

Study area

The study area was the upper Durance valley in the Southern French Alps. The five selected restoration sites were degraded by the construction of a high-voltage transmission line involving the creation of access tracks and working platforms. The construction work locally destroyed the vegetation and affected the soil structure. The study zone extends over about 100 km, from L'Argentière-la-Bessée in the north to La-Bâtie-Neuve in the south (44°78'78''N, 6°59''41'E; 44°57'93''N, 6°20''77'E). The study sites are located on slopes above the Durance valley, at an elevation of 1060 m to 1320 m above sea level. They are characterised by a subcontinental climate, with an average annual temperature of 8.7°C and an annual rainfall of 740 mm (Embrun meteorological station, annual temperature corrected for an altitudinal difference of roughly 2°C). Dry to mesophilic grasslands occur at sites managed by grazing or mowing. Most of these surrounding non-degraded grasslands are priority habitats of Natura 2000: 'Semi-natural dry grasslands of Festuco-Brometalia and scrubland facies on limestone'(N6210, European habitat directive 92/43/EEC; [START_REF] Calaciura | -Brometalia) (*important orchid sites)[END_REF]. The five restoration sites used for the experiments were former access tracks and working platforms, and restoration goal was the re-establishment of typical Festuco-Brometalia grasslands. Traditional management by grazing was reintroduced.

Donor and reference sites

One typical non-degraded Mesobromion grassland of the study area was chosen as donor site for harvesting plant material. The site was located in Freissinières (44°73'61''N; 6°56'72''E) at an elevation of 1100 m and a 3° south-west-facing slope. Two harvest techniques were applied, brush harvesting and traditional haymaking. The brush material was harvested on 13 July 2018 using a brush harvester mounted on a quad and equipped with an integrated vacuum system. On a surface of 4000 m², 2.6 kg of brush material was obtained. The harvested material comprised seeds (60% of total mass) and vegetative parts (40%). Seeds were counted in 10 subsamples of 0.5 g harvested materials revealing an average density of 887 seeds/g. Hay was cut on 15 July 2018 on an area of 800 m² using a rotary mower. The hay was dried for two days on the harvested grassland and was baled in five bales of 7.5 kg (45 × 30 × 75 cm) for a total of 37.5 kg, with approximately 1% of seeds in the total raw material. Brush material and hay were kept for three months at room temperature under dry and dark conditions.

Although the donor site reflected the medium environmental conditions of the five restoration sites we identified "best references" for each site to evaluate restoration success. Since best reference sites are not always the closest ones [START_REF] Durbecq | Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition[END_REF], chapter 1), we surveyed eighteen non-degraded grasslands along the new electricity line. These potential references included the donor site and were located at the same altitudinal belt as restoration sites (1000 -1400 m). Soil and microclimatic conditions measured at restoration sites and potential reference communities were used to identify the best references of each restoration site according to Durbecq et al. (2020, methodology).

Experimental design

At the five restoration sites, we repeated the following five treatments in 4 m × 4 m plots: control without active seed addition (control), hay transfer ('Hay'), brush material transfer ('Seed'), wheat sowing ('Triticum') and both brush material and wheat seed addition together ('Triticum + Seed'). The restoration sites that were already colonized by spontaneous vegetation were mown before the set-up of the experiments and they were then extensively grazed in spring and autumn. The position of the five treatments was randomised within sites.

In October 2018, 6.5 g/m² of harvested brush material was spread in the brush material treatments, corresponding to 3.75 g/m² of seed and 5765 seeds/m². The chosen seed density is a little higher than recommended for lowland grasslands and lower than usually sown in alpine grasslands [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]Scotton et al. 2012;[START_REF] Koch | Les semences locales dans la restauration écologique en montagne. Production et utilisation de mélanges pour la préservation[END_REF]. The brush material was pressed to the ground using a lawn roller. In the hay transfer treatment, a bale of hay of 7.5 kg was spread (0.5 kg/m²) on each plot. The amount of hay was adjusted to an estimated seed content of 1% (Scotton et al. 2012) corresponding to roughly 5 g/m² of seeds. The hay was pressed to the ground by the wheels of tractor in order to maintain hay on the ground. The sown wheat treatment was a mixture of two local lowland winter wheat (Triticum aestivum) varieties, "Meunier d'Apt" and "Saissette de Provence". These early lowland varieties are not well adapted to high altitude resulting in low seed production and a rapid decline during mountain grassland succession. The seed density was adjusted to 9 g/m². Like for the brush material transfer, a lawn roller was used to improve seed adhesion to the ground. Controls were not sown but they were also rolled.

Data collection

Vegetation relevés in donor and reference grasslands

In the donor grassland, vegetation relevés were conducted on 29 June 2018, and the occurrence of mature seeds was evaluated for each plant species at the harvest date. Relevés were used to establish a list of 37 potentially transferred species (hereafter: transferred species). The vegetation relevés of the eighteen potential reference sites were conducted between 21 June and 5 July 2018 by visual estimation of plant cover for each species in quadrats of 2 m × 2 m. In the donor grassland, five quadrats were sampled, and three in each of the other potential reference grasslands.

Germination test in a greenhouse

In November 2018, brush material and hay were spread in an unheated greenhouse to evaluate and compare the germination potential of seeds in brush material and hay. Harvested brush material and dry hay were sown to ten trays (0.03 m²) filled with standard potting soil substrate (mixture of 1/3 vermiculite and 2/3 of sterile peat). Five trays received 0.54 g of brush material corresponding to 18g/m² of seeds, and five trays received 12.5 g of hay corresponding to 4 g/m² of seed material according to an estimated seed content of 1%. The trays were regularly watered, and germinations identified and removed until April 2019.

Seedling abundance and vegetation relevés in grasslands under restoration

To measure seedling abundance, plant number was counted in three quadrats of 40 × 40 cm placed along the diagonal of each plot in June 2019, at the five restoration sites. Since it was not possible to distinguish seedlings and plants re-sprouting from belowground organs, the seedling counts may include stems or ramets of vegetative regeneration. We considered all reproducing plant species of the donor site as transferred although some of them also occurred in the control plots of sites under restoration, indicating their presence in the soil seed bank or seed rain. In June 2020, the cover of plant species was measured following the same protocol as in the potential reference grasslands: in each plot, one quadrat of 2 m × 2 m was sampled.

Erosion monitoring

To evaluate the effect of the different treatments on soil erosion, we measured erosion according to [START_REF] Feret | Utilisation d'un appareil de mesure simple, et précis, pour l'étude de l'érosion à Mayotte[END_REF] at four restoration sites (at one site, the permanent points were accidentally removed in autumn 2019 not allowing any analysis). The device comprised a 1.5 m long aluminium bar and two 0.5 m long iron poles fixed in the ground of each plot. These iron poles were the permanent points of the measurements. They were adjusted to provide a horizontal support for the aluminium bar before measurements. Vertical holes were drilled in the centre of the aluminium bar every 10 cm resulting in a total of 13 regularly spaced holes (Fig. S3.1). To take the erosion measurements, a graduated rod was introduced in each of the holes. The scale of the graduated rods allowed a direct measurement of the space between the aluminium bar and the ground. An increase in space between measurements indicates erosion whereas a decrease suggests an accumulation of soil. Measurements were taken in June 2019, October 2019, June 2020 and September 2020, in order to assess the effect of restoration treatments on soil erosion.

Data analysis

We used plant abundance, cover and richness of the entire plant community and of transferred species to run statistical tests and to calculate restoration indices. Major soil and microclimatic variables of restoration sites and potential reference sites were fitted to multivariate models (NMDS) to identify the best references of each restoration site. The identification was based on dissimilarity coefficients using Euclidean distances and is detailed in Durbecq et al. (2020, chapter 1). We compared restoration sites and these best reference sites using the restoration indices CSIInorm (normalized Community Structure Integrity Index) and HAI (Higher Abundance Index). The CSII allows measuring which proportion of the species abundance of the reference communities is represented in the restored communities, and the HAI evaluates the proportions of the species abundance in the restored communities that are higher than in the reference communities [START_REF] Jaunatre | New synthetic indicators to assess community resilience and restoration success[END_REF].

We ran linear and generalized mixed models with transfer treatments as fixed effect and site as random effect to analyse response variables (including restoration indices) to the restoration treatments. In the analyses of plant abundance, quadrat within treatment was additionally included as a random effect. Plant cover, species richness and restoration indices were normally distributed and thus analysed using a Gaussian distribution with identity link. Plant abundance was fitted using a Poisson distribution with log-link function.

In the case of a significant treatment effect, pairwise comparisons were used to compare differences between transfer techniques (glht function in multcomp package; [START_REF] Hothorn | Simultaneous Inference in General Parametric Models[END_REF].

A NMDS (Non-Metric Multidimensional Scaling) based on Bray-Curtis similarity was applied to compare plant species composition of the five restoration sites and their best reference sites. A Permutational Multivariate Analysis of Variance (PERMANOVA) was used to analyse whether the community composition was significantly different between treatments (R package "vegan"). All analyses were performed using R 3.5.3 (R Core Team 2019).

Results

The greenhouse tests revealed a mean density of 197.4 viable seeds/g in the brush harvest material and 2.4 viable seeds/g in the hay. This corresponds to a density of 1283 viable seeds per m² and 1200 viable seeds per m² transferred with brush material and hay to the restoration sites, respectively.

Effects of transfer techniques and wheat sowing on target species establishment

Seedling emergence of transferred species was only 50/m² (without wheat) to 65/m² (with wheat) in the brush material transfer and 200/m² in the hay transfer corresponding to 4%, 5%, and 17% of the number of transferred viable seeds, respectively. Transferred species abundance was significantly different between the restoration treatments (χ² = 51.85, df = 4, P < 0.001; Fig. 3.1). We found the clearly highest density of transferred species in the 'Hay'treatment although seed density was similar to brush material according to germination tests. The 'Seed'treatment involving brush material transfer without wheat addition was neither significantly different to the control nor to the 'Triticum'treatment.

Transferred seedling abundance in the combined 'Triticum + Seed'treatment was significantly higher than in the control but lower than in the 'Hay'treatment. The treatments also had a significant effect on the second-year cover of transferred species (χ² = 16.28, df = 4, P = 0.002; Fig 3 .2.a). However, the strongly positive effect of the 'Hay'treatment vanished and the difference to the control was not significant any more. Transferred species cover was significantly higher in the combined 'Triticum + Seed'treatment than in the 'Seed'and 'Triticum'treatments whereas all other treatments were not significantly different.

Similarly to transferred species cover (Fig. 3.2.a), richness of transferred species varied significantly between treatments (χ² = 19.65, df = 4, P < 0.001; Fig. 2.b). The 'Seed'treatment was again only significantly different from the control if combined with wheat sowing ('Triticum + Seed'). Contrary to transferred species cover but in line with first-year abundance, species richness was significantly higher in the 'Hay'treatment than in the control. The 'Hay'and the combined 'Triticum + Seed'treatments comprised on average seven more transferred species (± 3 species) than the control. The 'Seed'treatment without wheat sowing was not significantly different from the control or the 'Triticum'treatment. Similar treatment effects were visible for the total species richness (Fig. S3.2). However, only the difference between the combined 'Triticum + Seed'treatment and the control was significant.

Comparison with best references of each restoration site

The plant species composition of the restoration sites was very different from that of reference sites in all treatments (Fig. 3.3). Restoration and reference sites were particularly separated on the first axis. This axis represented a disturbance gradient with annual and ruderal species occurring on the left together with restoration sites (Arctium minus (Hill) Bernh. Polygonum aviculare L. Anisantha sterilis (L.) Nevski, etc.) and perennial grassland species on the right together with reference sites (Stipa pennata L. Helianthemum nummularium (L.) Mill. Pilosella officinarum F.W.Schultz & Sch.Bip. etc.) . The differences between reference sites reflect the heterogeneity of plant communities according to different environmental conditions that are independent of degradation. They were particularly separated on the second NMDS axis representing a productivity gradient from stony, xeric sites in the lower part of the biplot to more humid, mesophilic sites in the upper part. There was a large overlap between the five treatments within restoration sites, but the treatment effect was still significant (F = 1.432, df = 4, P = 0.012). As expected, the unsown control showed the greatest distance to the references. The 'Hay'and 'Triticum + Seed'treatments were closest to the reference sites, followed by the 'Seed'treatment without wheat. However, the latter treatment was not much closer to the reference communities than wheat sowing alone. The 'Triticum'treatment showed the highest variation between sites resulting in a large NMDS polygon. and for sites corresponding to reference n°16 (same reference for the two restoration sites), to reference n°12, to reference n°6, and to reference n°9.

The CSIInorm calculated for each restoration site compared with their respective best reference showed a significant difference between treatments (χ2 = 18.02, df = 4, P < 0.01; Fig. 3.4a and 3.5). CSIInorm was significantly higher in the combined 'Triticum + Seed'treatment than in the control, the 'Triticum'and the 'Seed'treatments. The 'Hay'treatment was not significantly different from other treatments.

The HAI was also significantly different between treatments (χ² = 11.44, df = 4, P < 0.05, Fig. 3.4b and 3.5). The lowest HAI was found in the 'Seed'treatment being significantly lower than the control and the 'Triticum'treatment, indicating significantly lower non-target abundances in the 'Seed'treatment. Differences between other treatments were not significant.

Ecosystem function: erosion control

Erosion monitoring did not show significant treatment effects at the end of the observation period. Lowest erosion was observed in the 'Hay'treatment but due to high between-site variation, the difference to other treatments was not significant (Fig. 3.6). Intermediate measurements demonstrated a high temporal variability of erosion and of treatment effects on erosion (Fig. S3.3). Positive values show higher erosion, thus a lesser amount of material on the ground 14 months after the beginning of the experiment.

Discussion

Both techniques, brush-harvested propagule transfer and hay transfer, allowed the introduction of high numbers of viable seeds. However, first-year seedling recruitment was much higher in hay transfer than in brush material transfer. Additional wheat sowing had a particularly positive effect on seedling establishment from brush material resulting in a second-year cover of transferred species similar to that of hay. Brush material transfer alone did neither increase transferred species cover nor plant species richness. According to transferred species abundance and cover, hay transfer and brush material transfer combined with wheat sowing were also most successful in restoring the plant species composition of the reference. Soil erosion was not affected by any restoration treatments.

Effect of seed-addition techniques on transferred species recruitment

Brush harvesting has many technical advantages compared to hay cutting allowing selection and cleaning of seeds, drying and particularly easy storage (lower volume than hay) under optimum temperature and humidity [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF][START_REF] Vitis | Seed storage: Maintaining seed viability and vigor for restoration use[END_REF][START_REF] Frischie | Ensuring seed quality in ecological restoration: Native seed cleaning and testing[END_REF]. Contrary to hay cutting, it is possible to brush-harvest several times a season thus maximizing the number of transferred species [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF][START_REF] Scotton | Efficiency of mechanical seed harvesting for grassland restoration[END_REF]. The possibility to use grasslands for hay making after brush harvesting is another advantage reducing losses for livestock farmers. However, several studies obtained a low efficiency in plant establishment from brush material compared to hay transfer [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF]Sengl et al. 2017;Albert et al. 2019). Albert et al. (2019) explained the lower seed number per harvested area by the higher working height of brushes compared to mowing machinery missing the seeds of low-growing species.

In our study, the brush material contained as many viable seeds as the hay suggesting that the low recruitment compared to hay transfer was due to lower germination and seedling survival. The mulch layer resulting from hay transfer may improve seedling recruitment through an increase in soil moisture retention (Donath et al. 2007;[START_REF] Mollard | Impacts of mulch on prairie seedling establishment: Facilitative to inhibitory effects[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF]. Soil moisture retention and temperature buffering are important factors influencing germination in our study region characterized by shallow soils, spring frosts and high solar radiation. Furthermore, hay mulch protects seeds from predation, and animal trampling (Scotton et al. 2012;[START_REF] Vanderburg | Trampling and Cover Effects on Soil Compaction and Seedling Establishment in Reseeded Pasturelands Over Time[END_REF]) and limits competition of ruderal species in early stages of grassland restoration [START_REF] Kiehl | Effect of Hay Transfer on Long-Term Establishment of Vegetation and Grasshoppers on Former Arable Fields[END_REF]. Additionally, a hay layer may reduce surface runoff [START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF]) and improve seed adherence to the soil [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF]. High recruitment in the hay transfer treatment suggests that such positive effects prevailed in our study over well-known negative effects of a mulch layer, such as light limitation [START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF].

Effects of temporary wheat cover on seedling recruitment may be similar to hay mulch effects, such as protection against drought and solar radiation [START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF]. Additionally, belowground effects may facilitate seedling recruitment and establishment since wheat roots stabilize the soil and foster the recolonization of the soil fauna (Faivre 2000). In modifying soil structure and composition [START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF][START_REF] Liu | The effects of vegetation on runoff and soil loss: Multidimensional structure analysis and scale characteristics[END_REF], wheat can even be considered as an ecosystem engineer (Jones et al. 1996). Many high-altitude restoration studies aimed at improving recruitment conditions and reducing soil erosion using "nurse species" or wheat straw addition to stabilize the soil [START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF][START_REF] Kavian | Impact of Wheat Residue on Soil Erosion Processes[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]. However, propagule run-off, germination and early survival are less likely affected by wheat sowing than by hay mulch since wheat cover develops later. In our study, this was probably the reason for an absence of a wheat sowing effect on first-year abundance of species transferred with brush material whereas the effect on second-year cover was strongly positive. Like hay transfer, facilitative effects of wheat sowing prevailed over negative effects of competition for light and water observed in other studies on additional sowing of nurse species [START_REF] Donath | Influence of competition by sown grass, disturbance and litter on recruitment of rare flood-meadow species[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF].

Soil preparation before sowing has often been recommended to improve seedling recruitment in sowing of brush-harvested seeds or seed mixtures [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF][START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF]Klaus et al. 2017;Shaw et al. 2020). Soil preparation increases seed and water retention as well as seed adhesion [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF] and may compensate for the absence of a hay mulch layer limiting seed run-off. In our study area, a previous experiment testing soil preparation effects on seedling recruitment showed a increase in firstyear abundance of transferred species (Durbecq et al. 2021, chapter 2). Thus, the plots that received brush material in our study may particularly benefit from previous soil tillage and a combination with additional wheat sowing may be the optimum strategy for seedling recruitment and establishment.

Successful restoration of reference communities and erosion protection

Seed addition using brush material and hay of the donor site was successful in increasing the similarity to best references at plant community level. As in the analysis of cover and number of species transferred from the donor site, best restoration results were obtained for hay transfer and the combination of brush material with wheat sowing, showing highest similarity to references in plant species composition and highest target abundance (higher CSIInorm = abundance of species occurring in the reference). This finding confirmed that a dead (hay) or living (wheat) "nurse cover" improves seedling establishment of target (reference communities) and transferred (donor community) species [START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF][START_REF] Kavian | Impact of Wheat Residue on Soil Erosion Processes[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]. The brush material transfer without wheat sowing did not increase similarity to references nor target abundance. However, HAI (proportion of non-target abundances) of this treatment was lower compared to the control whereas the HAI of brush material with wheat and hay transfer were not significantly different from the control. These results indicated that hay mulch and wheat cover did not only provide better conditions for target species but also for non-target species recruitment.

In mountain and alpine grasslands with more or less steep slopes, soil stabilization processes are slow, which increases soil erosion [START_REF] Wiesmair | Relationships between plant diversity, vegetation cover, and site conditions: implications for grassland conservation in the Greater Caucasus[END_REF]). Erosion control is thus essential to restore these ecosystems [START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF][START_REF] Löbmann | Towards a better understanding of shallow erosion resistance of subalpine grasslands[END_REF]. Without plant cover, erosion, depletion of organic matter and gully formation may compromise restoration approaches (Dupin et al. 2019). Several authors have shown a negative linear relationship between vegetation cover and soil runoff [START_REF] Peratoner | Organic seed propagation of alpine species and their use in ecological restoration of ski runs in mountain regions[END_REF][START_REF] Liu | The effects of vegetation on runoff and soil loss: Multidimensional structure analysis and scale characteristics[END_REF]Gu et al. 2020). Hence, immediate revegetation by locally adapted propagules is widely recommended [START_REF] Scotton | Seed Harvesting for Ecological Restoration: Efficiency of Haymaking and Seed-Stripping on Different Grassland Types in the Eastern Italian Alps[END_REF][START_REF] Wiesmair | Relationships between plant diversity, vegetation cover, and site conditions: implications for grassland conservation in the Greater Caucasus[END_REF]Dupin et al. 2019). Using well-adapted temporary nurse plant cover may have a stronger positive effect on soil erosion control than dead plant material, such as hay or straw, since developing roots allow a belowground stabilisation of the soil [START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF]. According to [START_REF] Liu | The effects of vegetation on runoff and soil loss: Multidimensional structure analysis and scale characteristics[END_REF] and Gu et al. (2020), an average total vegetation cover of 60% observed in our study may reduce soil erosion by up to 80% compared to bare soil. However, our restoration treatments did not sufficiently influence total plant cover compared to control and thus the treatment effect on soil erosion was not significant. Contrary to our results, [START_REF] Kavian | Impact of Wheat Residue on Soil Erosion Processes[END_REF] found a longterm reduction in soil runoff using wheat residues as "nurse cover". Our restoration sites were not characterized by steep slopes reducing soil run-off and the probability to find differences between treatments. Additionally, spontaneous vegetation emerging from the soil seed bank dominated in the beginning limiting the treatment effect.

Conclusions

All applied transfer techniques were successful in establishing plant species of the donor community. However, the transfer of brush-harvested seed material was only efficient together with wheat sowing. Our results demonstrated that restoration of grasslands of the mountain altitudinal belt with shallow and stony soils clearly benefit from dead (hay) or living (wheat) vegetation cover providing a nurse effect. Facilitation effects prevail over competition and/or reduction in resources such as light [START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF][START_REF] Padilla | The role of nurse plants in the restoration of degraded environments[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF]. Hay transfer was the best method to increase germination and seedling recruitment but the difference to brush material transfer with wheat sowing vanished in the second year indicating higher seedling mortality in the hay mulch [START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF]. The lower seedling recruitment in brush material transfer may be compensated by harrowing the soil before brush material transfer to improve recruitment conditions [START_REF] Kiehl | Species introduction in restoration projects -Evaluation of different techniques for the establishment of semi-natural grasslands in Central and Northwestern Europe[END_REF]Klaus et al. 2017;Durbecq et al. 2021, chapter 2). Propagule transfer did not improve the soil erosion control. Short-lived species spontaneously emerging from the soil seed bank dominated in the beginning reducing the magnitude of treatment effects. Long-term monitoring is required to evaluate the replacement of shortlived ruderals by the transferred perennial species often observed in restoration studies (Albert et al. 2019;[START_REF] Kiss | Establishment gaps in species-poor grasslands: artificial biodiversity hotspots to support the colonization of target species[END_REF]Shaw et al. 2020). In chapter 3, hay transfer was the most efficient technique in transferring species of the donor grassland. The transfer of brush-harvested material was only efficient when combined with wheat sowing.

Supporting information

(3.b) Does sowing of wheat facilitate the establishment of brush-harvested seeds?

Wheat sowing clearly facilitated seedling establishment of seeds transferred with brush material. In mountain grasslands with shallow and stony soils, recruitment of harvested seeds clearly benefited from a facilitative effect of living (wheat) or dead (hay) vegetation cover.

(3.c) Do transfer techniques and wheat sowing increase the similarity between restored and reference communities?

Hay transfer and brush material transfer combined with wheat sowing were the most successful techniques in restoring mountain grasslands, showing highest similarity to references in plant species composition and highest target abundances.

(3.d) Does propagule addition limit soil erosion?

Soil erosion was not affected by any restoration treatment at the end of the observation period. Intermediate measurements demonstrated a high temporal variability of erosion. and of treatment effects on erosion.

As in the chapters 2 and 3 experiments, most restoration projects using seed addition involved transferring all target species at the same time, resulting in strong plant-plant interactions between transferred species, and therefore, the prioritization of the most competitive among them. In chapter 4, I focus on the analysis of priority effects varied by sequential sowing of different species groups (Dominants or Subordinates), each comprising three species.

Chapter Four

Consider sequential sowing to use priority effects in plant community restoration

Aure Durbecq, Armin Bischoff, Elise Buisson, Emmanuel Corcket, Renaud Jaunatre

Introduction

Priority effects (hereafter PEs) occur when the order of species arrival in a habitat modifies biotic and abiotic properties of the micro-environment and thus affects the recruitment, establishment, growth and/or reproduction of late-arriving species [START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF][START_REF] Temperton | Assembly Theory for Restoring Ecosystem Structure and Functioning: Timing is Everything[END_REF][START_REF] Weidlich | Priority effects caused by plant order of arrival affect below-ground productivity[END_REF] (Levine 2004). Furthermore, the strength of PEs can be mediated by both biotic and abiotic factors, such as species identity [START_REF] Cleland | Priority effects vary with species identity and origin in an experiment varying the timing of seed arrival[END_REF][START_REF] Stuble | Priority effects: natives, but not exotics, pay to arrive late[END_REF][START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF][START_REF] Wilsey | Exotic grassland species have stronger priority effects than natives regardless of whether they are cultivated or wild genotypes[END_REF][START_REF] Werner | Persistent asymmetrical priority effects in a California grassland restoration experiment[END_REF] or overlapping niches [START_REF] Vannette | Historical contingency in species interactions: towards nichebased predictions[END_REF], predation pressure [START_REF] Chase | Predators temper the relative importance of stochastic processes in the assembly of prey metacommunities[END_REF]) and habitat size or productivity [START_REF] Fukami | Assembly History Interacts with Ecosystem Size to Influence Species Diversity[END_REF][START_REF] Olsen | Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. U.S. Department of Agriculture[END_REF][START_REF] Chase | Stochastic Community Assembly Causes Higher Biodiversity in More Productive Environments[END_REF], Kardol, et al. 2013). In productive environments, PEs may be very strong resulting in alternative stable states [START_REF] Chase | Community assembly: when should history matter?[END_REF][START_REF] Hobbs | Ecological Filters, Thresholds, and Gradients inn Resistance to Ecosystem Reassembly[END_REF]Kardol et al. 2013;[START_REF] Weidlich | The importance of Being First: Exploring Priority and Diversity Effects in a Grassland Field Experiment[END_REF]. PEs are, however, poorly studied in unproductive environments (Wisley 2020). Finally, PEs do not only influence the early community assembly but also shape the potential future community trajectories, including their composition and associated functions (Grmann et al 2010;[START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF]Wermer et al 2016;[START_REF] Stuble | Priority Treatment Leaves Grassland Restoration Vulnerable to Invasion[END_REF].

Translating the concepts of community assembly and trajectories into relevant methods to drive ecosystem dynamics is a core approach in restoration ecology. If a habitat has been damaged or destroyed, restoration managers try to re-establish the predisturbance ecosystem including species composition and ecosystem functions (Gann et al. 2019). Changing initial abiotic conditions and species pools may help to accelerate restoration towards the reference plant community. Despite growing evidence of the importance of PEs in early plant succession [START_REF] Van Der Putten | Plant species diversity as a driver of early succession in abandoned fields: a multi-site approach[END_REF][START_REF] Vaughn | Short-term priority over exotic annuals increases the initial density and longer-term cover of native perennial grasses[END_REF] and their long-term consequences [START_REF] Fukami | Assembly History Interacts with Ecosystem Size to Influence Species Diversity[END_REF][START_REF] Švamberková | The legacy of initial sowing after 20 years of exarable land colonisation[END_REF]; García-Girón 2021) most restoration approaches using seed-addition involved transferring all target species at the same time [START_REF] Török | Beyond the species pool: modification of species dispersal, establishment, and assembly by habitat restoration[END_REF]). However, using PEs by establishing different sequences of species arrival has been identified as a promising tool to increase the establishment success of target species for ecological restoration [START_REF] Fukami | Community assembly: alternative stable states or alternative transient states?[END_REF]Young et al. 2001, Vaughn and[START_REF] Vaughn | Short-term priority over exotic annuals increases the initial density and longer-term cover of native perennial grasses[END_REF]Wermer et al. 2016;Young & Stuble 2017).

Consequently, there is an increasing number of field and greenhouse studies testing PEs as a restoration tool. Tests of PEs involve sequential sowing of different species or species groups in order to test their influence on plant community dynamics. Species groups may be based on their origin (native vs. exotic, [START_REF] Grman | Within-Year Soil Legacies Contribute to Strong Priority Effects of Exotics on Native California Grassland Communities[END_REF][START_REF] Hess | Priority effects: Emerging principles for invasive plant species management[END_REF]Goodale & Wilsey 2019;[START_REF] Ploughe | Priority effects: How the order of arrival of an invasive grass, Bromus tectorum, alters productivity and plant community structure when grown with native grass species[END_REF][START_REF] Yu | Priority effects and competition by a native species inhibit an invasive species and may assist restoration[END_REF], on their life cycle type (annual vs. perennial, [START_REF] Schantz | Role of propagule pressure and priority effects on seedlings during invasion and restoration of shrub-steppe[END_REF]Vauchn & Young 2015), or on their functional group (grasses vs. forbs vs. legumes, [START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF][START_REF] Stuble | Priority effects: natives, but not exotics, pay to arrive late[END_REF][START_REF] Stuble | Every restoration is unique: testing year effects and site effects as drivers of initial restoration trajectories[END_REF][START_REF] Weidlich | The importance of Being First: Exploring Priority and Diversity Effects in a Grassland Field Experiment[END_REF][START_REF] Weidlich | Priority effects caused by plant order of arrival affect below-ground productivity[END_REF][START_REF] Delory | When history matters: The overlooked role of priority effects in grassland overyielding[END_REF]. Other studies examined PEs by mixing these groups [START_REF] Werner | Persistent asymmetrical priority effects in a California grassland restoration experiment[END_REF]Shantz et al 2018;[START_REF] Mason | Arrival order among native plant functional groups does not affect invasibility of constructed dune communities[END_REF][START_REF] Young | Using priority effects to manipulate competitive relationships in restoration[END_REF]Cleland et al. 2014;[START_REF] Lang | Suppression of an invasive legume by a native grass -High impact of priority effects[END_REF][START_REF] Stuble | Priority Treatment Leaves Grassland Restoration Vulnerable to Invasion[END_REF]Wisley et al. 2020). Alternatively, species may be assigned to groups according to their importance in reference plant communities, for example in distinguishing dominant (species frequently occurring in high abundance) and subordinate species (frequently occurring in low abundance; Grime 1998). Subordinate species may have a greater impact on ecosystem functions than dominant species [START_REF] Mariotte | Subordinate plant species enhance community resistance against drought in semi-natural grasslands[END_REF][START_REF] Mariotte | Do subordinate species punch above their weight? Evidence from aboveand below-ground[END_REF], particularly in semi-natural grasslands [START_REF] Gibson | Grasses and Grassland Ecology[END_REF][START_REF] Werner | Persistent asymmetrical priority effects in a California grassland restoration experiment[END_REF]. They can increase plant community resistance against drought [START_REF] Mariotte | Subordinate plant species enhance community resistance against drought in semi-natural grasslands[END_REF], probably via mycorrhizal fungal associations that improve plant productivity and positive plant-soil feedback (Mariotte et al. 2012;De Vries et al. 2011, 2018). Furthermore, dominant species are competitive and thus less sensitive to PEs than subordinate species [START_REF] Sarneel | importance of priority effects for riparian plant community dynamics[END_REF]. Delaying dominant species arrival may thus improve the establishment of the subordinate species (Mariotte et al. 2012;[START_REF] Young | Using priority effects to manipulate competitive relationships in restoration[END_REF]) and promote the coexistence of both. Contrary to dominant species, the subordinate species are expected to show a low competitive response (tolerance to competition of other species) and low competitive effect (competition pressure exerted on other species; [START_REF] Goldberg | Competitive Effect and Response: Hierarchies and Correlated Traits in the Early Stages of Competition[END_REF]Wang et al. 2010;Keddy et al. 2002).

We first identified sets of dominant or subordinate species in our study area using abundances in vegetation releves. Then we tested PEs by sowing dominants and subordinates at the same time and sequentially. According to the theoretical competitive abilities exerted for subordinate and dominant species, we hypothesized that the order of arrival of the dominants and subordinates changes the species composition of the plant community (Fig. 4.1a). More specifically, as dominants have a strong competitive ability (both competitive effect and response), we expected that sowing dominants first or together with subordinates leads to competitive exclusion of subordinates. In contrast, subordinates are expected to be less competitive than dominants. Therefore, sowing subordinates first should favour their establishment without preventing the establishment of dominants. Competitive response (proportion of focal species cover or biomass when grown with other species compared to monoculture) and effect (i.e. proportion of cover or biomass of other species when they are grown with the focal species compared to monoculture) are displayed according to the hypotheses drawn for each species group (dominant or subordinate). Hypothesis n°1: Subordinates show a low competitive response and therefore establish less well in pre-established communities, but resist if sown at first (b, purple line). Hypothesis n°2: Subordinates show a low competitive effect and therefore, do not or hardly affect other species regardless of arrival date (c, purple line). Hypothesis n°3: Dominant species show a highly competitive response and are therefore not affected by other species, even if sown later (b, red line). Hypothesis n°4: Dominant species show a strong competitive effect and therefore strongly affect other species when arriving first, but to a lesser degree when arriving later (c, red line).

Material and Methods

Site description

The experiment was set up in a degraded montane grassland at La-Bâtie-Neuve, in the upper Durance valley ('Haute-Durance') of the Southern French Alps (44°57'93''N, 6°20''77'E). It is located at 1270 meters above sea level (asl), on an approximately 20° west-facing slope. The annual precipitation is 947 mm on average. July is the warmest month with an estimated mean temperature of 15.5°C, and January is the coolest with a mean temperature of -3°C. Frost can occur from September to May (estimated from Chorges meteorological station, 863 m asl, 10 km from La-Bâtie-Neuve). The soil is stony on calcareous bedrock. Grasslands are extensively grazed by cattle. The recent construction of a high-voltage transmission line in the valley involved a degradation of the soil structure affected by stripping, compacting and decompacting the soil to create transitory access tracks and construction platforms. At the end of the work, the stockpiled soil was moved back to recreate the original slope.

Experimental set up

To test the effect of date of species arrival on community assembly, we applied sequential sowing using two communities of different perennial plant species. Both communities included one Poaceae, one Plantaginaceae and one Fabaceae representing different functional groups. The first community comprised one dominant species of each family, and the second one subordinate species of each family (Table 4.1). These sown species are common in grasslands of the study area belonging to the habitat type "Semi-natural dry grasslands of Festuco-Brometalia and scrubland facies on calcareous substrates" (N6210, EU directive habitat 92/43/EEC, [START_REF] Calaciura | -Brometalia) (*important orchid sites)[END_REF]. The species were assigned to the community of dominant and subordinate species according to their relative rank of plant cover in 54 plots of typical undisturbed grasslands of the 'Haute-Durance'valley [START_REF] Durbecq | Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition[END_REF], chapter 1).

Experimental design

We tested four treatments: The 2018 sowing date was October 19 th , and the 2019 sowing date was October 9 th . Autumn sowing was chosen to allow dormancy breaking by cold stratification. Seeds of local origin according to the French label 'Végétal local' (Malaval et al. 2018) were obtained from a local seed company (Phytosem, Gap, France). The number of sown seeds was adjusted to a potential density of 100 individuals/m² for each sown species, based on seed producer expertise (Table 4.1). Plot size was 2m × 2m plots, spaced by 50 cm from one another. To prepare the seed bed and to cover seeds the plots were manually raked before and after each sowing. The experimental site was fenced to prevent seedling damage by cattle grazing. The plots were neither watered nor fertilized.

Data collection

Plant community surveys were carried out on all plots. The percentage cover of all vascular species was estimated in sub-plots of 1m × 1m placed in the centre of each experimental plot (n = 40; Fig. 4.2a) in June 2019 and 2020.

To assess individual growth response, we harvested aboveground biomass of each sown species in two separate 30 × 53.5 cm sub-plots (0.16 m²; Fig. 4.2b), one in 2019 and one in 2020. We also counted the number of individuals. The harvest dates were 278 days and 264 days (9 months) after the first and the second sowing, respectively. To avoid an impact on plant community surveys, biomass was sampled outside the 1 × 1 m sub-plots (Fig.

Statistical analyses

To explore the effect of sequential sowing on the community assembly, linear models (LM) were fitted with sowing treatment (control, D+S, D1 st , S1 st ) and year as fixed effects and plant species cover as response variable. We tested the significance of the LM using the 'anova'function of the R package 'stats'with F-test (Chambers & Hastie 1992). In case of a significant treatment effect, multiple least-square mean comparisons were run using a Tukey adjustment in order to test differences between treatment levels ('emmeans'package, Lenth 2020). Data that did not comply with the assumptions of linear models (normal distribution, homoscedasticity) were sqrt-transformed before analysis.

In order to compare plant communities between the four treatments, Non-Metric Multidimensional Scaling (NMDS) was performed based on Bray-Curtis distance (Borcard et al. 2011) using R package 'vegan' [START_REF] Oksanen | vegan: Community Ecology Package. R package version 2[END_REF]. A permutation multivariate analysis of variance (PERMANOVA, Anderson 2001) with 9,999 permutations was used to analyse whether the community composition was significantly different between treatments (R package 'vegan').

We then tested for each sown species and the group of unsown species the differences between sowing one year before other species and simultaneous sowing. The proportion of individual species cover to total plant cover was used as response variable. Since transformation were not successful to comply with the assumptions of linear models, differences between the three sequential sowing treatments were analysed using Kruskal-Wallis and pairwise Wilcoxon tests. P-values of pairwise Wilcoxon tests were adjusted for multiple testing using the conservative Bonferroni correction (Jafari & Ansari-Pour 2019).

To evaluate facilitative versus competitive advantage, we calculated a priority index (PI) and a secondary index (SI) comparing the performance effects of arrival before, after and at the same time as other species [START_REF] Cleland | Priority effects vary with species identity and origin in an experiment varying the timing of seed arrival[END_REF]. Both PI and SI were calculated for aboveground biomass, abundance and cover of each sown species. For PI, we compared sowing first and simultaneous sowing, and for SI, sowing second and simultaneous sowing. The PI and SI results calculated on individual aboveground biomass are shown in the main document (Fig. 4.6) while indices on abundance and cover are presented in supporting information (Fig. S4. 3 and S4.4). Individual biomass production is a particularly reliable indicator to assess plant competitive ability, particularly in studies with few species and with small biomass range [START_REF] Gaudet | A comparative approach to predicting competitive ability from References 118 plant traits[END_REF]. Positive values of PI and SI indicate that the Fi benefited from being sown first, or second, respectively (i.e. before or after the other species group) compared to being sown simultaneously with the other group (for PI: Biomass2020(Fi)1st exceeding mean Biomass2020(Fi)simultaneously). Negative values indicate that the Fi performed less well when being sown first or second. According to criteria for linear models (normality, homoscedasticity), the difference between the indices and zero of each was either determined using t-test for PI or Mann-Whitney U test for SI, with a Bonferroni p-value adjustment (Jafari & Ansari-Pour 2019). All analyses were performed using R 3.5.3 (R Core Team 2019).

Results

Community assemblages

Total species cover significantly increased between 2019 and 2020 in the four treatments (df = 1, F = 105.5, p < 0.001, Fig. S4.1a), with a lower species cover in the control than in the other treatments in 2020 (df = 3, F = 5.589, p < 0.001). Compared to the control, unsown species cover was not significantly affected by sequential sowing, neither in 2019 nor in 2020. As the total species cover, unsown species cover significantly increased in 2020 (df = 1, F = 13.25, p < 0.001,

Community composition driving by time-advanced

The dominant Plantaginaceae P. media did not germinate in any treatment. However, the dominant Poaceae B. erecta dominated when sown first (two years prior to the final survey) in both D+S and D1 st treatments, contributing 45% to total community cover (± 2%). Its contribution to total cover was less than 5% when sown secondly (S1 st treatment; Fig. 4.4). The contribution of the dominant Fabaceae A. vulneraria was similar (± 10%) when sown secondly (S1 st ) or simultaneously with the other group (D+S), and 20% greater when sown first (D1 st ). The contribution of the three subordinate species (i.e. F. cinerea, P. lanceolata and O. viciifolia) reached each approximately 20% when sown first (S1 st ) whereas it did not exceed 2% when sown secondly (D1 st ). The cover of the Poaceae F. cinerea and the Plantaginaceae P. lanceolata did not exceed 6% when sown simultaneously with the other species (D+S), but the subordinate Fabaceae (O. viciifolia) contributed more than 15% to total cover in the D+S treatment. Total cover contribution of unsown species was 80% in the control and drastically decreased in D+S, D1 st and S1 st treatments to reach respectively 16%, 26% and 24%.

The total cover of the dominant Poaceae B. erecta was significantly higher in both D+S and D1 st treatments with an average cover of 25%, compared to the S1 st treatment showing a cover of less than 5% (Fig. 4.5, Table S4.1). No significant difference was found between the three sequential sowing treatments for the dominant Fabaceae A. vulneraria. The three subordinate species (F. cinerea, O. viciifolia and P. lanceolata) performed significantly better in S1 st than in D1 st . However, the cover of the subordinate Fabaceae O. viciifolia cover was not significantly different between the D+S and S1 st treatment whereas the cover of F. cinerea and P. lanceolata was significantly lower in the D+S than in the S1 st treatment.

Priority and secondary indices

For none of the species, priority indices were significantly different from zero, neither for aboveground biomass (Fig. 4.6a, Table S4.1), nor for species abundance (Fig. S4.3a) or cover (Fig. S4.4a). However, significant differences in the secondary index showed that the establishment of the dominant B. erecta and A. vulneraria, and the subordinate O. viciifolia were significantly affected by sequential sowing. O. viciifolia biomass and cover were lower (Fig. 4.6b; S4.3b, S4.4b) and B. erecta biomass was lower when sown second (Fig. 4.6b, Table S4.1). In contrast, the dominant A. vulneraria significantly benefited from being sown second (Fig. 4.6b, S4.3b, S4.4b, Table S41). The secondary index of F. cinerea and P. lanceolata was not significantly different from zero.

Discussion

Manipulating the order of arrival by simultaneous or sequential sowing influenced the community composition two years after first sowing. As expected, subordinate species (i.e. F. cinerea, O. viciifolia and P. lanceolata) were favoured by priority effects (hereafter PEs) as they all best developed when sown first. However, dominant species (B. erecta, A. vulneraria and P. media) showed differences in their establishment. P. media did not germinate at all, regardless of the treatment. B. erecta known as a stress-tolerant competitor [START_REF] Grime | Plant strategies and vegetation processes[END_REF]) showed a significant disadvantage when sown one year later and performed significantly better when sown before or at the same time as the subordinates (i.e. D1 st or D+S treatments respectively). This result suggests that B. erecta was affected by direct competition or indirect effects of already established vegetation. Similarly, Corcket et al. (2003) found a strong effect of competition on this species, and Harper (1961) identified a competitive priority effect of Bromus madritensis on Bromus rigidus when sown three weeks before. In contrast, the dominant Fabaceae A. vulneraria showed a higher performance when sown after other species, which indicated that it benefited from the presence of already established vegetation. Thus, we did not find a general pattern of priority effects in dominant versus subordinate species. However, dominant species were on average less affected by sowing together with subordinate species than subordinate species sown together with dominants.

PEs demonstrated interesting contrasted effects between species, which can be explained by the niche components hypothesis (Vanette & Fukami 2014). This hypothesis is a robust tool to analyse priority effects even if large differences in speciesspecific strategies and interactions among environments increase unexplained variation. In this study, the dominant Poaceae B. erecta and the subordinate Fabaceae O. viciifolia showed the same trends. The two species both established well, when sown at the same time as species of the other group (simultaneously with subordinates or dominants) or only with the other two species of the same group (D1 st for B. erecta and S1 st for O. viciifolia). Both species were clearly disadvantaged when sown in 2019 (as second species group) suggesting that both species show a strong recruitment ability if the niche is not occupied. However, the priority index did not confirm this result. Both species, B. erecta and O. viciifolia, showed a significant secondary effect. These both late-arriving species recruitment were probably hampered by niche modification and sizeasymmetric competition of the pre-established vegetation (Vanette & Fukami 2014;Wisley 2020), or by niche pre-emption [START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF].

The dominant Fabaceae A. vulneraria well established in the three seed-addition treatments and was not negatively affected by the pre-established community when sown after subordinate species. A. vulneraria even benefited from a secondary effect, demonstrating thus a high recruitment capacity in established vegetation (Vanette & Kukami 2014). Positive effects of already established vegetation on the recruitment niche (e.g. living cover protecting young seedlings and improving water retention, [START_REF] Donath | Influence of competition by sown grass, disturbance and litter on recruitment of rare flood-meadow species[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF]; Chapter 3) may have favoured A. vulneraria. Alternatively, a positive plant-soil feedback may have contributed to the secondary effect (Bever 2003;De Vries et al. 2011, 2018;[START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF][START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF][START_REF] Goldstein | Applying competition theory to invasion: resource impacts indicate invasion mechanisms in California shrublands[END_REF]. The association of plant and soil organisms may have been favoured by the previous establishment of the Fabaceae O. viciifolia. Species-specific functional traits were also suggested to play an important role in the establishment of the subordinate Fabaceae. As a late-successional species, A. vulneraria is a dispersal-limited but fast-growing species that may take advantage from being sown in a pre-established plant community (Erschbamer 2007;Marcante et al. 2009). Furthermore, its roots are longer and deeper than those of the sown subordinate Fabaceae O. viciifolia and of the other sown species (Jungk 1993), avoiding thus competition for soil resources. Our results are similar to that of [START_REF] Hess | Restauration écologique des communautés végétales après éradication d'espèces invasives : Rôle de la dynamique de colonisation et des effets de priorité[END_REF] who found a particularly weak competitiveness of O. viciifolia when sown after exotic species in an experiment on priority effects. Furthermore, our results are in agreement with the hypotheses of Vanette & Fukami (2014) who suggested that the strength of PEs varies according to the niche components (overlap, impact and requirements). Such niche requirements differ between species, and may influence (negatively or positively) late-arriving species recruitment and/or establishment.

However, the mechanisms of underlying PEs need to be better investigated, with a particular focus on plant-soil feedback and belowground interactions (Bever 2003;[START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF][START_REF] Weidlich | Priority effects caused by plant order of arrival affect below-ground productivity[END_REF][START_REF] Hess | Restauration écologique des communautés végétales après éradication d'espèces invasives : Rôle de la dynamique de colonisation et des effets de priorité[END_REF].

To conclude, our study provides evidence that plant the community assembly was influenced by the order of arrival, potentially changing community composition and trajectories. Although our results did not show consistent responses for dominant and subordinate species, they were in agreement with other studies indicating strong variations between species and thus high species-specific PEs [START_REF] Cleland | Priority effects vary with species identity and origin in an experiment varying the timing of seed arrival[END_REF][START_REF] Stuble | Priority effects: natives, but not exotics, pay to arrive late[END_REF]. Using the priority and secondary indices of target species sown in ecological restoration may be a relevant method to improve management actions. Species showing a strong priority effect but a low recruitment in early succession may be sown in the beginning, and species that become dominant when sowing first or species showing a positive secondary effect may be sown later. To be applied in a restoration context, further studies are needed to assess both priority and secondary indices of restoration target species in different habitat types. This would thus enable practitioners to establish lists of species to be sown first and second in order to develop plant community structure and composition towards references identified prior to restoration.

General discussion 5 Discussion

The thesis aimed at better understanding the factors limiting the restoration of mountain grasslands after soil disturbance, and to identify successful restoration techniques to compensate the degradation. In the first part of the general discussion, I relate the main findings of the thesis in terms of seed-addition technique success (5.1.1) and their underlying ecological mechanisms and processes (5.1.2), and I conclude on the importance of priority effects in shaping plant community assembly (5.1.3). The second part of the discussion focuses on the limits and perspectives of the framework used to identify reference communities (5.2.1), and on the trade-off between extensive grazing as a requirement to maintain systems open and as a constraint in seedling recruitment (5.2.2). Finally, given the outcomes of chapters 3, and of both chapters 2 and 4, respectively, I develop suggestions to improve our understanding in soil control measures (5.2.3) and in plant-soil interactions (5.2.4).

General outcome

Success of seed-addition techniques to accelerate restoration

Using a filter-based integrated community concept is a useful and reliable tool to improve knowledge in restoration ecology [START_REF] Hulvey | Using filter-based community assembly models to improve restoration outcomes[END_REF][START_REF] Halassy | The application of a filter-based assembly model to develop best practices for Pannonian sand grassland restoration[END_REF][START_REF] Temperton | Assembly Theory for Restoring Ecosystem Structure and Functioning: Timing is Everything[END_REF][START_REF] Chang | Integrating succession and community assembly perspectives[END_REF]. Examining dispersal, biotic and abiotic filters allow identifying different factors driving community assembly and dynamics of mountain grasslands. Chapters 1 and 2 mainly focused on abiotic drivers. In agreement with [START_REF] Pykälä | Plant species richness and persistence of rare plants in abandoned semi-natural grasslands in northern Europe[END_REF] and [START_REF] Srinivasan | Vegetation-environment relationships in a South Asian tropical montane grassland ecosystem: restoration implications[END_REF], both slope and aspect driving direct incident solar radiation were identified as major factors influencing species richness and composition in mountain grasslands. Elevation is also known as an important factor influencing microclimate and plant community composition [START_REF] Callaway | Positive interactions among alpine plants increase with stress[END_REF][START_REF] Körner | Small differences in arrival time influence composition and productivity of plant communities[END_REF][START_REF] Sundqvist | Community and Ecosystem Responses to Elevational Gradients: Processes, Mechanisms, and Insights for Global Change[END_REF], but in in my study, elevation was standardized according to the restoration project (difference < 400 m) resulting in a non-significant effect. Edaphic factors such as soil moisture, soil pH and exchangeable phosphorus were also important parameters structuring plant communities of the 'Haute-Durance'valley.

The existence of a dispersal filter was demonstrated in chapters 2 and 3. Adding seeds from a nearby source community clearly improved ecological restoration of mountain grasslands, largely promoted by additional treatments influencing both the biotic and abiotic filters. These additional treatments were successful in accelerating restoration since they increased the recruitment of transferred species (chapters 2 and 3) and shaped the restored community towards the references identified in chapter 1, suggesting that the restoration trajectories are on the right track. Despite several technical advantages (harvest, storage, spread), the transfer of brush-harvested propagules was poorly successful in establishing species of the source community when sown alone (without any other treatment, chapters 2 and 3). However, its combination with soil preparation in chapter 2, and the addition of wheat in chapter 3, largely increased the recruitment and establishment of brush-harvested propagules. I also observed significant differences in the recruitment rate of transferred species between brush and hay transfer (chapter 3). When both harvest techniques were applied at the same donor site and the quantity of sown seeds was similar, the seedling number of transferred species was much higher in the hay transfer treatment. Moreover, a comparison with greenhouse germination tests of propagules in hay and brush material demonstrated a low field emergence, suggesting that seedling recruitment is a bottleneck in ecological restoration of mountain grasslands (Öster et al. 2009). Brush material transfer alone did neither increase transferred species cover nor plant species richness, suggesting that the dispersal limitation filter of mountain grasslands can only be overcome, if this transfer is combined with other community assembly filter manipulations to improve the recruitment of transferred species.

Towards a better understanding of community assembly filters to improve restoration success

Manipulating abiotic (soil preparation, chapter 2) and biotic factors (hay mulch and wheat addition, chapter 3) increased the recruitment and establishment of transferred species. Several underlying mechanisms may explain this improvement, suggesting a modification of the niche in favour of the transferred species establishment: (i) Positive effects of treatments on soil conditions; (ii) Positive effects of treatments on seedling recruitment (germination, seedling growth and survival); (iii) Negative effects of treatments on the recruitment of spontaneous species (seed bank and rain) limiting competition with transferred species.

Both the mulch layer of the hay transfer and the temporary living cover of the sown wheat (hereafter collectively referred to as VC for 'Vegetation cover') may facilitate seedling recruitment by protecting seeds and seedlings. VC reduces direct solar radiation that may damage seedlings and prevents evaporation reducing drought stress (Donath et al. 2007;[START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF][START_REF] Mollard | Impacts of mulch on prairie seedling establishment: Facilitative to inhibitory effects[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF]. VC may also limit seed removal by granivores such as arthropods and vertebrates, predation of young seedlings, damage by livestock herbivory and trampling, because seeds and seedlings are less visible (Scotton et al. 2012;[START_REF] Linabury | Insects remove more seeds than mammals in first-year prairie restorations[END_REF][START_REF] Vanderburg | Trampling and Cover Effects on Soil Compaction and Seedling Establishment in Reseeded Pasturelands Over Time[END_REF]. Moreover, VC is increasingly recognized to provide a shield against wind pressure and seed run-off, particularly in mountain grassland with steep slopes [START_REF] Huc | SEM'LESALPES -Des semences d'origine locale pour la restauration de milieux ouverts en montagne alpine[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]. Several restoration techniques have been developed in alpine grasslands to limit propagule run-off such as hydroseeding, hydromulching or geotextiles (Fattorini Marzio 2001;[START_REF] Krautzer | Restoration of Arctic-Alpine Ecosystems[END_REF][START_REF] Shao | Effectiveness of geotextile mulches for slope restoration in semi-arid northern China[END_REF][START_REF] Tamura | Effectiveness of seed sowing techniques for sloped restoration sites[END_REF]). However, these techniques are expensive and less cost-efficient in mountain grasslands. VC improves seed adhesion to the soil [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF], particularly the hay layer that contains cellulose promoting mucilage adhesion [START_REF] Western | The sticky tale of seed coat mucilages: production, genetics, and role in seed germination and dispersal[END_REF]. Similarly, soil preparation before sowing had a facilitative recruitment effect as it significantly favoured transferred species establishment suggesting seed adhesion improvement by micro-relief creation [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF][START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF]. VC and tillage affected the soil component indirectly and directly suggesting strong niche modification effects [START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF]. Acting as a windscreen, VC stabilizes also the soil by decreasing surface run-off [START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF][START_REF] Kavian | Impact of Wheat Residue on Soil Erosion Processes[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF], while increasing soil moisture retention by reducing solar radiation (Donath et al. 2007;[START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF][START_REF] Mollard | Impacts of mulch on prairie seedling establishment: Facilitative to inhibitory effects[END_REF][START_REF] Havrilla | RestoreNet: An emerging restoration network reveals controls on seeding success across dryland ecosystems[END_REF].

All the three techniques may also have negative effects on seedling establishment via reduced vegetation cover (soil preparation) or increased competition (hay and brush material transfer). However, facilitation effects prevailed over competition and/or reduction in resources such as light or water [START_REF] Eckstein | Interactions between litter and water availability affect seedling emergence in four familial pairs of floodplain species[END_REF][START_REF] Donath | Influence of competition by sown grass, disturbance and litter on recruitment of rare flood-meadow species[END_REF][START_REF] Padilla | The role of nurse plants in the restoration of degraded environments[END_REF][START_REF] Wright | Living close to your neighbors: the importance of both competition and facilitation in plant communities[END_REF], indicating that parameters of the chosen methods were well adapted (thickness of hay layer, depth of harrowing, date of wheat sowing). Soil tillage increased transferred species abundance but reduced the abundance of ruderal and competitive early-successional species [START_REF] Grime | Benefits of plant diversity to ecosystems: immediate, filter and founder effects[END_REF]) that may have hampered the establishment of the transferred late-successional species [START_REF] Edwards | Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands[END_REF]Donath et al. 2007;[START_REF] Schmiede | Enhancing plant biodiversity in species-poor grassland through plant material transfer -the impact of sward disturbance[END_REF][START_REF] Jaunatre | Topsoil removal improves various restoration treatments of a Mediterranean steppe (La Crau, southeast France)[END_REF][START_REF] Harvolk-Schöning | Floodplain meadow restoration revisited: Long-term success of large scale application of diaspore transfer with plant material in restoration practice[END_REF]. Contrary to [START_REF] Kiehl | Effect of Hay Transfer on Long-Term Establishment of Vegetation and Grasshoppers on Former Arable Fields[END_REF] who suggested that a mulch layer limits competition of ruderal species in early stages of grassland restoration, VC did not have negative effects on spontaneous colonisation while simultaneously promoting establishment of transferred species in my study. The proportion of non-target abundances of brush material combined with wheat and hay transfer were not significantly different from the control, indicating that VC did not only provide better conditions for transferred species but also for nontransferred species recruitment, probably due to their general protection effect against drought stress, solar radiation, predation, and soil/propagule run-off. As these techniques are complementary, soil preparation may be even beneficial in combination with VC and this combined effect of soil preparation and VC is worth testing in future studies.

Priority effects shape community assembly and influence restoration outcome

In chapter 4, I tested priority effects by sequential sowing using a one-year time difference. Soil preparation and the addition of vegetation cover in chapters 2 and 3 also shaped the communities to prioritize transferred species by modifying the conditions of germination. Soil tillage reduced the abundance of species spontaneously emerging from the seed bank, and wheat sowing had a nurse effect due to the rapid development of its belowground and aboveground organs, thus modifying the niche pre-emption [START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF]. Furthermore, comparing species ex situ versus in situ (chapter 2) revealed differences in germination patterns. For example, Plantago media L. Festuca marginata (Hack.) K.Richt. and Schedonorus pratensis (Huds.) P.Beauv. showed a much higher greenhouse germination whereas the difference between greenhouse and field was much smaller in Bromopsis erecta (Huds.) Fourr. Rhinanthus alectorolophus (Scop.) Pollich did not germinate in the greenhouse whereas it dominated in the early stages of succession after brush-harvested propagule transfer in the chapter 2 experiment. However, in chapter 4, I did not find a general pattern of priority effects neither in dominant versus subordinate species groups nor within the same family (i.e. A. vulneraria benefited from sowing after other species whereas O. viciifolia and B. erecta were disadvantaged). In agreement with other studies [START_REF] Cleland | Priority effects vary with species identity and origin in an experiment varying the timing of seed arrival[END_REF][START_REF] Stuble | Priority effects: natives, but not exotics, pay to arrive late[END_REF], these results indicate that priority effects are highly species-specific (chapter 4). Thus, transferred species may have changed the community trajectory in chapter 2 and 3 experiments according to the effects of soil tillage or different VC on seedling recruitment.

In conclusion, the manipulation of filters in the three experiments created or modified priority effects and feedback loops with abiotic conditions, which in turn changed community trajectories towards reference communities. As suggested in the last thesis chapters, several factors may have promoted the seedling recruitment, such as positive effects of already established vegetation on niche modification (e.g. CV), or on the soil compartment (e.g. plant-soil feedback ;Bever 2003;[START_REF] Vries | Land use alters the resistance and resilience of soil food webs to drought[END_REF], 2018;[START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF][START_REF] Goldstein | Applying competition theory to invasion: resource impacts indicate invasion mechanisms in California shrublands[END_REF][START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF]. Furthermore, as in the chapters 2 and 3 experiments, most restoration projects using seed addition involved transferring all target species at the same time [START_REF] Török | Beyond the species pool: modification of species dispersal, establishment, and assembly by habitat restoration[END_REF], resulting in strong plant-plant interactions between transferred species, and therefore, the prioritization of the most competitive among them. Long-term monitoring would allow the identification of species that survive and establish well, and on the opposite those species that disappear over time. Another implication of priority effects would be the identification of key facilitator species (ecological engineers, nexus or keystone species) Jones et al. 1996;[START_REF] Power | Challenges in the Quest for Keystones: Identifying keystone species is difficult-but essential to understanding how loss of species will affect ecosystems[END_REF][START_REF] Lockwood | Assembly Models and the Practice of Restoration[END_REF]. Such species should be sown first to favour the establishment of other species that may be added later. Finally, monitoring the plant community over several years is necessary to evaluate whether the observed initial differences in seedling recruitment significantly influence plant species composition and restoration success in the long run, and to analyse the replacement of ruderals by the transferred perennial species [START_REF] Auestad | Nature will have its way: local vegetation trumps restoration treatments in semi-natural grassland[END_REF]Albert et al. 2019;Shaw et al. 2020;[START_REF] Kiss | Establishment gaps in species-poor grasslands: artificial biodiversity hotspots to support the colonization of target species[END_REF].

Perspectives

Integrating theory for a more generalised application of restoration strategies

Researchers stressed the importance of integrating ecological theory in ecological restoration to allow the transfer of knowledge and methodologies between different restoration contexts (Walker et al. 2007;[START_REF] Temperton | Assembly Theory for Restoring Ecosystem Structure and Functioning: Timing is Everything[END_REF][START_REF] Palmer | Ecological Theory and Restoration Ecology[END_REF][START_REF] Lindenmayer | Improving Restoration Programs Through Greater Connection With Ecological Theory and Better Monitoring[END_REF]. Hence, recent studies suggested extending the surveys beyond the close vicinity of restoration sites and using statistical tools to identify and to rank structuring variables in order to select appropriate reference communities [START_REF] Suganuma | Indicators of restoration success in riparian tropical forests using multiple reference ecosystems[END_REF][START_REF] Mcmanamay | Identifying nonreference sites to guide stream restoration and long-term monitoring[END_REF][START_REF] Erskine | Using reference sites to guide ecological engineering and restoration of an internationally significant uranium mine in the Northern Territory, Australia[END_REF]. In chapter 1, I propose a methodological framework in four steps to identify the best references for ecological restoration. This framework may be appropriate for various ecosystems, provided that the restoration towards non-degraded references is possible. The aim of this framework is, first, to obtain a good understanding of the environmental factors structuring the plant communities of the study system, second, to analyse environmental similarities between several potential reference sites around the degraded sites, and finally, to conclude on the choice of the best references as target or source communities for ecological restoration. This method can be easily applied to restoration practice but requires a good knowledge of ecosystem ecology. As it focuses on environmental drivers of plant community assembly, limitations of the evaluation approach can be the natural variability of the factors driving the system and/or the identification of appropriate drivers that are resistant to degradation. Moreover, main drivers of the system may be overlooked. For instance, soil structure, soil fauna and the history of land management may be important but their measurement is timeconsuming. Additionally, historical data (e.g. wildfire records, historical land use changes) that complement data collected in situ [START_REF] Asbjornsen | Defining Reference Information for Restoring Ecologically Rare Tallgrass Oak Savannas in the Midwestern United States[END_REF][START_REF] Urgenson | Social perspectives on the use of reference conditions in restoration of fire-adapted forest landscapes[END_REF] are not always available at the study site scale. In my study, it would be useful to examine the evolution of community trajectories over time (for each treatment; Chapters 2, 3, 4) in order to check whether restored communities really develop towards "best references" identified in the chapter 1. Moreover, it would be interesting to (i) test whether the use of propagules harvested in best reference communities is more successful than the use of those harvested in other source grasslands, and (ii) to analyse the effectiveness of the method in other systems. For instance, if this method is applied to a lagoon restoration project, the water level, the duration of flooding and the degree of salinity would be major factors that may be used to identify the references [START_REF] Bouzillé | Gestion des habitats naturels et biodiversité. Concept, méthodes et démarches[END_REF].

Grazing: trade-off between seedling damage and grassland preservation

In chapters 2 and 3, the applied restoration techniques would be useful for mountain as well as for low altitude grasslands under extensive grazing, but also for the restoration of other systems that need to be revegetated, such as quarries [START_REF] Kirmer | Methodische Grundlagen und Ergebnisse initiierter Vegetationsentwicklung auf xerothermen Extremstandorten des ehemaligen Braunkohlentagebaus in Sachsen-Anhalt: mit 40 Tabellen[END_REF][START_REF] Sheoran | Soil Reclamation of Abandoned Mine Land by Revegetation: A Review[END_REF][START_REF] Chenot | Long-term effects of topsoil transfer assessed thirty years after rehabilitation of dry alluvial quarries in Southeastern France[END_REF]. In chapter 2, the effect of livestock grazing on transferred species recruitment was weakly negative. Grazing may hamper the establishment by biomass removal (Scotton et al. 2012;[START_REF] Buisson | Limiting processes for perennial plant reintroduction to restore dry grasslands: Perennial plant reintroduction in dry grasslands[END_REF]Vidaller et al. 2019) but also increase seed adhesion by trampling [START_REF] Chambers | Seed movements and seedling fates in disturbed sagebrush steppe ecosystems: implications for restoration[END_REF][START_REF] Isselin-Nondedeu | Contributions of vegetation cover and cattle hoof prints towards seed runoff control on ski pistes[END_REF]). As our study sites were first grazed before seedling emergence of transferred species, and as the following grazing period occurred seven months later, the first grazing probably had a positive effect (i.e. in reducing competition by pre-existing vegetation and creating micro-reliefs), but the second grazing period may have negatively affected young seedlings. Thus, the timing of grazing needs to be taken into account to limit the damage to seedlings, particularly if grazing exclusion is not possible.

While the livestock type may influence grazing effect [START_REF] Tóth | Livestock Type is More Crucial Than Grazing Intensity: Traditional Cattle and Sheep Grazing in Short-Grass Steppes[END_REF], I pooled the grazer species effect in an overall grazing effect including sheep and cattle. It would be useful for future restoration studies to distinguish cattle and sheep grazing effects on seedling recruitment and to separate positive microsite creation effect and negative seedling damage effects for both grazers. Moreover, it would be interesting to include effects of stocking rate (livestock density), grazing duration [START_REF] Boschi | The effect of horse, cattle and sheep grazing on the diversity and abundance of land snails in nutrient-poor calcareous grasslands[END_REF][START_REF] Critchley | Vegetation dynamics and livestock performance in system-scale studies of sheep and cattle grazing on degraded upland wet heath[END_REF], and to analyse whether their effects on seedlings translate into mid-or long-term effects on the plant community [START_REF] Mcdonald | Seed bank classification and its importance for the restoration of species-rich flood-meadows[END_REF].

How to measure erosion control in restoration studies?

In addition to the dispersal filter limiting the natural restoration of European semi-natural grasslands [START_REF] Münzbergová | Seed, dispersal, microsite, habitat and recruitment limitation: identification of terms and concepts in studies of limitations[END_REF][START_REF] Török | Fast restoration of grassland vegetation by a combination of seed mixture sowing and low-diversity hay transfer[END_REF][START_REF] Muller | Effect of topsoil removal and plant material transfer on vegetation development in created Mediterranean meso-xeric grasslands. N Hölzel (ed)[END_REF][START_REF] Halassy | Trait-based approach confirms the importance of propagule limitation and assembly rules in old-field restoration[END_REF][START_REF] Kövendi-Jakó | Three years of vegetation development worth 30 years of secondary succession in urban-industrial grassland restoration[END_REF], further important aims of mountain grassland restoration are the stabilisation of bare soils and erosion control. A rapid vegetation recovery helps to reduce erosion [START_REF] Peratoner | Organic seed propagation of alpine species and their use in ecological restoration of ski runs in mountain regions[END_REF][START_REF] Liu | The effects of vegetation on runoff and soil loss: Multidimensional structure analysis and scale characteristics[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]Gu et al. 2020), and many revegetation techniques focus on anti-erosion efficiency and persistence of vegetation cover [START_REF] Graiss | Soil Erosion and Surface Runoff on Slopes in Mountain Environment Depending on Application Technique and Seed Mixture -A Case-Study[END_REF][START_REF] Fattorini | Establishment of Transplants on Machine-Graded Ski Runs Above Timberline in the Swiss Alps[END_REF][START_REF] Krautzer | Restoration of Arctic-Alpine Ecosystems[END_REF][START_REF] Shao | Effectiveness of geotextile mulches for slope restoration in semi-arid northern China[END_REF][START_REF] Tamura | Effectiveness of seed sowing techniques for sloped restoration sites[END_REF][START_REF] Scotton | Mountain grassland restoration: Effects of sowing rate, climate and soil on plant density and cover[END_REF]). However, while immediate revegetation of degraded mountain grasslands by locally adapted propagules is widely recommended to avoid erosion [START_REF] Scotton | Seed Harvesting for Ecological Restoration: Efficiency of Haymaking and Seed-Stripping on Different Grassland Types in the Eastern Italian Alps[END_REF][START_REF] Wiesmair | Relationships between plant diversity, vegetation cover, and site conditions: implications for grassland conservation in the Greater Caucasus[END_REF]Dupin et al. 2019), few studies have investigated the direct effect of restoration techniques on the reduction of soil erosion. In chapter 3, I used a device adapted from [START_REF] Feret | Utilisation d'un appareil de mesure simple, et précis, pour l'étude de l'érosion à Mayotte[END_REF] in order to evaluate the effect of VC on soil erosion (Fig. S3.1 chapter 3). However, our restoration treatments did not sufficiently influence total plant cover compared to the control and thus the treatment effect on soil erosion was not significant. Contrary to our results, [START_REF] Kavian | Impact of Wheat Residue on Soil Erosion Processes[END_REF] found a reduction in soil runoff using wheat residues as "nurse cover" in a simulation experiment on a slope of 30%. In another in situ study, [START_REF] Chenot | Long-term effects of topsoil transfer assessed thirty years after rehabilitation of dry alluvial quarries in Southeastern France[END_REF]) measured soil erosion by placing empty boxes on slopes below restoration plots to collect eroded soil over several months. In mountain grasslands, such devices need to be fixed strongly and protected from grazing which complicates its application. Photogrammetry or terrestrial laser scanning are alternative indirect measurement techniques providing topographic models of soil erosion and their temporal dynamics [START_REF] Nadal-Romero | The application of terrestrial laser scanner and SfM photogrammetry in measuring erosion and deposition processes in two opposite slopes in a humid badlands area (central Spanish Pyrenees)[END_REF]. In my study, intermediate measurements demonstrated a high temporal variability of erosion (Fig. S3.3 chapter 3), suggesting a possible compensatory effect of hay and wheat decomposition increasing the humus layer and thus potentially raising the soil level. Finally, as wheat (chapter 3) shows a rapid development of roots and clonal structures such as rhizomes, wheat sowing may contribute to limit erosion [START_REF] Löbmann | Towards a better understanding of shallow erosion resistance of subalpine grasslands[END_REF] and to improve restoration success in favouring target species establishment. Moreover, as my chapter 3 experiment was not fenced, it would be interesting to analyse the effect of wheat addition without the potential negative effect of livestock grazing.

Ecological restoration overlooks soil interactions: trade-off between positive effects on vegetation and negative effects on soil organisms

Plant-soil interactions (and feedbacks) operate during all stages of the plant life cycle, as well as during plant succession (Bever 2003;[START_REF] Wardle | The influence of biotic interactions on soil biodiversity[END_REF][START_REF] Voorde Tfj Van De | Intra-and interspecific plantsoil interactions, soil legacies and priority effects during old-field succession[END_REF][START_REF] Putten Wh Van Der | Plant-soil feedbacks: the past, the present and future challenges[END_REF]. In grassland restoration studies, soil preparation is increasingly recommended to improve seedling recruitment (chapter 2). Therefore, different techniques of soil treatment have been tested, such as topsoil removal or harrowing, to remove the seed bank or belowground plant organs of undesired plant species [START_REF] Hölzel | Restoration of a species-rich flood meadow by topsoil removal and diaspore transfer with plant material[END_REF]Klimkowska et al. 2007;[START_REF] Schnoor | Effects of soil disturbance on plant diversity of calcareous grasslands[END_REF][START_REF] Schmiede | Enhancing plant biodiversity in species-poor grassland through plant material transfer -the impact of sward disturbance[END_REF][START_REF] Jaunatre | Topsoil removal improves various restoration treatments of a Mediterranean steppe (La Crau, southeast France)[END_REF][START_REF] Muller | Effect of topsoil removal and plant material transfer on vegetation development in created Mediterranean meso-xeric grasslands. N Hölzel (ed)[END_REF]Bischoff et al. 2018). However, few studies consider the consequences of these soil treatments on the communities of soil organisms [START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF][START_REF] Wardle | The influence of biotic interactions on soil biodiversity[END_REF][START_REF] Bever | Rooting theories of plant community ecology in microbial interactions[END_REF]). Yet, there is growing evidence that soil legacies and plant-soil interactions are major drivers of the community assembly dynamics [START_REF] Dam | How plants cope with biotic interactions[END_REF][START_REF] Grman | Within-Year Soil Legacies Contribute to Strong Priority Effects of Exotics on Native California Grassland Communities[END_REF][START_REF] Bever | Rooting theories of plant community ecology in microbial interactions[END_REF][START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF]Kardol et al. 2013;[START_REF] Putten Wh Van Der | Plant-soil feedbacks: the past, the present and future challenges[END_REF]. Moreover, interactions between wheat roots (chapter 3) and soil organisms such as bacteria, nematodes or mycorrhiza have been shown to modulate aboveground interactions [START_REF] Dam | How plants cope with biotic interactions[END_REF]. Thus, soil-mediated effects of wheat sowing on restoration would be worth studying. As plant-soil organism interactions range from mutually positive (e.g. mycorrhizal fungi, nitrogen-fixing bacteria) to strongly negative (e.g. pathogenic microbes, root-feeding invertebrates), and as soil organisms play an important role in determining the outcome of plant-plant interactions, they are essential for early plant colonization in grassland restoration (Bever 2003;[START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF]. Since soil treatments suggested to improve plant colonisation have a generally negative effect on soil organisms [START_REF] Chan | An overview of some tillage impacts on earthworm population abundance and diversity -implications for functioning in soils[END_REF][START_REF] Andrade | The soil microbial community and soil tillage[END_REF][START_REF] Holland | The environmental consequences of adopting conservation tillage in Europe: reviewing the evidence[END_REF]) positive plant-soil interactions may be compromised. This trade-off between positive effects on seedling recruitment and negative effects on soil organisms needs to be considered in ecological restoration. However, consequences of soil treatments on soil organisms are less well studied and the final outcome of this trade-off is unknown.

Finally, as plant-soil interactions are species-specific [START_REF] Bezemer | Plant species and functional group effects on abiotic and microbial soil properties and plant-soil feedback responses in two grasslands[END_REF][START_REF] Voorde Tfj Van De | Intra-and interspecific plantsoil interactions, soil legacies and priority effects during old-field succession[END_REF], and as the strength of priority effects depends on both the order of arrival and the identity of the species (chapter 4;[START_REF] Gillhaussen P Von | Priority Effects of Time of Arrival of Plant Functional Groups Override Sowing Interval or Density Effects: A Grassland Experiment[END_REF][START_REF] Cleland | Priority effects vary with species identity and origin in an experiment varying the timing of seed arrival[END_REF][START_REF] Wilsey | Exotic grassland species have stronger priority effects than natives regardless of whether they are cultivated or wild genotypes[END_REF][START_REF] Stuble | Priority effects: natives, but not exotics, pay to arrive late[END_REF][START_REF] Werner | Persistent asymmetrical priority effects in a California grassland restoration experiment[END_REF], one year advance of a particular species may change soil biota and productivity by niche modification. Such modification may subsequently affect ecological processes and restoration success (e.g. biogeochemical cycling, plant community assembly; Fukami 2015; [START_REF] Weidlich | Priority effects caused by plant order of arrival affect below-ground productivity[END_REF][START_REF] Gundale | Comparison of plant-soil feedback experimental approaches for testing soil biotic interactions among ecosystems[END_REF][START_REF] Hess | Restauration écologique des communautés végétales après éradication d'espèces invasives : Rôle de la dynamique de colonisation et des effets de priorité[END_REF]. Therefore, future research needs to pay greater attention to belowground effects of restoration actions and subsequent plant-soil interactions during early community assembly. This research would improve our understanding of plant community succession and represents a potential for increasing restoration success [START_REF] Young | The ecology of restoration: historical links, emerging issues and unexplored realms[END_REF][START_REF] Kardol | How understanding aboveground-belowground linkages can assist restoration ecology[END_REF]Kardol et al. 2013).

Au préalable

Ajoutez ici un texte de description afin de susciter l'attention de vos abonnés sur ce sujet.

En pratique

Ajoutez ici un texte de description afin de susciter l'attention de vos abonnés sur ce sujet.

Cas d'étude à grande échelle

Ajoutez ici un texte de description afin de susciter l'attention de vos abonnés sur ce sujet

AU PRÉALABLE

Plusieurs techniques de restauration écologique sont couramment employées, en fonction des superficies à traiter, de leurs contraintes topographiques et des moyens techniques à disposition.

Il est préférable d'envisager la récolte des graines localement.

Dans ce cas, deux zones sont impliquées dans la mise en oeuvre des travaux de restauration de prairie :

 la zone à restaurer, et  la zone source pour la récolte des graines ou du foin.

Le choix de la technique de restauration va dépendre de la surface et des contraintes de ces deux zones, à savoir, sont-elles mécanisables ?

Le transfert de foin est préférable si les surfaces de récolte et de restauration sont à proximité, mécanisables, grandes (>1ha) et peu pentues. Par contre, si les surfaces sont petites ou présentent de fortes pentes, la récolte des graines via la brosseuse sera plus adaptée.

Prairie de référence pour la restauration écologique -Embrun (05) -© A. Durbecq

Comment restaurer les prairies montagnardes ?  Expérimentation ponctuelle avec différentes modalités de restauration des trajectoires de communautés végétales (dispositif expérimental _ Thèse Avignon Université).

La restauration écologique est définie comme 'le processus qui assiste le rétablissement d'un écosystème qui a été dégradé, endommagé ou détruit ' (SER Primer 2004 ;McDonald et al. 2016 ;Gann et al. 2019). Son but est donc de rétablir l'écosystème dégradé en tâchant de retrouver sa structure, sa composition, ses fonctions et sa dynamique d'origine. Le but de la thèse (dispositif expérimental de la mesure de compensation MC2 : 'Restauration écologique des zones ouvertes remaniées ') est d'améliorer nos connaissances sur les mécanismes de succession végétale, de comparer les différentes méthodes utilisées pour la revégétalisation de zones ouvertes remaniées par les travaux de construction (prairies et pelouses), de développer des solutions techniques pour la restauration de prairies sèches à mésophiles en milieu montagnard, et donc de fournir des recommandations techniques en fonction de la gestion et des conditions pédoclimatiques du milieu pour mener à bien la restauration écologique (dispositif de déploiement de la mesure MC2). L'objectif de la phase de déploiement est donc de restaurer les milieux ouverts de Haute-Durance remaniés par des travaux de construction de la ligne électrique en accélérant la recolonisation par les espèces végétales. La restauration des prairies semi-naturelles de montagne est cruciale car on estime que la dynamique de recolonisation végétale naturelle peut être supérieure à 20 ans (Dupin et al. 2019) Il existe une autre possibilité adaptable aux petites surfaces pentues : la brosseuse tractée avec aspirateur intégré de Mr. Mouchet Nicolas ; SARL Mouchet Bois et Forêts (Fig. 2).

En 2019, 25,6 kg de fleur de foin sur 2,4 ha de prairies sources, soit environ 181 litres de graines ont été récoltés.

Pour la récolte du foin, une prairie d'une surface de 1 ha située à Puy-Saint-Eusèbe (44°6'67''N ; 6°41'66''E) a servi à récolter le foin utilisé pour ensemencer la piste refermée menant aux pylônes 38-39 de la ligne P6.

Le foin a été séché durant 72 heures après la récolte pour éviter l'échauffement et la pourriture pendant le stockage, puis mis en andain sous forme de 15 bottes rectangulaires de 45 x 30 x 75 cm, d'environ 7,5 kg chacune, soit un équivalent de 112 kg de foin, soit environ 1500 litres de foin.

Hormis la piste menant aux pylônes 38-39, toutes les pistes ont été restaurées par semi de fleur de foin, récolté dans le Queyras à l'aide d'une brosseuse avec aspirateur intégré.

RESULTATS

Les premiers sites restaurés ont été revégétalisés par semis de graines (fleur de foin) récoltées localement en juillet 2019 et semées en octobre 2019. Les suivis floristiques ont été effectués en juillet 2020 par ECO-MED sur les prairies sources, 9 sites restaurés et 9 sites non-dégradés s (ci-après 'sites de référence ') situés à proximité des sites dégradé (Figure 4).

En seulement 9 mois, les premiers résultats ont permis de mettre en évidence une bonne reprise de la végétation sur les sites restaurés. La revégétalisation par le semi de graines présentent d'ores et déjà un recouvrement végétal moyen supérieur à 30% sur les sites restaurés en 2019 (Figure 6).

  Figure index

  Fig. I.1. Integrated framework of ecological restoration and community assembly filters after a disturbance. The natural variability of the community before disturbance is characterised by the range of stable states. A range of reference communities encompass the range of stable states to consider the potential restored community trajectories under both deterministic and stochastic processes. At the end of the disturbance, non-linear community trajectories undergo the pressure of the three main assembly filters (different filter sizes indicate their hypothesized relative strength over time and double arrow indicates biotic interactions and environmental feedbacks). The recovery debt represents the gap between the community under restoration and the references (Moreno-Mateos et al. 2017; Strobl et al. 2019). Modified from Hobbs and Norton (1996), including elements of Chang and HilleRisLambers (2016) ……………….….……………………………………………………………………….…… 4 Fig. I.2. Location of the study area with the five degraded sites selected to set up restoration experiments (in light green) along the construction zone of the electricity line (in orange)..7 Fig. I.3. General schema of the thesis structure …………….………………………………………..….…… 13 Chap. 1. Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition .……….………………….…...…… 17 . Fig. 1.1. The four major steps of the proposed method to select references. ① Delimit an extend study area around restoration sites; ② Identify environmental factors structuring plant communities (data collection and driver analysis); ③ Evaluate environmental distances between restoration and potential reference sites (dissimilarity coefficient); ④ Select the most similar references to restoration sites (dissimilarity coefficient) ……………. 22

Fig. S1. 1 .

 1 Fig. S1.1. NMDS based on plant species composition showing groups of the reference sites according to a. geographical position (south; central; north) in comparison with groups according to significant environmental variables: b. P2O5; c. soil moisture; d. annual direct incident solar radiation (ASR) and e. soil pH ……………….……………………………………..…….….… 35

Figure index XVIChap. 2 .Fig. 2 . 1 .

 index221 Figure index

Fig. 2 . 4 .

 24 Fig. 2.4. Plant species composition in the four restoration treatments using NMDS. Polygons indicate the position of the outmost plots in each treatment (NMDS stress: 0.190) ….…...… 49 Fig. 2.5. Final seedling survival of (a) Bromopsis erecta and (b) Rhinanthus alectorolophus.The direction of main effects is indicated using "<" (P<0.05) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05) ……………………………………..……………………………….…….…………………………..…………… 50

Fig. S2. 2 .

 2 Fig. S2.2. Seedling of (a) Rhinanthus alectorolophus and (b) Bromopsis erecta after tagging with a coloured ring and a stick. The photos were taken on May 3rd, 2019 ……………………. 57 Fig. S2.3. Percentage of reproductive individuals of Rhinanthus alectorolophus in the first year.The direction of main effects is indicated using "<<" (P<0.01), "<" (P<0.05), "(<)" (P<0.1) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05) ……………………………………..………………..…….… 57

Fig. 3 . 1 .Fig. 3 . 2 .Fig. 3 . 3 .

 313233 Fig. 3.1. Effect of seed transfer techniques on the abundance of transferred species estimated in quadrats of 40 × 40 cm in 2019. Error bars represent ±SE and different letters indicate significant differences between treatments (P < 0.05) ……………………………………..…....…….… 69 Fig. 3.2. Effect of seed transfer techniques on (a) transferred species cover, and (b) transferred species richness, both based on 2020 cover values and estimated in quadrats of 2 × 2 m. Error bars represent ±SE and different letters indicate significant differences between treatments (P < 0.05) ………………………………………………………………………....…….….… 70 Fig. 3.3. NMDS of plant species composition in the five transfer technique treatments compared to plant species composition in the best reference for each restoration site previously identified in Durbecq et al. (2020). Polygons indicate the position of the outmost plots in each treatment (stress = 0.20). Different symbols indicate the five different restoration sites and corresponding references: and for sites corresponding to

Fig. 3 . 5 .Fig. 3 . 6 .Fig. S3. 2 .Fig. S3. 3 .Chap. 4 .Fig. 4 . 1 .

 353623441 Figure index

Fig. 4 .

 4 Fig. 4.6 (a) Priority Index (PI), and (b) Secondary Index (SI), both calculated on aboveground biomass of sown species. Error bars are ±SE and asterisks indicate significant effects with * (p-adj < 0.05) and ** (p-adj < 0.01). p-adj are p-values with Bonferroni adjustment. (D): dominant species, (S): subordinate species ……………………………………………………………....…… 96 Fig. S4.1 (a) Total species cover and (b) Unsown species cover (species from the seed bank/seed rain) in the four treatments. D+S: dominant and subordinate species sown simultaneously, D1 st : dominant species sown 1 st (subordinates 2 nd ), and S1 st : subordinate species sown 1 st (dominants 2 nd ). (D): dominant species, (S): subordinate species. Error bars are ±SE and different letters indicate significant differences between treatments ………..…. 99 Fig. S4.2. Species biomass when sown alone one year earlier (First 2019) or simultaneously with the other group (Simultaneously 2019), and when sown two years alone two years earlier (First 2020) or simultaneously with the other group (Simultaneously 2020). The years correspond to the year of the biomass survey (not to the year of sowing). PI: Priority Index, SI: Secondary Index ……………………………………..………………………………………………...… 100 Fig. S4.3. (a) Priority Index (PI), and (b) Secondary Index (SI), both calculated on the abundance of the sown species. Error bars are ±SE and asterisks indicate significant effects with * (p-adj < 0.05) and ** (p-adj < 0.01). p-adj are p-values with Bonferroni adjustment …………………………………………………………………………………………………………………………………. 101 Fig. S4.4. (a) Priority Index (PI), and (b) Secondary Index (SI), both calculated on sown species cover. Error bars are ±SE and asterisks indicate significant effects with * (p-adj < 0.05) and ** (p-adj < 0.01). p-adj are p-values with Bonferroni adjustment ………...…….…… 101

Fig. I. 1 .

 1 Fig. I.1. Integrated framework of ecological restoration and community assembly filters after a disturbance. The natural variability of the community before disturbance is characterised by the range of stable states. A range of reference communities encompass the range of stable states to consider the potential restored community trajectories under both deterministic and stochastic processes. At the end of the disturbance, non-linear community trajectories are driven by three main assembly filters (different filter sizes indicate their hypothesized relative strength over time and double arrow indicates biotic and abiotic filters feedbacks).The recovery debt represents the gap between the community under restoration and the references[START_REF] Moreno-Mateos | Anthropogenic ecosystem disturbance and the recovery debt[END_REF][START_REF] Strobl | Integrated assessment of ecosystem recovery using a multifunctionality approach[END_REF]. Modified from[START_REF] Hobbs | Towards a Conceptual Framework for Restoration Ecology[END_REF], including elements of[START_REF] Chang | Integrating succession and community assembly perspectives[END_REF].

Fig. I. 2 .

 2 Fig. I.2. Location of the study area with the five degraded sites selected to set up restoration experiments (in light green) along the construction zone of the electricity line (in orange).

  (1.a) How should we determine the references? (1.b) Which factors influencing species composition are appropriate for the choice of references? Chapter 1 Taking into account the main filters limiting plant recruitment, I asked: (2.a) Does soil preparation by harrowing before sowing improve recruitment and survival of the transferred species? (2.b) What is the effect of early extensive grazing on seedling recruitment and survival of the transferred species? Chapter 2

Fig. I. 3 .

 3 Fig. I.3. Illustration of the thesis structure (see Fig. I.1. for further explanations).

I. 1 )

 1 corresponding to the chapters 2 and 3, respectively. Another experiment manipulating the arrival of dominant and subordinate species to test priority effects (question (4) and chapter 4) was set up on one of the five degraded sites with ten spatially randomized replicates. Different types of propagule transfer techniques were carried out in the three experiments to overcome the dispersal filter (Fig.I.3, Table

Fig. 1 . 1 .

 11 Fig. 1.1. The four major steps of the proposed method to select references. ① Delimit an extend study area around restoration sites; ② Identify environmental factors structuring plant communities (data collection and driver analysis); ③ Evaluate environmental distances between restoration and potential reference sites (dissimilarity coefficient); ④ Select the most similar references to restoration sites (dissimilarity coefficient).

Fig. 1 . 2 .

 12 Fig. 1.2. Location of the study area with restoration sites (1R, 2R, 3R, 11R and 20R, in red) and reference sites (numbered from north to south, without R, in blue) along the construction of a new electricity line.

Fig. 1 . 3 .

 13 Fig. 1.3. RDA biplot using Hellinger-transformed plant species cover (species names in blue) and reference sites (green) constrained by environmental variables (red arrows). Environmental variables: ASR = Annual Solar Radiation; P2O5 = plant available phosphorous; soil moisture; soil pH; elevation and K2O = plant available potassium. Full species names: TableS1.3.

Fig. 1 . 3 .

 13 Here the colors show examples for chosing only one reference for (a) one site (example of restoration site 2R: ); (b) 2 sites (example of 1R and 3R: ); (c) all the restoration sites (1R, 2R, 3R, 11R and 20R:

Fig. 1 . 4 .

 14 Fig. 1.4. NMDS based on environmental variables showing a) the three most similar nondegraded sites (without R) for each restoration site (with R) and b) the non-degraded site that present the lowest average environmental distance with all the restoration sites. Different restoration sites are represented by different colours. The grey numbers represent the environmental distance (dissimilarity coefficient). See Table1for more details.

Fig. S1. 1 .

 1 Fig. S1.1. NMDS based on plant species composition showing groups of the reference sites according to a. geographical position (south; central; north) in comparison with groups according to significant environmental variables: b. P2O5; c. soil moisture; d. annual direct incident solar radiation (ASR) and e. soil pH.

Fig. 2 . 1 .

 21 Fig. 2.1. Restoration site before (left) and after (right) experimental set-up involving exclosure by fencing and soil preparation by harrowing.

  . S2.1).

Fig. 2 . 4 .

 24 Fig. 2.4. Plant species composition in the four restoration treatments using NMDS. Polygons indicate the position of the outmost plots in each treatment (NMDS stress: 0.190).

Fig. 2 . 5 .

 25 Fig. 2.5. Final seedling survival of (a) Bromopsis erecta and (b) Rhinanthus alectorolophus.The direction of main effects is indicated using "<" (P<0.05) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05).

Fig. S2. 2 .

 2 Fig. S2.2. Seedling of (a) Rhinanthus alectorolophus and (b) Bromopsis erecta after tagging with a coloured ring and a stick. The photos were taken on May 3rd, 2019.

Fig. S2. 3 .

 3 Fig. S2.3. Percentage of reproductive individuals of Rhinanthus alectorolophusin the first year. The direction of main effects is indicated using "<<" (P<0.01), "<" (P<0.05), "(<)" (P<0.1) and "=" (not significantly different). Significant grazing-by-soil preparation interactions are presented using "*" (P<0.05).

Fig. 3 . 1 .

 31 Fig. 3.1. Effect of seed transfer techniques on the abundance of transferred species estimated in quadrats of 40 × 40 cm in 2019. Error bars represent ±SE and different letters indicate significant differences between treatments (P < 0.05).

Fig. 3 . 2 .

 32 Fig. 3.2. Effect of seed transfer techniques on (a) transferred species cover, and (b) transferred species richness, both based on 2020 cover values and estimated in quadrats of 2 × 2 m. Error bars represent ±SE and different letters indicate significant differences between treatments (P < 0.05).

Fig. 3 . 3 .

 33 Fig. 3.3. NMDS of plant species composition in the five transfer technique treatments compared to plant species composition in the best reference for each restoration site previously identified in Durbecq et al. (2020, chapter 1). Polygons indicate the position of the outmost plots in each treatment (stress = 0.20). Different symbols indicate the five different restoration sites and corresponding references:and for sites corresponding to reference n°16 (same reference for the two restoration sites), to reference n°12, to reference n°6, and to reference n°9.

Fig. 3 .

 3 Fig. 3.4. (a) CSIInorm and (b) HAI, comparing the plant communities of transfer technique treatments of each restoration site with their respective best reference site. Error bars represent ±SE and different letters indicate significant differences between treatments (P < 0.05).

Fig. 3 . 5 .

 35 Fig.3.5. Mean target plant cover of abundant species (> 9 samples, 41 of 259 species in total) in restoration treatments and the best reference community. Different colours represent the mean cover proportion in references (black), the mean cover proportion in communities under restoration up to the mean cover in the reference communities (green), and the mean cover proportion exceeding that of the reference communities (orange). Asterisks indicate transferred species. For readability, the x-axis is limited to 10% cover (only Bromopsis erecta (Huds.) Fourr. Schedonorus arundinaceus (Schreb.) Dumort. and Galium molugo agg. exceeded).

Fig. 3 . 6 .

 36 Fig. 3.6. Effect of seed transfer techniques on soil erosion. Error bars represent ±SE.Positive values show higher erosion, thus a lesser amount of material on the ground 14 months after the beginning of the experiment.

Fig. S3. 1 .

 1 Fig. S3.1. Picture of erosion measurement device

Fig. S3. 3 .

 3 Fig. S3.3. Temporal differences in the effect of seed transfer techniques on soil erosion between (a) June 2019 and October 2019, (b) October 2019 and May 2020, and (c) May 2020 and September 2020. Error bars represent ±SE and letters indicate significant differences between treatments (P < 0.05). Positive values show a lesser amount of material on the ground, thus higher erosion.

Fig. 4 . 1 .

 41 Fig. 4.1. Hypothesized priority effects. (a) Hypothesized response of the species interactions according to both (b) competitive response and (c) competitive effect[START_REF] Goldberg | Competitive Effect and Response: Hierarchies and Correlated Traits in the Early Stages of Competition[END_REF]. Dominants are displayed in red and subordinates in purple. Competitive response (proportion of focal species cover or biomass when grown with other species compared to monoculture) and effect (i.e. proportion of cover or biomass of other species when they are grown with the focal species compared to monoculture) are displayed according to the hypotheses drawn for each species group (dominant or subordinate). Hypothesis n°1: Subordinates show a low competitive response and therefore establish less well in pre-established communities, but resist if sown at first (b, purple line). Hypothesis n°2: Subordinates show a low competitive effect and therefore, do not or hardly affect other species regardless of arrival date (c, purple line). Hypothesis n°3: Dominant species show a highly competitive response and are therefore not affected by other species, even if sown later (b, red line). Hypothesis n°4: Dominant species show a strong competitive effect and therefore strongly affect other species when arriving first, but to a lesser degree when arriving later (c, red line).

  simultaneous sowing (the six species were sown together in 2018) (iii) D1 st : the three dominant species (D) were sown in 2018 and the three subordinate species (S) in 2019 (iv) S1 st : the three subordinate species were sown in 2018 and the three dominant species in 2019. Each treatment was replicated in ten spatially randomized plots (total n = 40; Fig. 4.2a).

Fig. 4 . 2 .

 42 Fig. 4.2. Experimental design with a. the 40 plots including the ten replicates of the four randomly distributed treatments: D+S: dominant and subordinate species were sown simultaneously, D1 st : dominant species were sown 1 st (subordinates 2 nd ), S1 st : subordinate species were sown 1 st (dominants 2 nd ) and Control (no sowing); b. Zoom on one 2 × 2 m plot (and its sub-plots).

  'Focal individual'(Fi) is the individual sown species considered in the calculation. The two indices are defined as follow:PI = _____________________________ Biomass2020(Fi)1st -Biomass2020(Fi)simultaneously ______________________________ Biomass2020(Fi)1st + Biomass2020(Fi)

  Fig. S4.1b). The NMDS ordination showed a clear separation of the community composition between the four treatments(df = 3; F = 11.29; p < 0.001, Fig. 4.3). Along the first axis of the NMDS, the three sequential sowings were characterized by the sown species on the far right, while ruderal species such as Daucus carota L. Lactuca serriola L. Convolvulus arvensis L. Stachys recta L. Arenaria serpyllifolia L. dominated the control (left). The second axis separated the three sowing treatments, placing the simultaneous sowing (D+S) in the middle of the two sequential sowings with D1 st on the upper and S1 st on the lower part of this axis.

Fig. 4 . 3 .

 43 Fig. 4.3. NMDS of plant species composition in the four sowing treatments in July 2020. Polygons indicate the position of the outmost plots in each treatment with D+S: dominant and subordinate species sown simultaneously, D1 st : dominant species sown 1 st (subordinates 2 nd ), S1 st : subordinate species sown 1 st (dominants 2 nd ) and Control (no sowing). Sown species are in bold. NMDS stress = 0.24.

Fig. 4 . 4 .

 44 Fig. 4.4. Species cover contribution to the total cover in the four treatments. (D): dominant species; (S): subordinate species. D+S: dominant and subordinate species sown simultaneously, D1 st : dominant species sown 1 st (subordinates 2 nd ), S1 st : subordinate species sown 1 st (dominants 2 nd ) and Control (no sowing). P. media did not germinate.

Fig. 4 .

 4 Fig. 4.6. (a) Priority Index (PI), and (b) Secondary Index (SI), both calculated on aboveground biomass of sown species. Error bars are ±SE and asterisks indicate significant effects with * (p-adj < 0.05) and ** (p-adj < 0.01). p-adj are p-values with Bonferroni adjustment. (D): dominant species, (S): subordinate species.

Fig. S4. 2 .

 2 Fig. S4.2. Species biomass when sown alone one year earlier (First 2019) or simultaneously with the other group (Simultaneously 2019), and when sown two years alone two years earlier (First 2020) or simultaneously with the other group (Simultaneously 2020). The years correspond to the year of the biomass survey (not to the year of sowing). PI: Priority Index, SI: Secondary Index.

Fig

  Fig. S4.3. (a) Priority Index (PI), and (b) Secondary Index (SI), both calculated on the abundance of the sown species. Error bars are ±SE and asterisks indicate significant effects with * (p-adj < 0.05) and ** (p-adj < 0.01). p-adj are p-values with Bonferroni adjustment.

Fig. S4. 4 .

 4 Fig. S4.4. (a) Priority Index (PI), and (b) Secondary Index (SI), both calculated on sown species cover. Error bars are ±SE and asterisks indicate significant effects with * (p-adj < 0.05) and ** (p-adj < 0.01). p-adj are p-values with Bonferroni adjustment.

Faivre

  P (2000) Revégétalisation et sols reconstitués au niveau des pistes de ski. Bilan sommaire de 3 campagnes de terrain (97-99) et perspectives de recherche. Centre Interdisciplinaire Scientifique de la Montagne, Université de Savoie, Val Thorens, France. Falk DA, Palmer MA, Zedler JB (2013) Foundations of Restoration Ecology. Island Press.

  PERSPECTIVES L'utilisation des effets de priorité pourrait être un atout en restauration écologique. Bien que cette approche soit encore préliminaire, il serait intéressant de poursuivre les recherches pour établir les listes d'espèces qui bénéficieraient à être semées de manière séquentielle pour restaurer la structure de la communauté. RTE a lancé le 'Programme Haute-Durance'consistant à la rénovation des lignes électriques à haute tension dans les Hautes-Alpes. Ce programme ayant fait l'objet d'une demande de dérogation à l'interdiction de destruction d'espèces protégées, RTE s'est engagé sur la mise en oeuvre de mesures compensatoires, dont la mesure intitulée 'Restauration expérimentale de zones ouvertes remaniées'. Le présent rapport (annexe #) présente l'état d'avancement de la mesure et les résultats de la première année de suivi du dispositif de déploiement avec transfert de foin et semis, projet de restauration mis en oeuvre par le bureau d'étude ECO-MED, sur une surface d'environ 8 ha. OBJECTIFS DE LA RESTAURATION ÉCOLOGIQUE  Restauration écologique des milieux ouverts de Haute-Durance remaniés par des travaux de terrassement (revégétalisation des plateformes de travail et des pistes d'accès transitoires) par transfert de foin et de fleur de foin (dispositif de déploiement _ ECO-MED).

  , et un milieu mis à nu est d'autant plus exposé aux risques d'érosion du sol. La revégétalisation par des espèces locales améliore ainsi le contrôle des processus érosifs des sols et limite la progression de plantes invasives. Elle participe également au maintien de la continuité des milieux favorisant ainsi le retour et la circulation d'espèces patrimoniales. A l'automne 2020, l'ensemencement a concerné 64 pistes et plateformes associées, 11 sur la ligne Grisolles -MontDauphin (P6), 48 sur la ligne Argentière -Serre Ponçon et 1 sur la ligne Argentière -Briançon. Au total 6,3 km de pistes soit 4,5 ha ont été ensemencés. En plus de ces lignes, 4 tronçons d'enfouissement de ligne souterraine ont été ensemencés sur une longueur de 521 mètres soit 0,25 ha. temporaires ont été tracées pour le projet RTE-HD, dont 131 pistes tout ou en partie en milieu ouvert. En incluant les cultures et semis agricoles, 118 pistes ont été revégétalisées. Les 13 pistes restantes seront ensemencées en 2021. La mesure MC2 est donc achevée à 90%.OBJECTIFS DU SUIVILe suivi se déroulera chaque année sur les 3 années après l'ensemencement, avec les indicateurs suivants :  Suivi de la composition floristique (sites source, sites restaurés et sites de référence). Bon recouvrement végétal et similarité avec la référence. Diversité de composition des communautés végétales (en Poacées, Fabacées, et autres familles végétales).126METHODOLOGIEProtocole de mise en oeuvre de la restauration écologiqueRécolte des graines et du foinPlusieurs techniques complémentaires de revégétalisation sont couramment employées, en fonction des superficies à traiter, de leurs contraintes topographiques et des moyens techniques à disposition. Nous présentons ici les deux techniques qui ont été mises en oeuvre pour la mesure MC2 : le transfert de foin sec et de graines récoltées par une brosseuse (nommé couramment et ci-après 'fleur de foin ').La praire source pour la récolte en graine doit correspondre à la zone à restaurer en termes d'habitat, de composition floristique et de facteurs environnementaux. Le ratio surface d'ensemencement / surface de prélèvement est d'environ 1 / 1 pour des prairies de fauche jusqu'à 1300-1400 m d'altitude(Dupin et al. 2019).Pour la récolte de graines (fleur de foin), différentes récoltes ont été effectuées en juillet 2019 et 2020 par Yvon Sindzingre et Aure Durbecq dans les communes de Ceillac, Puy-Sanières et Ristolas (05), à l'aide de la brosseuse avec aspirateur intégré (Fig.2).Il existe différent type de brosseuse pour récolter la fleur de foin. Si la surface est grande et peu pentue il est possible d'utiliser une brosseuse tractée par un tracteur (Fig.1), mais cela demande d'effectuer un passage avec un aspirateur portatif (ex. : aspirateur-broyeur Stihl modèle SH86) pour ramasser les graines tombées au sol après le passage de la brosseuse.

Fig. 1 .

 1 Fig. 1. Brosseuse tractée classique.

Fig. 2 .

 2 Fig. 2. Brosseuse avec aspirateur intégré.

Fig. 3 .

 3 Fig. 3. Epandage du foin sur une piste à restaurer.

Figure 4 .

 4 Figure 4. Carte de localisation du projet et des sites de suivi de la restauration écologique.

(Fig. 6 )

 6 , nos premiers résultats indiquent un potentiel de réduction de l'érosion des sols de 60% par rapport à 100% de sol nu (Gu et al. 2019 : Annexe 1 ; Figure. 9). Par ailleurs, l'analyse des compositions des communautés végétales a permis de comparer les recouvrements végétaux entre les prairies sources et les prairies restaurées. A travers la distinction en trois groupes de familles végétales (les fabacées, les poacées, et les autres familles ; Figure 7), nos résultats mettent en évidence la capacité plus lente des fabacées et des poacées à s'installer dans un milieu dégradé. Cependant, les sites restaurés en 2019 présentent un recouvrement végétal d'au moins 5% dans chacune de ces deux familles de plantes. Ces résultats indiquent une dynamique en faveur d'une bonne recolonisation de ces familles végétales dont les espèces annuelles pionnières sont souvent peu représentées sans action de restauration.

Fig. 6 .

 6 Fig. 6. Pourcentage de recouvrement végétal moyen en juillet 2019 sur les sites de référence (sites non-dégradés) et sur les sites revégétalisés.

  Figure 7. Pourcentage de recouvrement végétal moyen distingué en trois groupes : fabacées, poacées et autres familles, relevés en juillet 2019 sur les sites sources (pour la récolte en graines) et sur les sites revégétalisés.

Figure 8 .

 8 Figure 8. Richesses floristiques moyenne, minimal et maximale, en juillet 2019 sur les sites de référence (sites non-dégradés) et sur les sites revégétalisés.

…………………………… 55 Chapter 4. Consider sequential sowing to use priority effects in plant community restoration

  ……………………………..………………………….…………………………………………….…...…… 83

Table 4 . 1 .

 41 Characteristics of dominant and subordinate species sown in the experiment.

  D+S: dominant and subordinate species were sown simultaneously, D1 st : dominant species were sown 1 st (subordinates 2 nd ), S1 st : subordinate species were sown 1 st (dominants 2 nd ) and Control (no sowing); b. Zoom on one 2 × 2 m plot (and its sub-plots) ………………………….……………………………………..…………………………….……..… 90 Fig. 4.3. NMDS of plant species composition in the four sowing treatments in July 2020. Polygons indicate the position of the outmost plots in each treatment with D+S: dominant and subordinate species sown simultaneously, D1 st : dominant species sown 1 st (subordinates 2 nd ), S1 st : subordinate species sown 1st (dominants 2 nd ) and Control (no sowing). Sown species are in bold. NMDS stress = 0.24 …………………………………..……………… 93 Fig. 4.4. Species cover contribution to the total cover in the four treatments. (D): dominant species; (S): subordinate species. D+S: dominant and subordinate species sown simultaneously, D1 st : dominant species sown 1 st (subordinates 2 nd ), S1 st : subordinate species sown 1 st (dominants 2 nd ) and Control (no sowing). P. media did not germinate ………………. 94 Fig. 4.5. Sown species cover in the three sequential sowing treatments with D+S: dominant and subordinate species sown simultaneously, D1 st : dominant species sown 1 st (subordinates 2 nd ), and S1

	Figure index
	randomly distributed treatments:
	XVIII

st : subordinate species sown 1 st (dominants 2 nd ). (D): dominant species, (S): subordinate species. Error bars are ±SE and different letters indicate significant differences between treatments (a separate analysis was run for each species with Bonferroni adjustment of p-values) ……………………………………..…………………………………..…… 95

Table I .

 I 1. Filter-based restoration actions carried out during the thesis

	Thesis chapter	Restoration actions	Associated assembly filter
	1	Reference identification	Environmental drivers of plant community assembly
		Soil preparation	Abiotic
	2	Transfer of brush-harvested material	Dispersal
		Livestock grazing management	Biotic (herbivory) & Abiotic (trampling)
		Transfer of brush-harvested material	Dispersal
	3	Hay transfer (dead plant cover) Wheat addition (living plant cover)	Dispersal, Biotic (plant-plant interactions) & Abiotic (micro-climate) Biotic (plant-plant interactions) & Abiotic (micro-climate)
		Erosion measurment	Abiotic
		Sequential seeding	Dispersal
	4	Priority effect	Biotic (plant-plant interactions) & Abiotic (micro-climate change)

Chapter One Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition

	This chapter was published in 2020:
	Durbecq A, Jaunatre R, Buisson E, Cluchier A, Bischoff A (2020) Identifying

reference communities in ecological restoration: the use of environmental conditions driving vegetation composition. Restoration Ecology, 28(6), 1445-1453. https://doi.org/10.1111/rec.

13232 Identifying reference communities in ecological restoration: the use of environmental conditions driving vegetation composition

  slope, aspect) = 0.339 + 0.808 * cos(latitude) * cos(slope) -0.196 * sin(latitude) * sin(slope) -0.482 * cos(aspect) * sin(slope) [Eq. 3 in McCune et Keon 2002] c) f returns ln(Rad, MJ.cm-2.yr-1). It is therefore returned to an arithmetic scale with the EXP(x) function. We obtain Annual solar radiation: ASR = EXP(f) Table S1.3. Complete names of plant species represented in the RDA biplot (Fig. 1.3)

	RDA names		Full names	RDA names	Full names
	Ach_millefolium Achillea millefolium	Lot_corniculatus Lotus corniculatus
	Ant_vulneraria	Anthyllis vulneraria	Med_lupulina	Medicago lupulina
	Ara_hirsuta		Arabis hirsuta	Pil_offcinarum	Pilosella officinarum
	Are_serpyllifolia Arenaria serpyllifolia	Pla_lanceolata	Plantago lanceolata
	Ast_danicus		Astragalus danicus	Pla_media	Plantago media
	Bra_rupestre		Brachypodium rupestre Poa_pratensis	Poa pratensis
	Bro_erecta		Bromopsis erecta	Pot_verna	Potentilla verna
	Dac_glomerata	Dactylis glomerata	Sal_pratensis	Salvia pratensis
	Fes_marginata	Festuca marginata	San_minor	Sanguisorba minor
	Table S1.1. Correlations between environmental variables. Gal_verum Galium verum Tar_Taraxacum Taraxacum officinale agg.
	Hip_comosa Kna_arvensis	Elevation Hippocrepis comosa ASR Knautia arvensis	Teu_chamaedrys Teucrium chamaedrys Soil moisture Soil pH P2O5 K2O The_divaricatum Thesium divaricatum
	Elevation Lat_pratensis	1.000 Lathyrus pratensis 0.081	0.035 Tra_pratensis -0.017	0.207 Tragopogon pratensis 0.247
	ASR	0.081	1.000	0.071	0.442	-0.367	0.175
	Soil moisture	0.035	0.071	1.000	0.073	0.450	0.104
	Soil pH	-0.017	0.442	0.073	1.000	-0.330	0.398
	P2O5	0.207	-0.367	0.450	-0.330	1.000	0.118
	K2O	0.247	0.175	0.104	0.398	0.118	1.000
	Table S1.2. Relationships between grassland plant composition (as represented in Fig. 1.2)
	and six environmental gradients using "envfit" with N = 999 permutations (R package
	"vegan"). The significant variables are in bold.
		R²	P
	Elevation	0.19	0.201
	ASR	0.59	0.004
	Soil moisture 0.39	0.023
	Soil pH	0.36	0.026
	P2O5	0.40	0.025
	K2O	0.02	0.830

  1, * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.Fig. 2.2. Number of seedlings per m 2

		df	Seedling number	Species richness	Survival
			All	Transferred	All	Transferre	Bromopsis	Rhinanthus
			species	species	species	d species	erecta	alectorolophus
	Harrowing 1 7.097 ** 6.435 *	3.476 .	6.308 *	2.484 NS 0.741 NS
	Grazing	1 3.726 .	3.440 .	2.661 NS 2.804 .	1.799 NS 2.585 NS
	H x G	1 0.560 NS 0.011 NS	0.014 NS 1.095 NS	5.089 *	4.851 *

Table S2 . 1 .

 S21 Number of seedlings of transferred species from brush material measured in the greenhouse and in the field. Nomenclature follows[START_REF] Tison | Flore de la France méditerranéenne continentale[END_REF].

	Transferred species	Greenhouse	Field
	Achillea millefolium	6.7	0
	Alyssum alyssoides	0	0
	Arabis hirsuta	0.4	0
	Bromopsis erecta	6.7	1.6
	Bunium bulbocastanum	0	0.2
	Dactylis glomerata	5.9	0.2
	Festuca marginata	35.6	0.1
	Festuca valesiaca	2.6	0
	Festuca sp.	4.4	0
	Galium sp.	17.8	0.4
	Heracleum sphondylium 0	0.2
	Leontodon hispidus	0	0
	Onobrychis viciifolia	1.1	0.1
	Plantago lanceolata	0.4	0
	Plantago media	95.6	0
	Poa pratensis	3.7	0
	Poa trivialis	1.1	0
	Potentilla sp.	2.6	0
	Ranunculus bulbosus	0.7	0
	Rhinanthus	0	1.3
	alectorolophus
	Rumex acetosa	0.4	0
	Schedonorus pratensis	6.7	0.2
	Silene nutans	0	0.1
	Silene vulgaris	1.1	0
	Taraxacum sp.	2.2	0
	Trisetum flavescens	1.5	0
	Veronica sp.	0.4	0

Fig. S2.1. Seedling emergence of transferred species from brush material measured in the greenhouse and in the field. For the field data, species occurring in control plots of a given site were excluded from measurements of this respective site. Number of seedlings are detailed in the table above. Reference: Tison JM, Jauzein P, Michaud H (2014) Flore de la France méditerranéenne continentale. Naturalia Publications.

  . PEs can be either positive (i.e. facilitative;[START_REF] Bertness | Competition and Facilitation in Marsh Plants[END_REF] or inhibitory(i.e. competitive; Cole 1983;[START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF] but usually, a competitive advantage for the first arriving species prevails because of their greater size or density compared with late-arriving species (i.e. size-asymmetric competition;[START_REF] Grman | Within-Year Soil Legacies Contribute to Strong Priority Effects of Exotics on Native California Grassland Communities[END_REF][START_REF] Wainwright | Seasonal priority effects: implications for invasion and restoration in a semi-arid system[END_REF]. Many other mechanisms can also drive PEs, such as niche pre-emption or niche modification(Helsen et al 2015;[START_REF] Fukami | Historical Contingency in Community Assembly: Integrating Niches, Species Pools, and Priority Effects[END_REF], plant-soil feedbacks and soil legacies(Bever 2003;[START_REF] Putten Wh Van Der | Plant-soil feedbacks: the past, the present and future challenges[END_REF][START_REF] Fukami | Complex plant-soil interactions enhance plant species diversity by delaying community convergence[END_REF][START_REF] Grman | Within-Year Soil Legacies Contribute to Strong Priority Effects of Exotics on Native California Grassland Communities[END_REF] or even allelopathic effects

Table 4 .

 4 1. Characteristics of dominant and subordinate species sown in the experiment. Competitive, stress-tolerant and ruderal strategies according to[START_REF] Grime | Benefits of plant diversity to ecosystems: immediate, filter and founder effects[END_REF]), Erschbamer (2007), and[START_REF] Pierce | Allocating CSR plant functional types: the use of leaf economics and size traits to classify woody and herbaceous vascular plants[END_REF]. Number of seeds sown/m² adjusted according to seed producer expertise on field germination (Phytosem, Gap, France). Frequency of occurrence and average cover according toDurbecq et al. (2020, chapter 1).

	Species	Composition group	Family	CSR strategy	No. of seeds/ m²	Frequency of occurrence	Average cover (%)
	Bromopsis erecta	Dominant (D)	Poaceae	S/SC	555	0.93	32.13
	Festuca cinerea	Subordinate (S)	Poaceae	S/SR	2000	0.26	21.51
	Plantago media	Dominant (D)	Plantaginaceae	C/CR	1250	0.68	5.42
	Plantago lanceolata	Subordinate (S)	Plantaginaceae	C/SC	533	0.40	1.22
	Anthyllis vulneraria	Dominant (D)	Fabaceae	CS/CSR	400	0.28	3.07
	Onobrychis viciifolia	Subordinate (S)	Fabaceae	CS/CSR	710	0.14	1.04

  EN PRATIQUELe transfert des graines récoltées à la brosseuse et le transfert du foin sont des techniques efficaces pour restaurer les prairies de montagne. Cependant, les résultats de la thèse ont mis en évidence que le transfert de graines récoltées à la brosseuse est amélioré par un léger hersage antérieur au semis, ou par l'ajout d'un semis supplémentaire de blé. Cela permet de favoriser le recrutement et l'établissement de plantules cibles pour la restauration, à condition que le blé ne perdure pas dans l'écosystème.

	Par ailleurs, bien que le transfert de foin
	semble mieux convenir aux surfaces
	étendues	et	peu	pentues,	la
	contractualisation avec les agriculteurs
	pour ramasser le foin, le stockage et le
	transport des bottes de foin compliquent
	la mise en oeuvre de cette technique, en
	particulier sur les projets de restauration à
	grande	échelle	(cf.	rapport
	Revégétalisation des zones ouvertes
	remaniées, ci-après).
	Le choix de la technique à sélectionner
	doit donc tenir compte de ces facteurs.

  Plusieurs études ont mis en évidence l'importance de la restauration des prairies dans les processus de stabilisation des sols et du contrôle de l'érosion, spécialement en milieu montagnard. Dans ces milieux, les conditions topographiques et climatiques rendent ces processus extrêmement lents, et la présence d'un couvert végétal est un facteur essentiel, d'où la recommandation d'une revégétalisation immédiate à partir de semences prélevées localement. Avec un recouvrement de 30% la première année après la revégétalisation

La récolte de foin a rapidement été abandonnée au profit de la récolte de graines. En effet, les premiers résultats de thèse et la recherche bibliographie laissent à penser une bonne efficacité avec le semi de graines (aussi appelé semi de fleur de foin). De plus, l'épandage de foin s'avère inadapté aux petites surfaces difficiles d'accès car il nécessite une logistique lourde (apport de foin, épandage…) ; à l'inverse, la fleur de foin est facilement transportable et épandable sur tout type de surface ; seule la récolte engendre un surcoût, cependant elle permet à l'agriculteur de conserver un fourrage quasiment indemne.A l'automne 2019, l'ensemencement a été effectué sur 29 pistes et plateformes associées, 16 sur la ligne Grisolles -MontDauphin (P6) et 13 sur la ligne Argentière -Serre Ponçon. Au total, 4,1 km de pistes soit 2,9 ha ont été ensemencés.En juillet 2020, la récolte de fleur de foin s'est déroulée sur 1 ha à Château-Ville-Vieille et sur 6,6 ha à Ristolas.

© A. Durbecq © P. Haslgrübler

Epandage du foin et Semis de fleur de foinLe foin est réparti de manière homogène sur environ 4 cm d'épaisseur (environ 500 grammes de foin/m²) à l'aide des outils disponibles. Exemples : pailleuse auto-chargeuse, épandeur à fumier, rouleau doseur, fourche à foin. Le passage de rouleau est recommandé mais pas indispensable.En restauration écologique de prairies de montagne, il est conseillé d'effectuer le semis des graines récoltées en automne (entre octobre et décembre), juste avant les premières neiges, ceci afin de favoriser la levée de dormance rompue par l'effet du gel nécessaire aux espèces de graminées d'altitude.En 2019, ECO-MED a effectué un épandage de 1500 litres de foin sur 2276 m² de piste de manière manuelle à l'aide d'une fourche à foin (ligne P6 pylônes 38-39).Sur le reste des pistes refermées en 2019, un semis par fleur de foin a été réalisé dans les quantités suivantes : 143 litres semés au total (semis de 1 g/m²) sur 26150 m², revégétalisant ainsi la totalité des pistes refermées par RTE (c'est-à-dire disponibles pour la mise en oeuvre de la phase de compensation) soit 2,6 ha de milieux ouverts compensés en 2019.L'usage d'un semoir mécanique portatif a été essayé. La fleur de foin ne s'avère pas adaptée à ce type d'outil, du fait de la légèreté des graines, de leur taille très variable et de la présence d'épis et brins dans le mélange qui bloque le mécanisme. Le semis à la main, dit à la volée, s'avère être le plus efficace.

Remerciements

Cette thèse n'aurait pu voir le jour sans le financement de RTE (Réseau de Transport d'Electricité), du bureau d'étude ECO-MED et de l'ANRT (

Chapter Three

Mountain grassland restoration using hay and brush material transfer combined with temporary wheat cover This chapter was submitted to Ecological Engineering on February, 26 th 2021.

Consider sequential sowing to use priority effects in plant community restoration Abstract

Priority effects have been identified as a core concept in restoration ecology since their influence in early community succession may have long-term consequences for plant succession and community assembly. Therefore, the trajectory of the restored community can be modified by the sequence of plant species arrival, thus modifying such priority effects.

In order to test the effect of species arrival on community assembly after soil disturbance in French mountain grasslands, we applied sequential sowing using two communities, each comprising three different perennial plant species: one community of species dominant in the study area, the other one comprising subordinate species. We tested four sowing treatments: control (without any sowing), two sequential sowing treatments and simultaneous sowing. In the first sequential sowing treatment the three dominants were sown in 2018 and the three subordinates in 2019, in the second treatment the three subordinates were sown in 2018 and the three dominants in 2019. We analysed plant cover, abundance and aboveground biomass, and calculated priority and secondary indices for each sown species.

Our results showed that manipulating the order of arrival shaped the community composition. Both the dominant Poaceae Bromopsis erecta and the subordinate Fabaceae Onobrychis viciifolia showed significantly negative secondary index. The latearriving species recruitment of B. erecta and O. viciifolia was probably hampered by niche modification and size asymmetric competition of the pre-established vegetation. Interestingly, the dominant Fabaceae Anthyllis vulneraria was clearly favoured when sown in second, demonstrating a high recruitment capacity even when arriving late. We suggested that the strength of priority effects varies according to the niche characteristics which differ between species and may influence (negatively or positively) the late-arriving species establishment. Our study provided evidence that plant community assembly was influenced by the order of arrival but highlighted strong response variations between species suggesting high species-specific priority effects.

Keywords: dominant species, subordinate species, time-advanced, niche modification, stepwise sowing, grassland restoration, late-arriving species, early-arriving species

Supporting information

AVANCEMENT DES ACTIONS DE RESTAURATION

Sur la base des premiers résultats de thèse menée par Aure DURBECQ, le déploiement de la mesure de compensation MC2 'Restauration des zones ouvertes remaniées 'a débuté au printemps 2020.

En juillet 2019, 1 hectare de foin a été récolté en botte carré sur la commune de Puy-Sanières et 2,8 ha de fleur de foin ont été récoltés sur la commune de Ristolas.

Il s'est avéré difficile de trouver d'importante surface à récolter dans la vallée de la Haute-Durance. En effet, la surface de prairie de fauche est relativement faible vis-à-vis de la surface de prairie pâturée en Haute-Durance et le cheptel de bétail élevé amène les éleveurs à conserver l'intégralité de leur foin. Le Queyras, qui présente une richesse floristique similaire à la vallée de la Haute-Durance, s'est avéré propice à la récolte du foin.  Revégétalisation naturelle de la piste très rapide, achevée avant notre passage.

Restauration des pistes par commune

 Piste en culture ou prairie temporaire, l'agriculteur préférant alors généralement labourer après la fermeture pour semer une culture céréalière ou fourragère de type luzerne.

Piste refermée en attente du labour

Prairie en cours de restauration (N+1)

A. DURBECQ, 01/07/2020, Chorges (05)

Stockage

Les graines et le foin doivent être correctement séchées avant d'être stockées. Pour préserver la capacité germinative des semences, le stockage doit être effectué à l'abris de la lumière et de l'humidité, protégé des animaux et insectes susceptibles de manger les graines. Le temps de stockage doit être le plus court possible. Temps maximum : 1 à 2 ans (Scotton et al. 2012).

Préparation de la zone à restaurer avant semis

Avant le semis : Le substrat doit être meuble en surface au moment du semis. Si la terre est très compacte, il est possible de la décompacter juste avant le semis avec un passage de herse ou le godet d'une pelle mécanique.

En pratique : Si la remise en état après les travaux de terrassement est récente, le sol est encore meuble et donc opérationnel pour recevoir un semis de graine et/ou un transfert de foin. Il n'est donc pas nécessaire d'effectuer un décompactage. Cependant, un léger griffage (hersage) du sol est conseillé pour favoriser l'adhésion des graines au sol. Par ailleurs, s'il y a suffisamment de terre végétale, il n'y a pas besoin d'enrichir le sol avec du fumier, du compost ou de l'engrais. De manière générale, plus le milieu est en altitude, plus la végétation est adaptée à des substrats pauvres en matières organiques et nutriments.

Protocole de suivi de la restauration écologique

Localisation des zones suivies

La zone d'étude se situe dans la vallée de la Haute-Durance, dans le département des Hautes-Alpes, en France. Elle s'étend sur plus de 100 km, du Briançonnais (commune de L'Argentière-la-Bessée : 44°79'29''N ; 6°55'90''E) au Gapençais (commune de La-Bâtie-Neuve : 44°56'53''N ; 6°19'61''E) en passant par l'Embrunais (Figure 4). (b) Dans la zone à proximité non-impactée, dite de référence : 1 quadrat de 2 x 2 mètres et relever tous les recouvrements/espèces (uniquement la première année de suivi).

6.2.2. Sur la piste restaurée par transfert de foin (ligne P6 pylônes 38-39) (a) Dans la zone restaurée avec foin : 8 quadrats de 2 x 2 mètres et relever tous les recouvrements/espèces (b) Dans la zone à proximité non-impactée, dite de référence : 1 quadrat de 2 x 2 mètres et relever tous les recouvrements/espèces (ici à mutualiser avec le relevé de fleur de foin en continue du foin ; uniquement la première année de suivi).

CONCLUSION

Les mesures compensatoires mises en place afin de restaurer les prairies de Haute-Durance montrent, dès la première année de suivi, des résultats favorables à une reprise de la végétation par l'apport de graines via le foin ou graines récoltés localement. Les sites dégradés qui ont été restaurés en octobre 2019 présentaient un couvert végétal supérieur à 30% en juillet 2020, dont la présence d'espèces prairiales d'intérêt telles que des poacées ou des fabacées autochtones d'origine local. Même si l'analyse de ces deux groupes ne permet qu'une analyse approximative de la réussite des mesures de restauration, ce résultat est encourageant et représente une solution alternative au semis des mélanges commerciaux de fabacées et graminées qui sont souvent peu diversifiés et mal adaptés aux conditions locales. Le nombre d'espèces recensées dans les sites restaurés variait entre 18 et 40 espèces, représentant une richesse floristique moyenne équivalente à celle rencontrée sur les sites de référence. Bien que le suivis à n+1 présentent des résultats positifs, évaluer de façon certaine les effets écologiques des opérations de restauration par ensemencement nécessite un suivi à plus long terme (Klimbowska et al. 2007 ;Sengl et al. 2017 ;Bischoff et al. 2018 ;Albert et al. 2019 ;Shaw et al. 2020).

Relation entre le couvert végétal et l'érosion du sol

Tableau récapitulatif des pistes restaurées

Abstract

Large-scale ecological degradation of grasslands is increasing worldwide mainly due to land use changes and habitat destruction. The construction of high-voltage transmission lines in my study area, the Haute-Durance valley in the Southern French Alps, involves a degradation of grassland soil and plant communities along transitory access tracks and construction platforms. In these degraded ecosystems of the montane altitudinal belt, restoration is limited by a lack of available propagules and low seedling recruitment on stony and shallow soils characterised by summer drought. I focus on the restoration of species-rich dry to mesophilic grasslands covering a large part of the construction zone between 1000 and 1400 m. Most of these grasslands are Natura 2000 priority habitats, such as N6210 "Semi-natural dry grasslands of Festuco-Brometalia and scrubland facies on calcareous substrates" (EU directive habitat 92/43/EEC).

My thesis aims to better understand the factors limiting the restoration of mountain grasslands after soil disturbance, and to identify successful restoration techniques to compensate the degradation. Chapter 1 is focused on the identification of appropriate reference communities, which is a crucial step in ecological restoration. In order to understand the mechanisms favouring target species establishment, five degraded semi-natural grasslands were used to set up two different experiments, using a filter-based community assembly approach of community dynamics to analyse the restoration outcome. In the first one (Chapter 2), I examined whether soil preparation is required to improve soil conditions and to reduce competition of pre-existing vegetation, and whether traditional extensive grazing should be excluded in initial stages of grassland restoration. In the second experiment (Chapter 3), I focused on different techniques of propagule transfer increasingly used in mountain grasslands (brush-harvested propagules and hay transfers), and I analysed the potentially facilitative effect of dead (hay) and living (wheat) plant cover on the recruitment of transferred brush-harvested propagules, and on soil erosion. Finally, as community assembly is also highly influenced by dispersal that may lead to priority effects, I tested sequential sowing of different combinations of plant species (dominants vs. subordinates) (Chapter 4) and I analysed how they affect community assembly.

The results demonstrated that the analysis of regional plant-environment interactions provides a useful tool to identify reference communities or donor sites for seed transfer, particularly if donor sites are not available adjacent to degraded habitats. The restoration techniques applied indicated that the reduction of competition by soil preparation before seed addition of brush material had a positive effect on the transferred species recruitment and can thus clearly be recommended in my study system. Furthermore, I found that the weak negative effect of grazing may not always justify fencing costs.

Hay mulch favoured seedling recruitment of target species, but propagule transfer without mulch layer needed to be compensated by additional temporary plant cover, suggesting that restoration of mountain grasslands with shallow and stony soils clearly benefits from a facilitative effect of dead (hay) or living (wheat) vegetation cover. The chapter 4 provided evidence that plant community assembly is influenced by the order of arrival but highlighted strong response variations between species suggesting high species-specific priority effects. The strength of priority effects probably varies according to the niche characteristics of different species and may influence (negatively or positively) the late-arriving species establishment.