Florence Volaire
email: florence.volaire@cefe.cnrs.fr

Karim Barkaoui

David Grémillet

Guillaume Charrier

Olivier Dangles

Laurent J Lamarque

Nicolas Martin-Stpaul

Isabelle Chuine

Is a seasonally reduced growth potential a convergent strategy to survive drought and frost in plants?

Keywords: Dehydration tolerance, dormancy, growth-stress survival trade-off, embolism resistance, mortality threshold, phenology, fast-slow economics spectrum, seasonality, strategy, drought survival, frost survival

Background Plants have adapted to survive seasonal life-threatening frost and drought. However, the timing and frequency of such events are impacted by climate change, jeopardizing plant survival. Understanding better the strategies of survival to dehydration stress is therefore timely and can be enhanced by the cross-fertilization of research between disciplines (ecology, physiology), models (woody, herbaceous species) and types of stress (drought, frost).

• Scope We build upon the 'growth-stress survival' trade-off, which underpins the identification of global plant strategies across environments along a 'fast-slow' economics spectrum. Although phenological adaptations such as dormancy are crucial to survive stress, plant global strategies along the fast-slow economic spectrum rarely integrate growth variations across seasons. We argue that the growth-stress survival trade-off can be a useful framework to identify convergent plant ecophysiological strategies to survive both frost and drought. We review evidence that reduced physiological activity, embolism resistance and dehydration tolerance of meristematic tissues are interdependent strategies that determine thresholds of mortality among plants under severe frost and drought. We show that complete dormancy, i.e. programmed growth cessation, before stress occurrence, minimizes water flows and maximizes dehydration tolerance during seasonal life-threatening stresses. We propose that incomplete dormancy, i.e. the programmed reduction of growth potential during the harshest seasons, could be an overlooked but major adaptation across plants. Quantifying stress survival in a range of non-dormant versus winter-or summer-dormant plants, should reveal to what extent incomplete to complete dormancy could represent a proxy for dehydration tolerance and stress survival.

• Conclusions Our review of the strategies involved in dehydration stress survival suggests that winter and summer dormancy are insufficiently acknowledged as plant ecological strategies. Incorporating a seasonal fastslow economics spectrum into global plant strategies improves our understanding of plant resilience to seasonal stress and refines our prevision of plant adaptation to extreme climatic events.

INTRODUCTION

In their natural environments, living organisms have adapted to biotic and abiotic stresses that impact their growth, reproduction, survival and overall fitness [START_REF] Grime | Plant strategies, vegetation processes, and ecosystem properties[END_REF]. However, the current and predicted climate change is likely to expose organisms to more intense and less predictable heat, drought, frost and other stresses, thus jeopardizing their survival [START_REF] Ummenhofer | Extreme weather and climate events with ecological relevance: a review[END_REF]. Consequently, many organisms already experience or will soon experience extreme environmental conditions outside their current tolerance range [START_REF] Botero | Evolutionary tipping points in the capacity to adapt to environmental change[END_REF]. It is therefore crucial to improve our knowledge of the stress survival strategies of plants to anticipate and manage the impacts of climate change on ecosystems. Comparative approaches can help identify convergent research avenues on processes and tradeoffs underlying stress survival [START_REF] Taborsky | Towards an evolutionary theory of stress responses[END_REF].

Firstly, a major and convergent ecological pattern in the plant kingdom is the generally observed negative relationship between growth and stress survival (see Glossary,Box 1). This relationship has been well documented for plants facing various abiotic stresses, including drought and frost stress [START_REF] Grime | Evidence for existence of three primary strategies in plants and its relevance to ecological and evolutionary theory[END_REF][START_REF] Grubb | A reassessment of the strategies of plants which cope with shortages of resources[END_REF]. The extent to which a species can combine growth and resilience to stress remains an ongoing question that merits further investigation, especially under climate change [START_REF] Doerner | Extreme environments: crucibles of potent abiotic stress tolerance[END_REF]. We here discuss the relationship between stress survival and growth, which is usually assessed as the maximum annual growth potential in optimal conditions [START_REF] Perez-Harguindeguy | New handbook for standardised measurement of plant functional traits worldwide[END_REF]. We show the generality of this relationship across various model species to understand plant survival strategies. Furthermore, we propose extending the scope of these global strategies by including phenological adaptations allowing plants to tolerate seasonal stress, such as seasonal dormancy. Doing so, growth should also be assessed, but not only as the maximum annual growth potential and not as the actual plant growth (or growth reduction) during the most stressful season [START_REF] Grubb | A reassessment of the strategies of plants which cope with shortages of resources[END_REF]. We propose to assess growth as the reduced growth potential (and not absolute growth) during the same most stressful season (see Box 1) since we demonstrate that the anticipated reduction in seasonal growth potential (relative to the maximum annual growth potential) may be a key strategy for survival of dehydrative stress.

Secondly, as drought and frost are abiotic stresses, both of which decrease the availability of water to plant cells [START_REF] Pearce | Plant freezing and damage[END_REF][START_REF] Verslues | Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status[END_REF], the strategies of dehydration avoidance and tolerance of plants subjected to either severe drought or freezing involve convergent physiological adaptations [START_REF] Verslues | Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status[END_REF][START_REF] Lata | Role of DREBs in regulation of abiotic stress responses in plants[END_REF][START_REF] Nakashima | The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat[END_REF]. Hence, solute accumulations, osmotic adjustment and modification of the properties of cell walls help plants to tolerate the dehydration induced by low water potential. These protective mechanisms for preventing or repairing cell damage are observed under various dehydrative stresses and for many species [START_REF] Close | Dehydrins: a commonality in the response of plants to dehydration and low temperature[END_REF][START_REF] Hare | Dissecting the roles of osmolyte accumulation during stress[END_REF][START_REF] Krasensky | Drought, salt, and temperature stressinduced metabolic rearrangements and regulatory networks[END_REF]. Thus, a plant facing a primary stress (e.g. drought) can acquire the benefit of the ability to face a secondary abiotic or biotic stress (e.g. frost), i.e. have cross-stress tolerance [START_REF] Liu | Priming crops for the future: rewiring stress memory[END_REF]. Cross-stress tolerance has been recently described in trees [START_REF] Charrier | Interaction of drought and frost in tree ecophysiology: rethinking the timing of risks[END_REF], crops [START_REF] Pushpavalli | Cross-tolerance for drought, heat and salinity stresses in chickpea (Cicer arietinum L.)[END_REF], grasses (Kong andHenry, 2016, 2019) and some resurrection plant species with double tolerance, able to recover after complete desiccation and be freezing-tolerant [START_REF] Fernández-Marín | Born to revive: molecular and physiological mechanisms of double tolerance in a paleotropical and resurrection plant[END_REF]. Consequently, in this review we highlight that the relationship between growth and stress survival can be better understood when considering the convergent physiological mechanisms ensuring plant survival, namely the strategies related to dehydration tolerance under severe summer drought and winter frost. These mechanisms should be common among vascular plant species living in climates where severe summer drought and/ or winter frost are the most limiting factors and life-threatening at given seasons. We focus here on seasonal dormancy, which minimizes water flows and maximizes dehydration tolerance in many plant species, both in winter and summer.

At the crossroads of ecological and physiological approaches, this viewpoint argues that seasonal dormancy is a key strategy illustrating the relationship between growth potential and stress survival. (Complete) dormancy is a programmed inability to grow (Box 1). Incomplete dormancy (Box 1) can be defined as a programmed reduction in growth potential during the most stressful season [START_REF] Volaire | Summer dormancy in perennial temperate grasses[END_REF]. We argue that this little-explored phenological adaptation could underpin a continuum of dehydration tolerance and resilience ability of plants living in bioclimatic areas with severe seasonal frost or drought. Moreover, assessing the intra-and inter-specific variability of incomplete dormancy could highlight whether and which plant species anticipate periodic seasonal stresses.

THE GROWTH-STRESS SURVIVAL TRADE-OFF UNDERPINS THE FAST-SLOW CONTINUUM OF PLANT STRATEGIES

A generalized fast-slow continuum should include seasonality

The trade-off between growth, or more generally any physiological activity related to fitness (growth, development, reproduction) and stress survival, usually and hereafter called the 'growth-survival trade-off', is a central tenet in ecology across all types of organisms [START_REF] Grime | Vegetation classification by reference to strategies[END_REF][START_REF] Sibly | A life-cycle theory of responses to stress[END_REF][START_REF] Turbill | Hibernation is associated with increased survival and the evolution of slow life histories among mammals[END_REF]. This trade-off underpins the identification of global plant ecological strategies along a plant economics spectrum, reflecting a gradient of resource use [START_REF] Reich | The world-wide 'fast-slow' plant economics spectrum: a traits manifesto[END_REF]. This gradient ranges from slow growth strategies associated with a high survival capacity and energy/carbon conservation in plants of species usually living in low-resource environments, to fast growth strategies associated with a high resource acquisition capacity but at the expense of poor stress survival, in plants of species usually living in richer environments [START_REF] Grime | Plant strategies, vegetation processes, and ecosystem properties[END_REF][START_REF] Wright | Functional traits and the growth-mortality trade-off in tropical trees[END_REF]. Accordingly, the survival probability of any species/population should be predictable by its position along the fast-slow plant economics spectrum in a given environment [START_REF] Reich | The world-wide 'fast-slow' plant economics spectrum: a traits manifesto[END_REF].

Here we illustrate that this fast-slow continuum is underpinned by the growth-survival trade-off. At the fast end of the continuum, highly productive and resource-acquisitive species (Fig. 1A), such as annual crop species [START_REF] Claeys | The agony of choice: How plants balance growth and survival under water-limiting conditions[END_REF], are usually dehydration-sensitive and poorly adapted to severe drought and frost stresses. By contrast, species with lower growth rates are more dehydration-tolerant and survive notably better in response to severe stress. The slowest end of the spectrum (Fig. 1A) can be exemplified by resurrection plants, which are among the most resource-conservative and slow-growing herbaceous species, with long life spans [START_REF] Tebele | Two decades of desiccation biology: a systematic review of the best studied angiosperm resurrection plants[END_REF]. They can survive months or even years of desiccation, i.e. <30% water content while maintaining tissue moisture levels in equilibrium with ambient atmospheric humidity [START_REF] Zhang | Molecular responses to dehydration and desiccation in desiccation-tolerant angiosperm plants[END_REF], illustrating the negative relationship between desiccation tolerance and growth [START_REF] Alpert | Constraints of tolerance: why are desiccation-tolerant organisms so small or rare?[END_REF].

Ecological strategies of the fast-slow plant economics spectrum have been described based on integrative plant functional traits [START_REF] Wright | The worldwide leaf economics spectrum[END_REF] that are usually measured under the expected least limiting conditions. Doing so, traits reflect the strategies underlying the maximum annual growth potential (e.g. usually measured during the peak of vegetation growth), a key component of plant fitness, and allow reliable comparisons between species/populations living in environments with contrasting conditions and stress intensities. However, these conditions may poorly describe the temporal variations and adjustments in plant responses to seasonally fluctuating conditions [START_REF] Volaire | What do you mean 'functional' in ecology? Patterns versus processes[END_REF]. Yet most species show seasonal activity and alternate periods of active growth (resource acquisition) and periods of restricted growth (resource conservation) whenever the environmental conditions are limiting. For example, some resurrection plants are desiccation-sensitive at certain times while desiccation-tolerant at others [START_REF] Farrant | Desiccation tolerance in the vegetative tissues of the fern Mohria caffrorum is seasonally regulated[END_REF]. Likewise, some plant species can reduce their metabolic rates, becoming quiescent under unpredictable stress while recovering when the conditions become favourable again [START_REF] Velappan | Cell cycle arrest in plants: what distinguishes quiescence, dormancy and differentiated G1?[END_REF]. Moreover, species with a seasonal dormancy periodically anticipate the incoming stress and 'know when not to grow' [START_REF] Bielenberg | Knowing when not to grow[END_REF], although at given seasons only (Fig. 1B). This raises the following question: is the reduction of seasonal growth potential during the most life-threatening seasons (Box 1, Fig. 1B) as, or even more, meaningful than the maximum annual growth potential to describe plant strategy and overall adaptation to seasonal stress? In this paper we propose that seasonal dormancy, whether complete or incomplete, be considered a key strategy to refine the fast-slow continuum economics spectrum by adding a temporal dimension to it [START_REF] Grubb | A reassessment of the strategies of plants which cope with shortages of resources[END_REF], and we suggest a methodology to do so.

Seasonal dormancy is a key strategy to refine the fast-slow continuum

Dormancy is an adaptive response, enabling survival during seasons when environmental conditions are most life-threatening [START_REF] Vegis | Dormancy in higher plants[END_REF], such as winter in extratropical regions and summer in the Mediterranean and semi-arid regions. Regardless of the season, complete dormancy is associated with a loss of access to growth-promoting signals, generally induced by photoperiodic and thermal cues, resulting in decreased metabolic and cellular activities in anticipation of forthcoming stresses such as frost or drought [START_REF] Horvath | Knowing when to grow: signals regulating bud dormancy[END_REF][START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF]. Dormancy can be regarded as a prolonged 'gearing down' strategy [START_REF] Grubb | A reassessment of the strategies of plants which cope with shortages of resources[END_REF] without possible rapid regrowth when conditions become briefly favourable to physiological activity. Thus, dormancy release requires a prolonged exposure to particular environmental conditions.

Notwithstanding the diversity of adaptive mechanisms to cope with frost and drought, which both generate tissue dehydration [START_REF] Verslues | Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status[END_REF][START_REF] Kong | Does cross-acclimation between drought and freezing stress persist over ecologically relevant time spans? A test using the grass Poa pratensis[END_REF], we propose a unique framework to describe them (Fig. 2). Among the strategies of dehydration avoidance and tolerance shared by most vascular plant species, complete dormancy facilitates survival of the highest dehydration risk, either during winter or summer due to a programmed inability of meristems to grow, despite possible suitable transitionary environmental conditions [START_REF] Lang | Preface[END_REF].

If winter dormancy is a widespread strategy in extratropical woody and herbaceous species, summer dormancy is a common strategy among herbaceous perennials in Mediterranean and semi-arid areas [START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF]. In tropical tree species, slow-growing habits [START_REF] Oliveira | Linking plant hydraulics and the fast-slow continuum to understand resilience to drought in tropical ecosystems[END_REF] and drought deciduousness [START_REF] Vargas | Beyond leaf habit: generalities in plant function across 97 tropical dry forest tree species[END_REF] confer a high resilience to drought, suggesting there may be some level of incomplete dormancy during the stress period. Interestingly, summer dormancy has rarely been identified in woody species [START_REF] Lei | Ecotypic variation in summer dormancy of blackbrush (Coleogyne ramosissima) and its ecological significance[END_REF], contrary to winter dormancy. Both summer and winter dormancy are yet also common in many animal species [START_REF] Wilsterman | A unifying, ecophysiological framework for animal dormancy[END_REF], suggesting that dormancy may be a strategy shared by most living organisms to survive stress.

Complete dormancy is the strategy exhibited, for instance, by herbaceous plants that rely on underground perennating organs (tubers, rhizomes, bulbs, …) to survive periods of unfavourable conditions [START_REF] Lubbe | Winter belowground: changing winters and the perennating organs of herbaceous plants[END_REF]. However, dormancy can be regarded in many cases as a cryptic strategy (Albert et al., 2019) that may be difficult to detect and monitor. So far, the only method is to measure meristem growth under favourable conditions, i.e. under additional summer water supply for summer dormancy [START_REF] Laude | The nature of summer dormancy in perennial grasses[END_REF] and high temperature for winter dormancy [START_REF] Campoy | Dormancy in temperate fruit trees in a global warming context: a review[END_REF]. Following this method in experimental conditions, recent studies [START_REF] Bristiel | Is there a constant trade-off between stress tolerance and growth potential in the perennial grass Dactylis glomerata?[END_REF][START_REF] Keep | To grow or survive: which are the strategies of a perennial grass to face severe seasonal stress?[END_REF]) demonstrated a strong continuous trade-off between seasonal growth potential and stress survival in European populations of Dactylis glomerata and Lolium perenne (Fig. 3). Mediterranean populations of these two perennial grasses exhibited the highest and lowest growth potential in winter and summer, respectively. Consequently, they had a low and high survival rate to winter frost and summer drought, respectively (Fig. 3). The opposite pattern was observed for populations of these species originating from northern European areas. Considering that incomplete dormancy should be mainly cryptic, it is possible that programmed seasonal reduction of growth potential could have remained largely unnoticed so far (Fig. 1B) and be considered as quiescence. We propose here that this endogenously controlled and anticipated seasonal reduction of growth potential, i.e. levels of incomplete dormancy (Box 1) could be included as a modality of the dormancy strategy (Fig. 2). Hence, we propose in the following that, as for species with complete dormancy, the relative seasonal growth potential, i.e. the reduced growth potential of incompletely dormant species measured during the usually life-threatening season (Fig. 1B) relative to the maximum annual growth potential, should be negatively correlated with dehydration stress survival.

PHYSIOLOGY OF THE 'GROWTH-STRESS SURVIVAL' TRADE-OFF

In the light of ecological strategies, it is logical that growing slowly, either permanently in a chronically harsh environment or as a response to an anticipated or accidental threatening stress, is the general cost to pay for ensuring survival. It nevertheless requires maintaining core physiological activity, albeit at a low rate, to prevent death caused by a shortage of energy (carbon starvation) and/or water supply (dehydration). The downregulation of metabolic activity contributes to adjusting the metabolism to cope with stress and reach basal metabolic rates compatible with self-maintenance while saving carbon reserves for 'better days' [START_REF] Asami | Saving for a rainy day: control of energy needs in resurrection plants[END_REF]. Under extended periods of drought-impairing photosynthesis, plants may indeed face a carbon reserve depletion, known as carbon starvation [START_REF] Mcdowell | Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF]. However, the role of carbon supply, notably to minimize cellular dehydration by osmotic adjustment, strongly interacts with plant water content and flows since drought-associated mortality originates from a whole-plant water-relation backbone [START_REF] Mcdowell | Mechanisms of woodyplant mortality under rising drought, CO2 and vapour pressure deficit[END_REF].

The understanding of plant survival to drought and frost has therefore recently focused on hydraulic adaptations, for which recent advances raise comparative questions across multiple plant growth forms and types of stress. Here we review the physiological thresholds associated with survival versus mortality in a large range of species under severe drought and frost.

We demonstrate that these adaptations may be better understood when considering seasonal growth regulations at the whole plant level, exemplified by the dormancy strategy.

Defining physiological thresholds for plant survival to frost and drought

With the exception of resurrection plants, which can survive desiccation (Fig. 2), maintaining water supply is essential for most plant species. Under highly negative water potential, water flow may be interrupted by the embolization of the xylem, i.e. gas formation and spreading within and across xylem conducting elements. Various types of stress, including drought and frost, can induce the breakage of the water column by embolism, interrupting water transport and potentially affecting the viability of above-ground distal organs during severe stress [START_REF] Charrier | Interaction of drought and frost in tree ecophysiology: rethinking the timing of risks[END_REF]. Hence, embolism resistance, and therefore water transport capacity, has long been viewed as a key trait for stress survival (Fig. 4), although the relative importance of this strategy depends on plant growth forms. The high levels of embolism (>50 or 90 % according to studies) that can be reached during extreme drought and heat events can result in a lack of recovery of plant functioning and/or meristem reactivation once the stress is released [START_REF] Urli | Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees[END_REF][START_REF] Hammond | Dying on time: traits influencing the dynamics of tree mortality risk from drought[END_REF]. Embolism resistance is often estimated by water potential leading to 50 or 90 % embolism (P 50 or P 90 ). Plants present a large variation in P 50 , from -1 MPa for hygrophilous species such as Populus to almost -19 MPa for Callitris tuberculata, which is adapted to extreme droughts and reaches the physical limit of water transport [START_REF] Larter | Aridity drove the evolution of extreme embolism resistance and the radiation of conifer genus Callitris[END_REF]. The range of variation of P 50 within temperate grasses (-0.7 to -7.5 MPa) is similar to that found in woody angiosperm species from the same biogeographical areas [START_REF] Lens | Herbaceous angiosperms are not more vulnerable to drought-induced embolism than angiosperm trees[END_REF].

The resistance to embolism is enhanced by many strategies allowing the control of water potential decline [START_REF] Choat | Triggers of tree mortality under drought[END_REF], such as the maintenance of sufficient water uptake 4. Main processes determining plant survival or death from the onset to the cessation of a drought or cold period. Seasonal dormancy triggers the adaptive processes before the onset of the stress (or even with no subsequent stress occurring), therefore inducing the dehydration of some key plant tissues and reinforcing their dehydration tolerance. The reduction of growth and metabolic activity is the first process associated with the induction of dormancy and/or dehydration stress. As dehydration stress increases, the minimum recovery threshold or no-return point (yellow star) depends on plant maintenance of water transport capacity and dehydration tolerance in the surviving organs, which are often meristematic tissues. This figure is relevant for major plant species with some level of dehydration tolerance but does not apply to desiccation-tolerant plant species. [START_REF] Martin-Stpaul | Plant resistance to drought depends on timely stomatal closure[END_REF][START_REF] Duursma | On the minimum leaf conductance: its role in models of plant water use, and ecological and environmental controls[END_REF] or hydraulic vulnerability segmentation [START_REF] Charrier | Evidence for hydraulic vulnerability segmentation and lack of xylem refilling under tension[END_REF]. These adaptations align with the growth-stress survival trade-off since the reduction of metabolic activity and leaf shedding [START_REF] Martin-Stpaul | The temporal response to drought in a Mediterranean evergreen tree: comparing a regional precipitation gradient and a throughfall exclusion experiment[END_REF][START_REF] Charrier | Evidence for hydraulic vulnerability segmentation and lack of xylem refilling under tension[END_REF] contribute to minimizing growth and water flows and hence reduce the risk of embolism [START_REF] Oliveira | Linking plant hydraulics and the fast-slow continuum to understand resilience to drought in tropical ecosystems[END_REF][START_REF] Vargas | Beyond leaf habit: generalities in plant function across 97 tropical dry forest tree species[END_REF].

Water transport is also affected by frost stress due to freezethaw cycles. In this case, embolism is generated by propagating air bubbles entrapped in an ice lattice during sap thawing. Air-water contact surfaces are likely to be displaced by the extremely low water potential at the liquid-solid interface, ~-1 MPa below the freezing point [START_REF] Hansen | Evidence for ideal and non-ideal equilibrium freezing of leaf water in frosthardy ivy (Hedera helix) and winter barley (Hordeum vulgare)[END_REF]. Vulnerability to freeze-thaw embolism is generally correlated with the mean diameter of the xylem conduits, with wider vessels containing more dissolved gas that would form larger bubbles on freezing being the most vulnerable to embolism on thawing [START_REF] Charrier | Freeze-thaw stress: effects of temperature on hydraulic conductivity and ultrasonic activity in ten woody angiosperms[END_REF]. Trade-offs between freezing tolerance and growth rate have been reported [START_REF] Koehler | Evidence for a freezing tolerance-growth rate trade-off in the live oaks (Quercus series Virentes) across the tropical-temperate divide[END_REF][START_REF] Yin | A trade-off between growth and hydraulic resilience against freezing leads to divergent adaptations among temperate tree species[END_REF] and are notably illustrated in the case of dormant overwintering plants with low metabolic activity and therefore minimal embolism (see next section).

Although embolism occurs under various stresses (drought, heat, cold), empirical data do not support a universal relationship between the loss of water supply capacity and stress survival, e.g. a high embolism can be observed without entailing mortality both in plants under extreme stress [START_REF] Hammond | Dying on time: traits influencing the dynamics of tree mortality risk from drought[END_REF] and in overwintering plants [START_REF] Mayr | Winter at the alpine timberline. Why does embolism occur in Norway spruce but not in stone pine?[END_REF]. Interestingly, water pools in plant tissues are a more relevant indicator of drought-induced mortality than water transport capacity [START_REF] Sapes | Plant water content integrates hydraulics and carbon depletion to predict drought-induced seedling mortality[END_REF][START_REF] Lamacque | Drought-induced mortality: branch diameter variation reveals a point of no recovery in lavender species[END_REF][START_REF] Mantova | The interplay of hydraulic failure and cell vitality explains tree capacity to recover from drought[END_REF]. It is increasingly recognized that the dehydration tolerance of surviving organs under drought, i.e. the meristematic tissues such as shoot apical meristem buds, underground buds of perennating organs or vascular cambium in trees [START_REF] Hammond | A thin line between life and death: radial sap flux failure signals trajectory to tree mortality[END_REF][START_REF] Mantova | The interplay of hydraulic failure and cell vitality explains tree capacity to recover from drought[END_REF], could represent a common and crucial factor to ensure survival across plants subjected to severe drought or frost. In particular, the requirements for water transport and the level of water storage is thus expected to be minimal for dormant perennial species, whether herbaceous [START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF] or woody [START_REF] Wolfe | Does leaf shedding protect stems from cavitation during seasonal droughts? A test of the hydraulic fuse hypothesis[END_REF], in which most leaves are shed before the stress. Overall, water transport capacity and cellular dehydration tolerance may therefore interact to define the 'no return' point (Fig. 4). Across organisms and types of dehydration stress, state variables in surviving organs (e.g. water and solute contents) and flow variables (e.g. hydraulic conductivity) should be complementary to define minimal physiological thresholds according to species, population and dormancy.

Dormancy minimizes water flows and maximizes dehydration tolerance

Dehydration can be triggered by a lack of water and any factor disrupting water transport, such as vessel embolism caused by frost. Dormancy plays an important role in tolerance to dehydration caused by drought or frost [START_REF] Vitasse | The interaction between freezing tolerance and phenology in temperate deciduous trees[END_REF][START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF]. Most dormant organs, i.e. seeds [START_REF] Kleinwächter | Dehydrin expression in seeds: an issue of maturation drying[END_REF], buds in winter [START_REF] Chuine | Process-based models of phenology for plants and animals[END_REF] or buds in summer [START_REF] Volaire | Drought survival and dehydration tolerance in Dactylis glomerata and Poa bulbosa[END_REF], exhibit minimal developmental and metabolic activities involving the upregulation of many compounds that protect the cells against dehydration, such as heat-shock proteins, late embryogenesis abundant (LEA) proteins, and some soluble carbohydrates. Dormancy can also be regarded as a prerequisite to avoid embolism [START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. If living cells of meristems, phloem and xylem parenchyma are prone to frost damage, xylem conduits are prone to embolism triggered by drought and frost [START_REF] Cochard | Xylem dysfunction in Quercus -vessel sizes, tyloses, cavitation and seasonal changes in embolism[END_REF][START_REF] Sperry | Xylem embolism in response to freeze-thaw cycles and water stress in ring-porous, diffuse-porous, and conifer species[END_REF]. Therefore, reduced cellular and metabolic activities during stressful seasons translate into reduced sap flows, water savings, and thus reduced tension, thereby limiting embolism risks. Both buds and the vascular system undergo major transformations during the life-threatening season, which are necessary to endure dehydration during these seasons [START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. Noticeably, all communication pathways undergo transformation to limit and tolerate dehydration [START_REF] Wisniewski | Adaptive mechanisms of freeze avoidance in plants: a brief update[END_REF]. The symplastic pathway in all living cells, including sieve elements of the phloem, is shut down by the accumulation of a β-glucan called callose in the plasmodesmata. This shutdown decreases the concentrations of water and also oxygen, nutrients and other molecules (e.g. hormones, transcription factors), and is responsible for the decreased metabolic and physiological activity [START_REF] Rinne | Plasmodesmata at the crossroads between development, dormancy, and defense[END_REF]. Water transport in the apoplast is limited by small intracellular spaces [START_REF] Quamme | Anatomical features facilitating supercooling of the flower within the dormant peach flower bud[END_REF]. Expression of aquaporins is downregulated, minimizing water movement between the symplast and the apoplast [START_REF] Yooyongwech | Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy[END_REF]. Finally, and most importantly, water transport in the xylem is disrupted at the junction between the bud and the twig, which minimizes any exchange through the vascular tissue and/or embolism spread into the bud [START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF].

These protective mechanisms are likely to play a role in all dormant plants, and should be particularly investigated in summer-dormant herbaceous species, which rely on meristems protected by layers of senescent sheath bases, usually at the ground surface or below, with minimal water flows to facilitate survival of severe heat and drought [START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF]. Indeed, we propose that research conducted on winter dormancy and frost survival could guide that on summer dormancy and drought survival, notably in the case of incomplete dormancy.

ASSESSING RELATIVE SEASONAL GROWTH POTENTIAL TO BETTER UNDERSTAND STRESS SURVIVAL

In the context of increasingly frequent extreme climatic events, including drought and frost, we urge more integrative research between ecological and physiological approaches to better understand major commonalities among plants that allow them to face a range of dehydration stresses. In particular, we argue that research on ecological strategies should account for not only spatial adaptations across environments but also seasonal adaptations within environments over seasons. We propose to test the scope of a generalized fast-slow continuum at the seasonal level. To do so, the main challenge is to evaluate/test the generality of the correlation between reduced seasonal growth potential and dehydration tolerance of plants under severe seasonal stress.

Assessing the occurrence of seasonal dormancy is a methodological challenge that requires measuring the decrease in potential metabolic activity during the expected period of dormancy compared with a stage when the plants are not dormant. We propose comparing the seasonal growth potential measured during the most life-threatening season and the maximum annual growth potential measured during the most favourable season in order to calculate an index of relative seasonal reduction of growth potential (Box 1, Fig. 5). Doing so implies relaxing the stress (by water supply or during a wet summer, by experimental warming or during mild winters) to enable measurement of plant growth potential when the conditions are usually highly stressful and limiting for growth (Fig. 5). The growth potential can be measured at the whole-plant or organ level (production of above-ground biomass, leaf extension, increase in plant size, biovolume, or total leaf area, etc.), depending on the species and the context (experimental garden, field, controlled conditions) and during the relevant period of the growing season. Moreover, stress survival (plant mortality, loss of plant cover) should be measured under harsh environmental conditions. Species or populations originating from climates with life-threatening seasons, having the lowest growth potential (in summer or winter depending on the least favourable season), relative to their maximum annual growth potential during the most favourable season (usually spring), should exhibit the greatest dehydration tolerance under severe seasonal dehydration stress (Fig. 5). Measuring seasonal levels of complete and incomplete dormancy requires growing plants in environments suitable for dormancy induction, notably regarding temperature and daylength [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms[END_REF][START_REF] Maurya | Photoperiod-and temperature-mediated control of growth cessation and dormancy in trees: a molecular perspective[END_REF]. For instance, complete summer dormancy of the perennial grass Dactylis glomerata could only be induced (and thus revealed) when winter and spring temperatures reached a defined threshold [START_REF] Shihan | Induction and potential role of summer dormancy to enhance persistence of perennial grasses under warmer climates[END_REF] while daylength was not limiting throughout most of Europe for summer dormancy expression. Ideally, growth patterns should be assessed at the provenance sites of the species/population and measured on plants artificially heated (winter) or irrigated (summer). Common gardens comparing a range of species or populations of various origins could be useful to analyse the variation of seasonal growth potential and dormancy expression along environmental gradients representative of the ecological niche of the species [START_REF] Keep | To grow or survive: which are the strategies of a perennial grass to face severe seasonal stress?[END_REF].

CONCLUSIONS

Surviving stress implies a drastic reduction of physiological activity. Seasonal dormancy represents one of the most efficient strategies to survive stress, especially dehydration stress occurring at the same period every year. By comparing [START_REF] Volaire | Plant traits and functional types to characterise drought survival of pluri-specific perennial herbaceous swards in Mediterranean areas[END_REF][START_REF] Bristiel | Is there a constant trade-off between stress tolerance and growth potential in the perennial grass Dactylis glomerata?[END_REF][START_REF] Keep | To grow or survive: which are the strategies of a perennial grass to face severe seasonal stress?[END_REF]. Species or populations originating from climates with life-threatening seasons, having the lowest growth potential (in summer or winter depending on the least favourable season), relative to their maximum annual growth potential during the most favourable season (usually spring), should exhibit the greatest dehydration tolerance under severe seasonal dehydration stress. Dehydration tolerance should be determined by the minimum water content thresholds in meristematic tissues and water transport capacity (embolism resistance), allowing plant survival and growth recovery after stress release.

ecological and physiological approaches, this viewpoint underlines a unique framework of common strategies for herbaceous and woody species under winter frost or/and summer drought, and thus raises three major points. Firstly, we suggest that the seasonally reduced growth potential represents an overlooked adaptation to stress during the harshest seasons. It is worth testing to what extent seasonal growth reduction represents a relevant proxy of the level of plant survival to dehydration stress. The following research questions are still open: Which species/populations show a clear and anticipated reduction or cessation of growth potential? Which level of growth potential reduction and survival can they reach under extreme stresses? Is the duration of programmed dormancy across species positively correlated with greater environmental seasonality, as found within the animal kingdom [START_REF] Wilsterman | A unifying, ecophysiological framework for animal dormancy[END_REF]? Do the thresholds of embolism and dehydration of meristematic tissues triggering plant mortality vary with the depth of dormancy? These questions have rarely been addressed, particularly for summer dormancy, which has been much less explored than winter dormancy.

Secondly, we propose that investigating the relationship between the global and the seasonal fast-slow economics spectrum of plants could reveal the contribution of phenological events to ecological plant strategies. The global and seasonal fast-slow continuums are expected to align when plants originate from environments with low seasonality or constant environmental limiting factors. Conversely, the seasonal fast-slow continuum may provide a fruitful and complementary characterization of within-environment strategies for plants undergoing contrasting seasonal dehydration stress.

Finally, it is worth questioning whether dormancy strategies can still protect organisms from lethal stress in regions where climatic conditions move away from their climatic niche. Indeed, climate change makes the timing, frequency and intensity of stressful conditions less predictable than before and cues triggering dormancy less reliable, e.g. shifts in experienced photoperiod due to temporal and geographic species shifts [START_REF] Ettinger | Spatial and temporal shifts in photoperiod with climate change[END_REF]. The timing of dormancy is cued by air temperature and photoperiod signals that may no longer align with temperatures as climate change increases [START_REF] Lubbe | Winter belowground: changing winters and the perennating organs of herbaceous plants[END_REF]. The possible modifications of dormancy expression and dormancy timing that have been already observed [START_REF] Asse | Warmer winters reduce the advance of tree spring phenology induced by warmer springs in the Alps[END_REF][START_REF] Beil | Late to bed, late to rise-warmer autumn temperatures delay spring phenology by delaying dormancy[END_REF] may affect the outcome of the trade-offs between physiological activity and stress survival probability. Moreover, out-of-season severe stress, e.g. late spring freezes [START_REF] Chamberlain | Climate change reshapes the drivers of false spring risk across European trees[END_REF], also represent an increasing risk for plant survival. Climate change can also affect overwintering organs by altering snow cover depth and duration, thus modifying the exposure of plants to frost and then the duration of winter dormancy [START_REF] Lubbe | The role of perennation traits in plant community soil frost stress responses[END_REF][START_REF] Lubbe | Winter belowground: changing winters and the perennating organs of herbaceous plants[END_REF].

Considering the more frequent occurrence and intensity of droughts in many areas, summer dormancy may become an increasingly valuable adaptive strategy [START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF][START_REF] Shihan | Induction and potential role of summer dormancy to enhance persistence of perennial grasses under warmer climates[END_REF]. Therefore, it is necessary to foster transdisciplinary collaboration to enhance the cross-fertilization of research questions. Investigating common strategies and processes involved in dehydration stress survival, along with genetical and evolutionary approaches, could help in understanding better plant resilience under severe seasonal abiotic stress and hence predicting more efficiently the effects of climate change from organism to ecosystem scales.

BOX 1-GLOSSARY

Stress survival: Ability to survive after growth cessation due to an environmental factor severely restricting plant functioning (for the considered species and population), e.g. extreme and prolonged drought or frost.

Stress resistance: Maintenance of plant growth under an environmental factor moderately restricting plant functioning (for the considered species and population), e.g. moderate drought or cold.

Dehydration tolerance: Strategy allowing plants to tolerate low tissue level of dehydration (>30% water).

Desiccation tolerance: Strategy allowing plants to tolerate leaf water content equal to ambient atmospheric humidity and revive upon rehydration after long periods, e.g. resurrection plants.

Complete dormancy (sensu stricto dormancy): Endogenous controlled strategy that stops meristem activity to render it insensitive to growth-promoting signals in order to enhance plant survival during seasons when environmental conditions are most life-threatening.

Incomplete dormancy (sensu lato dormancy): Endogenous controlled strategy that reduces meristem activity to render it less sensitive to growth-promoting signals in order to enhance plant survival during seasons when environmental conditions are most life-threatening.

Growth potential: Growth rate, relative growth rate, or biomass production under optimum environmental conditions (without environmental stress).

Maximum annual growth potential: Growth potential during the most favourable season (at the 'peak of vegetation'), usually under no environmental stress.

Seasonal growth potential: Growth potential during the most life-threatening seasons (winter and/or summer) when the stress is experimentally relieved to enable measurement of growth potential during a season when the conditions are usually stressful and limiting for growth.

Relative seasonal growth potential: The difference between maximum annual growth potential and seasonal growth potential indexed on the maximum annual growth potential. It measures the relative programmed reduction of growth potential during the harshest season (incomplete or complete dormancy). This dormancy is hypothesized in this paper to be correlated with dehydration tolerance and stress survival in summer and/ or in winter, depending on the environment.

Fig. 1 .

 1 Fig. 1. The growth-stress survival trade-off (blue lines) and the fast-slow continuum of strategies in plant species-adapted from (Sibly and Calow, 1989): (A) exemplified by dehydration-sensitive, dehydration-tolerant and desiccation-tolerant species/populations along a global fast-slow continuum irrespective of seasonality but usually measured during the most favourable season; and (B) for three types (1-3) of dehydration-tolerant species or populations, along a seasonal fast-slow continuum shown to illustrate the suggested relationship between levels of incomplete seasonal dormancy (reduction of seasonal growth potentials) and frost or drought survival. The open red star symbols show the theoretical position of a given dehydration-tolerant plant along the global fast-slow continuum. The closed red star symbols show the actual position of the same plant along the seasonal fast-slow continuum when considering the real seasonal growth potential. In case of no dormancy (3), open and closed red star symbols align. Downloaded from https://academic.oup.com/aob/article/131/2/245/6960802 by guest on 23 October 2023

Fig. 2 .Fig. 3 .

 23 Fig.2. Convergent plant adaptive strategies under increasing dehydration stress (due to drought or frost). The strategy of tissue dehydration avoidance (green) decreases as dehydration stress increases and underpins stress resistance (growth maintenance). As stress increases in severity, growth ceases, and stress survival is increasingly supported by dehydration tolerance (pale yellow), particularly in specific organs such as buds and meristems when plants exhibit a dormancy strategy (dark yellow). Dehydration tolerance is associated with increasing embolism resistance (blue) and decreasing tissue water content. The strategy of desiccation tolerance confers survival to the most severe stress (drought and frost) but does not imply hydraulic adaptations during the stress and therefore, no embolism resistance. Adapted from Volaire (2018).

  Fig.4. Main processes determining plant survival or death from the onset to the cessation of a drought or cold period. Seasonal dormancy triggers the adaptive processes before the onset of the stress (or even with no subsequent stress occurring), therefore inducing the dehydration of some key plant tissues and reinforcing their dehydration tolerance. The reduction of growth and metabolic activity is the first process associated with the induction of dormancy and/or dehydration stress. As dehydration stress increases, the minimum recovery threshold or no-return point (yellow star) depends on plant maintenance of water transport capacity and dehydration tolerance in the surviving organs, which are often meristematic tissues. This figure is relevant for major plant species with some level of dehydration tolerance but does not apply to desiccation-tolerant plant species.

Fig. 5 .

 5 Fig. 5. Testing the correlation between seasonal growth potential and dehydration tolerance. This figure provides a methodology illustrated by the documented phenological patterns of a range of herbaceous species: sp1, e.g. temperate populations with no summer dormancy of Dactylis glomerata, Lolium perenne or Medicago sativa; sp2, e.g. Mediterranean populations with some level of summer dormancy of Festuca arundinacea or D. glomerata; sp3, e.g. Festuca rubra, with low annual growth potential and no summer dormancy; sp4, e.g. semi-arid summer dormant populations of D. glomerata[START_REF] Volaire | Plant traits and functional types to characterise drought survival of pluri-specific perennial herbaceous swards in Mediterranean areas[END_REF][START_REF] Bristiel | Is there a constant trade-off between stress tolerance and growth potential in the perennial grass Dactylis glomerata?[END_REF][START_REF] Keep | To grow or survive: which are the strategies of a perennial grass to face severe seasonal stress?[END_REF]. Species or populations originating from climates with life-threatening seasons, having the lowest growth potential (in summer or winter depending on the least favourable season), relative to their maximum annual growth potential during the most favourable season (usually spring), should exhibit the greatest dehydration tolerance under severe seasonal dehydration stress. Dehydration tolerance should be determined by the minimum water content thresholds in meristematic tissues and water transport capacity (embolism resistance), allowing plant survival and growth recovery after stress release.

Downloaded from https://academic.oup.com/aob/article/131/2/245/6960802 by guest on 23 October 2023

ACKNOWLEDGEMENTS

Thanks to Luis Miguel Chevin and Mark Norton for insights and suggestions.


Elham Karimi
email: elham.karimi@sb-roscoff.fr

Simon M Dittami
email: simon.dittami@sb-roscoff.fr

Maintaining beneficial alga-associated bacterial communities under heat stress: Insights from controlled co-culture experiments using antibioticresistant bacterial strains

Keywords: Algal-bacterial interactions, metabolic networks, systems biology, holobionts

Inoculation with bacterial helper cultures that complement host metabolism increases the control over host-associated microbiome in the model brown alga Ectocarpus.

Introduction

As virtually all eurkaryotes, brown algae are colonized by a diverse microbial community, together forming so-called holobionts [START_REF] Dittami | A community perspective on the concept of marine holobionts: state-of-the-art, challenges, and future directions. Zendoo, peerreviewed and recommended by[END_REF][START_REF] Egan | The seaweed holobiont: understanding seaweed-bacteria interactions[END_REF]. The thallus of algae is more than a habitat for microbes; there are numerous dynamic inter-species interactions and metabolic exchanges between both compartments. Technological advancements in sequencing and metagenomics now permit the generation of valuable information about the composition of the host microbiome. Furthermore, computational models of metabolic networks can be used to predict complementarity sets between microbiome species and hosts [START_REF] Belcour | Metage2Metabo, microbiota-scale metabolic complementarity for the identification of key species[END_REF][START_REF] Frioux | Scalable and exhaustive screening of metabolic functions carried out by microbial consortia[END_REF][START_REF] Levy | NetCooperate: a network-based tool for inferring hostmicrobe and microbe-microbe cooperation[END_REF].

Brown algae (Phaeophyceae) are important components of the flora on rocky shores in temperate and cold marine environments around the world, establishing critical ecosystem habitats such as kelp forests [START_REF] Steneck | Kelp forest ecosystems: biodiversity, stability, resilience and future[END_REF]. Many of these kelp forests, especially along the European coast of the North Sea, Eastern Australia, and California, are declining [START_REF] Krumhansl | Global patterns of kelp forest change over the past half-century[END_REF][START_REF] Smale | Impacts of ocean warming on kelp forest ecosystems[END_REF]) -a phenomenon that is commonly attributed to anthropogenic impacts and global warming, though the precise causes are unknown [START_REF] Provost | Climate-driven disparities among ecological interactions threaten kelp forest persistence[END_REF]).

Among brown algae, Ectocarpus (a small filamentous alga) has been established as a genetic and genomic model [START_REF] Cock | The Ectocarpus genome and the independent evolution of multicellularity in brown algae[END_REF] and it is known to depend on the bacteria for proper development and for acclimation to low salinities [START_REF] Dittami | Host-microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures[END_REF][START_REF] Pedersen | Ectocarpus fasciculatus: marine brownalga requiring kinetin[END_REF][START_REF] Tapia | Microbiota influences morphology and reproduction of the brown alga Ectocarpus sp[END_REF]. According to a recent study, the co-culture of Ectocarpus with selected bacteria resulted in increased algal growth, and samples treated by different specific bacteria had diverging metabolite profiles and morphologies [START_REF] Burgunter-Delamare | Metabolic Complementarity Between a Brown Alga and Associated Cultivable Bacteria Provide Indications of Beneficial Interactions[END_REF].

Despite advances in our understanding of the diversity of bacterial symbionts and the prospective use of metabolic complementarity to test host-microbe interactions, there are still unanswered questions about symbiont acquisition and host-microbe balance in the face of abiotic stress. Furthermore, it is unresolved if and to what extent imbalances between algae and their associated microbiomes may play a role in ecologically important things like the decline of kelp forests. The establishment of controlled laboratory systems for brown algalbacterial interaction would constitute a big advance in this field, enabling the mechanistical study of bacterial algal interactions in a changing environment, and the generation of testable hypotheses [START_REF] Van Der Loos | The Macroalgal Holobiont in a Changing Sea[END_REF].

The aim of this study was to improve our control of the alga-associated microbiome. Given the strong interdependencies between the algal hosts and their associated microbiome, this challenge was addressed by providing the alga with a defined set of antibiotic-resistant 'helper bacteria'. These bacteria were selected from a collection of host-derived bacteria, based on their predicted metabolism, to be able to produce the maximum number of compounds that are missing for the predicted metabolism of the host (Dittami et al. 2014). In addition, the algae and bacteria were kept under continuous antibiotic treatment to reduce the proliferation of other non-target strains that might colonize the niche opened up by the antibiotic treatment. The suitability of this approach was tested in the algal host model Ectocarpus, and the stability of the microbiome was assessed under both control conditions and under heat stress.

Materials &Methods

Beneficial bacterial consortium selection

In silico selection of beneficial bacteria was performed in a previous study (Karimi et al. 2021a). Briefly, 81 bacterial genomes isolated from Ectocarpus [START_REF] Karimi | Genome Sequences of 72 Bacterial Strains Isolated from Ectocarpus subulatus: A Resource for Algal Microbiology[END_REF] were annotated using Prokka v 1.13 [START_REF] Seemann | Prokka: rapid prokaryotic genome annotation[END_REF]. Subsequently, bacterial metabolic networks were constructed using Pathway Tools version 23 and the mpwt wrapper implemented in the AuCoMe pipeline [START_REF] Belcour | Metage2Metabo, microbiota-scale metabolic complementarity for the identification of key species[END_REF]. The bacterial metabolic networks and the available host metabolic network [START_REF] Dittami | The genome of Ectocarpus subulatus -A highly stress-tolerant brown alga[END_REF] were submitted to MiSCoTo as SMBL files [START_REF] Frioux | Scalable and exhaustive screening of metabolic functions carried out by microbial consortia[END_REF]. The latter tool was first used to determine the scope of all algal compounds that, according to the metabolic models, and without limiting metabolite exchange, became producible via exchanges between the host and all 81 bacterial genomes together: a total of 506 compounds listed in Supplementary Table S8 of Karimi et al. (2021a).

The same tool was then used to determine the minimal bacterial consortia that enabled the production of these compounds. A total of 18 minimal communities was predicted each comprising 10 bacterial strains (listed in Supplementary Table S1), and the union of all minimal consortia contained 15 bacteria. All of these 15 bacteria and their taxonomic affiliations are listed in Supplementary Table S1, and we attempted to generate antibioticresistant strains for each one of them. The host and bacterial networks have also been deposited in the Zenodo repository [START_REF] Karimi | Supplementary materials for "Robustness analysis of metabolic predictions in algal microbial communities based on different annotation pipelines[END_REF].

Antibiotic sensitivity testing on algae

Filaments of the brown alga Ectocarpus subulatus strain Bft15b were cultured in 10-liter Nalgene™ Round Polycarbonate flasks with Provasoli-enriched autoclaved seawater for 15 days. Temperature was maintained constant at 16°C, and constant airflow and a light-dark cycle of 12:12h were applied in controlled culture chambers. The algal filaments were then placed in 90mm sterilized Petri dishes filled with Provasoli-enriched artificial seawater containing the following components: NaCl 450mM, MgSO 4 (7H2O) 16mM, MgCl 2 (6H2O) 30 mM, KCl 10mM, CaCl 2 9mM adjusted to pH 7.8 with 10mM Tris. Culture conditions remained identical, except for the absence of bubbling. To obtain axenic algae, the following antibiotic treatment was selected based on the work of [START_REF] Cottrell | Production of axenic cultures of micromonas pusilla (prasinophyceae) using antibiotic[END_REF] and preliminary experiments in our laboratory: three-day exposure to a combination of Chloramphenicol (CHL 9.6 μg/ml) and Neomycin (NEO 24.8μg/ml). This treatment efficiently reduced associated bacteria but caused no visible damage (discoloration, rupture) to the algal cells.

Antibiotic sensitivity tests for bacteria and the generation of antibiotic-resistant strains

Disk diffusion tests were carried out to determine the susceptibility of the selected beneficial bacteria to chloramphenicol (CHL) and neomycin (NEO). Freshly cultured bacterial isolates were spread evenly on the R2A agar plates prepared with natural seawater, and grown in the presence of 30 µg CHL and 30 UI NEO antibiotic discs (Bio-Rad, Nanterre, France [START_REF] Matuschek | Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories[END_REF]) for up to one week at 18°C. Next, we attempted to generate antibiotic resistance in the selected strains. To this means, bacteria were grown in 25 ml of liquid R2A medium in Corning® cell culture flasks at 25°C with agitation (50 rpm) supplemented with 0.3 μg/ml of CHL. Once bacterial growth was visible, 1 ml of culture was transferred to a new flask with a higher concentration and this process continued. Based on the "adaptation on demand" hypothesis [START_REF] Johnsen | Bacterial evolution on demand[END_REF], the increase in antibiotic concentration leads bacterial population to evolve resistance to the target antibiotic, until growth was observed at the target concentration of CHL (Supplementary Table S2). At this point, the disk diffusion test was repeated to check the obtained resistance (Table 1). Then, the same procedure was carried out starting with 3.93 μg/ml Neomycin (NEO) for the CHL-resistant strains. Finally, the identity of bacterial strains resistant to both antibiotics was confirmed by sequencing the 16S rRNA gene with the universal primers 27F and 1492R. After reaching the desired antibiotic resistance, the strains were preserved in 20% (vol/vol) glycerol at -80°C.

Co-cultures of algae and bacteria

For our co-culture experiments, we constructed a set that included all seven bacteria for which resistant strains were obtained. This set corresponds to the predicted minimal community 11 in Supplementary Table S1, but lacks Frigoribacterium sp. 9N, Bacillus sp. 9J, and Novosphingobium sp. 9U. These bacteria were grown in liquid R2A media with both antibiotics at their final target concentration. Once bacterial growth was visible with the naked eye (approximately 5 days), the optical density (OD) of the cultures was determined using Genova plus analyzer UV/Vis life science® spectrophotometer at 600 nm. All cultures were diluted to an OD of 0.1 for the inoculation of the algal cultures. For the co-culture setup, algal filaments were dispersed in Petri plates (125 ml cultures), prepared with the two liquid antibiotics as described above, and inoculated with 500 μl of each resistant cultured bacterium (OD = 0.1). These cultures, along with a set of controls, were then used in the stress response experiments described below.

Temperature stress experiment

The design of the stress response experiments, as well as the controls included are described in Fig. 1. Briefly, at the start of the experiment (t 0 ), algal-bacterial co-cultures were inoculated as described above (INOCUL), and in addition, a non-antibiotic treated positive control (NATUR), and an antibiotic-treated non-inoculated alga as a negative control (ATB) were included. For all three of these conditions, we prepared 8 replicate Petri dishes with 4 filaments in each (a total of 96 algal filaments). After the acclimation period of 10 days at 16°C (t 10 ) in a culture chamber at 16°C, 4 of the 8 replicates for each treatment were placed in a separate but otherwise identical culture chamber and subjected to a temperature increase of 2 °C every two days for 6 days (t 12 , t 14 , t 16 ). The other 4 replicates were maintained at 16°C. At each of these time points, algal samples (one filament per Petri dish) were collected in Eppendorf tubes and immediately frozen in liquid nitrogen, and later at -80 °C for downstream analyses. Furthermore, at all steps of the experiment (Fig. 1), the general algal morphology was observed. Finally, at t 16 , aliquots (15 ml) of the culture media were taken, centrifuged at 10,000 g for 30 min to pellet the bacterial cells present, and the pellets were stored at -80 °C for downstream analysis.

Metabarcoding profiling of co-culture experiments

A metabarcoding approach was implemented to investigate the composition of the bacterial community after the co-culture experiments and temperature disturbances. For each culture condition, frozen algal tissue was freeze-dried and ground (TissueLyserII; Qiagen, Hilden, Germany; 2×60 s, 30 Hz). The powder obtained was then used for DNA extraction (the Macherey-Nagel NucleoSpin 96 Plant II Kit). The DNA extraction for bacterial pellets collected from seawater media was performed using the Wizard® Genomic DNA Purification Kit (Promega, Madison, USA). DNA integrity was verified with gel electrophoresis. Two blank controls were included as negative controls in addition to the samples. Libraries were prepared according to the standard Illumina protocol for metabarcoding MiSeq technology targeting the V3-V4 region (Illumina, 2017). Briefly, the amplicon PCR was performed using NOCHL primer sets [START_REF] Thomas | Evaluation of a new primer combination to minimize plastid contamination in 16S rDNA metabarcoding analyses of alga-associated bacterial communities[END_REF]. The PCR products were then purified using AMpure® XP beads (Agencourt Bioscience, Beverly, MA, USA) followed by index PCR in KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA) to attach dual indices and Illumina sequencing adapters to each sample using the Nextera XT Index Kit (Epicentre, Madison, WI, USA). A second PCR clean-up was then carried out as described above. The resulting libraries were checked using a bioanalyzer (DNS 1000) to verify fragment size and a Qubit® fluorimeter (Life Technologies, Paisley, UK) for DNA quantification. An equimolar pool of all libraries was subjected to paired-end (2 × 300 bp) sequencing on a single flow cell of an Illumina MiSeq sequencer using the MiSeq v3 kit (Illumina, San Diego, CA, USA) on Platform GENOMER, FR2424, Roscoff.

Sequence analysis

The resulting raw sequences (10,508,975 read pairs) were processed using DADA2 within the R (v4.0.0) statistical computing environment [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF]. Reads underwent quality filtering as error rates were calculated and removed from the dereplicated reads. An initial Amplicon Sequence Variant (ASV) table was constructed, and later chimeras were identified using the removeBimeraDenovo function. Finally, taxonomy was assigned using DADA2's native RDP Bayesian classifier against the Silva database version 138 (McLaren 2020). Non-bacterial sequences as well as all ASVs with less than ten reads or which were more abundant in the negative control compared to the samples were removed. No rarefication was conducted but the 'calc_obs_props' function of the 'metacoder' package version 0.3.4 was used to calculate proportions and the abundance of taxa was computed using the 'calc_taxon_abund' function. For diversity analysis, the 'vegan' package was employed using the 'diversity' function. PICRUSt2 (v2.4.1) was used to predict functional traits of bacteria based on their marker gene sequences in each treatment with default settings [START_REF] Douglas | PICRUSt2 for prediction of metagenome functions[END_REF].

Statistical analyses

All statistical analyses and graphs were performed within the computing environment R (v4.0.0; R Core Development Team, 2005) using the packages phyloseq (McMurdie and Holmes 2013), phylosmith [START_REF] Smith | phylosmith: an R-package for reproducible and efficient microbiome analysis with phyloseq-objects[END_REF], vegan [START_REF] Oksanen | Vegan: Community Ecology Package. R package version 2[END_REF]), ggplot2 (Valero-Mora 2010), metacoder [START_REF] Foster | Metacoder: An R package for visualization and manipulation of community taxonomic diversity data[END_REF], microbiome [START_REF] Lahti | Tools for microbiome analysis in R. Version 1.9[END_REF]. Statistical tests were considered significant at α = 0.05. Differences in community composition (β-diversity) were computed using Bray-Curtis dissimilarity matrices and tested via permutational multivariate analysis of variance (PERMANOVA) and the statistical analysis of the predicted pathway data was performed using STAMP version 2.1.3 [START_REF] Parks | STAMP: statistical analysis of taxonomic and functional profiles[END_REF].

Results

Antibiotic resistance and sufficient growth obtained for 7 out of 15 bacteria.

To set up a controlled co-culture experiment, we continuously applied antibiotics. Therefore, we needed to generate antibiotic-resistant strains of the bacteria we had selected based on metabolic complementarity. All 15 of the initially bacteria developed resistance to CHL and subsequently to neomycin (NEO) (Supplementary Table S2). For 10 out of these 15 strains the resistance to both antibiotics (Supplementary Figure S1) was durable and 7 strains out of 10 exhibited growth rates that were sufficient to prepare co-culture experiments within a timescale of 5 days (Table1)

Metabarcoding survey of algal samples

In total, 71 samples from three algal treatment conditions were considered for the core analysis of the study: bacterial inoculation with ATB treatment (n = 24, labeled as INOCUL); no inoculation and no ATB treatment (n = 23, here known as NATUR), and no bacterial inoculation with ATB treatment (n = 24, labeled as ATB). One sample replicate from the ATB treatment was removed alongside the negative controls because insufficient reads were obtained. Of 10 million raw reads were made up 1 005 455 unique sequences belonging to 1 933 different ASVs. The ASVs and taxonomy tables are provided in Supplementary Table S3.

Six inoculated resistant bacteria out of seven persisted in cultures.

Six inoculated bacteria out of seven were detected in metabarcoding data (Fig. 2). Only Rhizobium had no reads although this strain had developed resistance to both antibiotics in a timely manner. Overall, the inoculated strains were detected specifically in the conditions they were inoculated in, except for Maribacter which was present in all cultures (Fig. 2). In the samples that underwent the INOCUL treatment, reads corresponding inoculated bacteria, ATB-treated, natural, and ATB-treated inoculated samples (two-way ANOVA, p < 0.001), but differences between temperature-stressed and control samples were not significant in the same analysis (p = 0.28; Fig. 3). Generally, the communities from samples inoculated with the selected bacteria (INOCUL) exhibited lower phylogenetic diversity than control (NATUR) and higher than the antibiotic treated (ABT).

Non-metric Multidimensional Scaling (nMDS) visualization of β-diversity using the Bray-Curtis dissimilarity index revealed a clear separation of co-culture conditions (Fig. 4; PERMANOVA, Bray-Curtis method, 999 permutations; R2 = 0.75, p < 0.001). However, no clear differences between stress-and control samples were observed for the overall community composition. PERMANOVA analyses carried out separately for ATB, INOCL, and NATUR samples after inculcation (t 10 -t 16 ) also revealed that the community composition changed slightly but significantly in response to stress for the ATB and INOCL treatments (p=0.013 and p=0.041, respectively) but not for the NATUR treatment.

Taxonomic composition of bacterial communities

The taxonomic composition of bacterial communities in ATB, INOCUL, and NATUR cultures is illustrated in Fig. 5A, where only the non-inoculated strains are considered (inoculated strains are shown in Fig. 2). In the natural samples, we observed a complex community dominated by sequences corresponding Flavobacteriales, Caulobacterales, Oceanospirellales, and Rhodobacterales. ATB treated samples including the INOCUL samples (without the inoculated bacteria), were dominated by sequences belonging to Flavobacteriales and more specifically the genus Winogradskyella, and only 5-10% of reads corresponded to other groups.

The pairwise comparisons in Fig. 5B display the relative abundance of each observed taxonomic group in stress versus control samples. Here, nodes represent taxonomic levels, and each branch represents the phylogenetic relationship between those entities. ASV richness is represented by node size and the relative abundance of reads corresponding to this node by color. For the natural microbiota, this heat tree shows few taxonomic differences in the composition in response to stress, notably an increase in the proportion Cytophagales including Fabibacter, and a decrease in Pseudomonadales. The ATB in INOCL treatment exhibit a few common responses to stress, such as the increase in the part of Bacillales in response to stress, and a decrease in Gammaproteobacteria and Rhodobacteriaceae.

Specifically, in the INOCL treatment, we also observed a relative increase in Micrococcales and Rhizobiaceae in response to stress.

Taxonomy-based functional profiling of bacterial communities in control and stressed samples

In addition to generating the taxonomic profiles, we used Picrust2 to functional profiles based on the metabarcoding data. During this process, 15 ASVs with Nearest Sequenced Taxon Index (NSTI) values of more than 2 were eliminated, along with six ASVs with poor alignment [START_REF] Barbera | EPA-ng: Massively Parallel Evolutionary Placement of Genetic Sequences[END_REF][START_REF] Czech | Genesis and Gappa: processing, analyzing and visualizing phylogenetic (placement) data[END_REF][START_REF] Louca | Efficient comparative phylogenetics on large trees[END_REF][START_REF] Mirarab | SEPP: SATé-Enabled Phylogenetic Placement Biocomputing[END_REF]. The average of NSTI values for our samples (Supplementary Table S5) was 0.0470.007 (mean + SD), with slightly higher NSTIs (0.0530.003) for the NATUR samples than the INOCUL samples (0.0440.009), indicating the availability of close reference genomes in the PICRUST2 database. Principal component analyses of these profiles revealed that ATB, INOCL, and NATUR samples were clearly associated with distinct functional profiles (Fig. 6A). Using a Kruskal-Wallis test and a Benjamini Hochberg correction, we found that 314 out of 349 predicted metabolic pathways differed significantly (padj<0.05 in at least one of these conditions; Supplementary Table S5). Interestingly, from a functional point of view, INOCL samples were closer to the NATUR samples than the ATB samples (Fig. 6B). However, none of the predicted metabolic pathways differed significantly in response to stress when analyzing the ATB, INOCL, and NATUR groups separately.

Discussion

The effect of antibiotic treatments on algal cultures Protocols to obtain truly axenic cultures using antibiotic treatments are available for several microalgal species (e.g., [START_REF] Cirri | Biofilm interactions-bacteria modulate sexual reproduction success of the diatom Seminavis robusta[END_REF][START_REF] Von Dassow | Transcriptome analysis of functional differentiation between haploid and diploid cells of Emiliania huxleyi, a globally significant photosynthetic calcifying cell[END_REF]. For macroalgae axenic cultures are, to the best of our knowledge, available only for Ulva mutabilis [START_REF] Califano | Preparation of axenic cultures in Ulva (Chlorophyta)[END_REF] and even these do not exhibit normal growth and morphology unless complemented with bacterial morphogens [START_REF] Spoerner | Growth and Thallus Morphogenesis of Ulva mutabilis (Chlorophyta) Depends on A Combination of Two Bacterial Species Excreting Regulatory Factors[END_REF]. In most cases, e.g. the filamentous brown alga Ectocarpus (Burgunter-Delamare et al. 2020) but also some microalgae [START_REF] Geng | Changes in the Structure of the Microbial Community Associated with Nannochloropsis salina following Treatments with Antibiotics and Bioactive Compounds[END_REF]) antibiotic treatments reduced bacterial abundance and led to a shift in bacterial community composition. The example of Ectoacrpus subulatus shows that this reduction may be temporary and that the relative bacterial abundance, as estimated by the number of bacterial vs. host reads in metatranscriptomic experiments, may even increase after recovery from strong antibiotic treatments [START_REF] Kleinjan | Insights into the potential for mutualistic and harmful host-microbe interactions affecting brown alga freshwater acclimation[END_REF]). In our study, the comparison of the NATUR and the ATB treatments confirms a change in the bacterial community. In our case, the community shifted from a diverse assembly of Proteobacteria to a community dominated by Bacteriodetes and notably the genus Winogradskyella (Flavobacteria). This is similar to previous results by [START_REF] Burgunter-Delamare | Metabolic Complementarity Between a Brown Alga and Associated Cultivable Bacteria Provide Indications of Beneficial Interactions[END_REF] where another, yet undescribed genus of Flavobacteria became dominant after antibiotic treatments.

Winogradskyella is frequently associated with marine mammals and algae, and uncultivable strains of the genus have been proposed to have specialized in the utilization of the brown algal polysaccharide laminarian while reducing other aspects of their metabolism (Alejandre-Colomo et al. 2021). Our current working hypothesis is that if there is an open niche created by the antibiotic treatment it will be filled by resistant or previously dormant bacteria that are well-adapted to live on macroalgae. We may furthermore hypothesize that the newly established bacteria may, at least in some cases, be less beneficial to the algal host, and in any case, we have no control over the communities that will establish themselves.

Inoculation stabilized bacterial communities and partially restores metabolic functions

In this study, we attempted to fill the empty niche created by an antibiotic treatment by inoculation with a controlled and beneficial bacterial community. At the same time, we gave this target community a selective advantage by maintaining an antibiotic treatment.

Our data show that this approach worked partiallyapproximately 63-67% of bacterial reads obtained corresponded to the inoculated bacteria, and only about 31%-37% to others.

Although this is still a considerable amount of non-target bacteria, in a comparable study without continuous antibiotic treatment, on average only 11% of the final reads corresponded to the inoculated bacteria (see [START_REF] Burgunter-Delamare | Metabolic Complementarity Between a Brown Alga and Associated Cultivable Bacteria Provide Indications of Beneficial Interactions[END_REF], notably Table 4

therein).

Furthermore, our Picrust2 analyses underline that the obtained bacterial communities are functionally more similar to the "natural" laboratory communities than the communities after only ATB treatment (Fig. 6). This indicates that the inoculation with selected bacteria was at least to some extent able to restore the functions naturally present in the microbiome.

Bacterial communities had little influence on host temperature stress response or vice versa

In Ectocarpus, bacterial communities have also been shown to have a significant impact on the holobiont salinity response and vice versa [START_REF] Dittami | Host-microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures[END_REF][START_REF] Kleinjan | Insights into the potential for mutualistic and harmful host-microbe interactions affecting brown alga freshwater acclimation[END_REF]). We, thus, wanted to test if our approach of using antibiotics and antibiotic-resistant cultures could help stabilize beneficial bacterial communities, and by extension increase holobiont stress tolerance. Unlike in previous experiments with strong changes in salinity, our results on temperature stress exhibited only weak changes in the bacterial community in both the INOCL and in the control (ATB, NATUR), thus making it impossible to conclude on stabilizing effect. Please note, that the absence of an impact of stress on the bacterial communities was observed, despite the strong intensity of the temperature stress. Indeed, at the end of the experiments, we had reached the limit of the thermal tolerance range of our strains: an additional increase in temperature by two degrees led to rapid algal bleaching and death regardless of the treatment (ATB, INOCL, NATUR; data not shown). One possible explanation for the discrepancy between the previous salinity tolerance experiments may be the different effects the stressor had on the host or the microbiome.

Ectocarpus subulatus is a euryhaline species frequently found in estuaries. It is, as such, welladapted to strong fluctuations in salinity [START_REF] Peters | Barcoding of Cryptic Stages of Marine Brown Algae Isolated from Incubated Substratum Reveals High Diversity in Acinetosporaceae (Ectocarpales, Phaeophyceae). Cryptogamie[END_REF] . The associated microbiome of the strain used in [START_REF] Dittami | Host-microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures[END_REF], however, might have impoverished and evolved over the 20 years of cultivation in the laboratory, since its original isolation in 1995 [START_REF] West | Ectocarpus siliculosus (Dillwyn) Lyngbye from the Hopkins River Falls, Victoria. The first record of a freshwater brown alga in Australia[END_REF] and marine-freshwater transitions in general, are considered rare among microbes [START_REF] Logares | Infrequent marine-freshwater transitions in the microbial world[END_REF]. Regarding temperature changes, the algal hosts, on the other hand, generally have narrow temperature tolerance windows, and maximum summer temperatures are one of the factors limiting their distribution [START_REF] Lüning | Temperature tolerance and biogeography of seaweeds: The marine algal flora of Helgoland (North Sea) as an example[END_REF] Regarding bacteria, even bacteria considered psychrophilic usually grow well at 20°C [START_REF] Morita | Psychrophilic bacteria[END_REF], and most (mesophilic) bacteria tolerate up to 45°C [START_REF] Willey | s Microbiology[END_REF] Although this is presently only a hypothesis, is important to keep in mind the possibility of differences in the stress response of the host and the different symbiotic partners. Depending on which component is affected different approaches may be appropriate when attempting to manipulate the system.

Bacterial manipulation with antibiotic-resistant bacteriaa promising but low throughput strategy

While our experiments show a clear increase in the control over the Ectocarpusassociated bacterial communities, we did not reach complete control over the alga-associated microbiome, and several aspects of these experiments could be improved. First, as illustrated by the metabolic distance between the INOCL and the NATUR communities, we are not yet completely substituting the natural communities in terms of metabolic capacities; This may be related in part to the fact that we were able to co-culture only six of the initially selected 16 bacterial strains. At the same time, we are also still limited to cultivable isolates.

Increasing our efforts on cultivating the "unculturable" [START_REF] Zengler | Cultivating the uncultured[END_REF] would therefore also help cover a larger portion of the bacterial metabolism in co-culture experiments. In parallel, additional effort, and the use of more or different antibiotics may help increase the control over the remaining alga-associated microbiota. Here, however, a balance needs to be struck, as antibiotic resistance for the bacteria also comes with a cost, and evolving resistances may further alter bacterial physiology [START_REF] Händel | Compensation of the metabolic costs of antibiotic resistance by physiological adaptation in Escherichia coli[END_REF]) thus potentially impacting interactions with the host. Another limitation of our approach is the time it takes to prepare these cultures and co-culture experiments. Experiments with one or two bacterial strains of interest are easily feasible, but they are difficult to upscale because each bacterium reacts differently to antibiotics, and sometimes resistances are not stable making repeated treatments necessary. It will thus be more difficult to envision this approach for medium-or high-throughput screening of bacteria in a co-culture context, especially if complex communities are to be tested. Despite these limitations, we believe that the development of a collection of antibioticresistant helper cultures for our algae constitutes one promising avenue towards fully controlled laboratory experiments with algae for which there are currently no working axenization protocols. If these cultures can fulfill the metabolic functions of the natural microbiome, their addition will thus enable increased experimental control over the microbiome during experiments, enhancing the reproducibility of microbiome-related work.

In the long run we could then progressively aim towards replacing the microbes by the metabolites or metabolic intermediates they provide. Fig. 1. A schematic illustration of the sampling design and experimental procedure. Ectocarpus subulatus was cultivated from stock populations of strain Bft15b. In the climate chamber (16°C) algae were grown for 7 weeks and divided into two series in petri dishes. One of the series was treated for three days with a mixture of two antibiotics (CHL & NEO), after which this group were divided into two series and one series received inoculum of 7 different bacteria. All samples stayed at the same temperature for 10 days. Later, replicates of each condition were moved to higher temperatures where the temperature was increased every 2 days in 2°C. Microbiota samples were taken directly after their separation (t-3), before inoculation (t0), after acclimation(t10), and after 4 and 6 days (t14 and t16). Parts of the figure were created with BioRender.com. Fig. 2. Heatmap of relative read abundances corresponding to inoculated bacteria vs. other bacteria. Rows are centered; unit variance scaling is applied to rows. 5. A) Bacterial community in three culture conditions at the order level. Taxon with abundances more than 0.1% for all three conditions (the remaining were grouped as "others"). Only non-inoculated taxa were considered for this filter (please refer to Figure 2 for the inoculated taxa), and only samples after T10 (= time of inoculation), to keep the three conditions comparable. B) Differential heat tree matrix depicting the change in relative read abundance for the different taxa over different conditions and cultures. The number of taxa identified at each taxonomic level is represented by the size of the individual nodes in the gray cladogram. The smaller cladograms show pairwise comparisons between each culture condition: a brown node indicates that the taxon is more abundant in culture condition labeled in brown than in the one labeled in green and vice versa (C stands for Control (16 oC) and S stands for stressed (20 and 22 oC).

Figures and Tables

Zone of inhibition (mm)

Growth in liquid media (OD600 after 5 days)

Fig. 3 .

 3 Fig. 3. Bacterial Alpha diversity. The dot-plot figures show the Shannon diversity of the bacterial communities in the three culture treatment groups, ATB, INOCUL, and NATUR, and at different time points in either control (C) or stress conditions (S). Calculations are based on the relative abundance of observed ASVs.

Table S4 :

 S4 Alpha diversity indices

		Before gaining	After gaining
		antibiotic-resistance	antibiotic-resistance
	Selected bacteria
	consortia	CHL	NEO	CHL	NEO	CHL & NEO
	Frigoribacterium sp. 9N	61	16	26	0	0.000
	Pseudoclavibacter sp. 8L 18	10	0	0	1.186
	Maribacter sp. 151	15	0	0	0	0.184
	Bacillus sp. 9J	19	22	0	0	0.000
	Aeromonas sp. 9A	43	18	0	0	0.671
	Erythrobacter sp. EC-
	HK427	23	21	0	0	0.128
	Novosphingobium sp. 9U 51	18	0	0	0.001
	Pantoea sp. 111	33	23	0	0	0.432
	Pseudomonas sp. 8O	0	22	0	0	0.301
	Rhizobium sp. EC-SD404 56	17	0	0	0.145

Acknowledgments

We would like to thank Guillaume Manat for helpful discussions on making bacteria resistant to antibiotics, Sébastien Colin for help exploring the algal bacterial community by microscopy, Elodie Rolland for providing facilities for algal culturing, Akira Peters for providing the Ectocarpus culture, Bertille Burgunter-Delamare for useful advice on using DADA2 scripts, and Gwenn Tanguy and Erwan Legeay from the GENOMER platform (FR2424, Station Biologique de Roscoff) for access to the sequencing platform and support during library preparation.

Funding

This work was supported by the Centre national de la recherche scientifique (CNRS) [Momentum call 2017].

Data availability

The Illumina sequence data generated in this study (Raw MiSeq data) have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB57188.

amounted to 66% of total reads (69% in control conditions and 63% in stress conditions, Supplementary Figure S2).

Antibiotic treatments reduce bacterial diversity and modify community structure.

The Shannon diversity indices per sample ranged from 3.33 to 4.77 (Fig. 3, See Supplementary Table S4 for additional diversity indices). They differed significantly between

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary materials

Figure S1. The appearance of normal and antibiotic resistant strains on R2A agar and liquid R2A medium. The antibiotic discs correspond to Neomycine and Chlorapheniclcol, respectively. The liquid ABT treatment corresponded to a mixture of Chloramphenicol (9.6 μg/ml) and Neomycin (24.8μg/ml). Photos were taken 5 days after inoculation Table S1. Overview of mininal bacteria consortia prediacted by MiSCoTo and of Antibiotic resistance survey.

Table S2. Overview of the growth rate of selected bacteria at different concentrations of examined antibiotics in this study. 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1

ProteobacteriaAlphaproteobacteria Sphingomonadales Erythrobacteraceae Erythrobacter99.1354 TRUE X X X X X X X X X X X X X X X X X X Inocul. Novosphingobium sp. 9U 9U ProteobacteriaAlphaproteobacteria Sphingomonadales Sphingomonadaceae Novosphingobium 99.59 TRUE X X X X X X X X X X X X X X X X X X Pantoea sp. 111 111 ProteobacteriaGammaproteobacteria Enterobacterales Erwiniaceae Pantoea 100 TRUE X X X X X X X X X X X X X X X X X X Inocul. Pseudomonas sp. 8O 8O ProteobacteriaGammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas99.7727 TRUE X X X X X X X X X X X X X X X X X X Inocul. Rhizobium sp. EC-SD404 404 ProteobacteriaAlphaproteobacteria Rhizobiales Rhizobiaceae Rhizobium 100 TRUE X X X X X X X X X X X X X X X X X X Inocul.