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

Frederic Lens
email: frederic.lens@naturalis.nl

Hervé Cochard
email: herve.cochard@inra.fr

Hueng Xu

Larissa Chacon
email: larissa.chacondoria@naturalis.nl

Pauline Bristiel
email: pauline.bristiel@cefe.cnrs.fr

Jennifer A Balachowski
email: jbalachowski@ucdavis.edu

Nick P Rowe
email: nrowe@cirad.fr

Cyrille Violle
email: cyrille.violle@cefe.cnrs.fr

Catherine Picon-Cochard
email: catherine.picon-cochard@inra.fr

L Florence Volaire

Embolism and mechanical resistances play a key-role in dehydration tolerance of a perennial grass Dactylis glomerata L

Keywords: cavitation resistance, Dactylis glomerata L., dehydration tolerance, drought survival, embolism threshold, plant mortality, leaf fracture toughness, vessel wall thickness, stem anatomy, intra-specific variability, perennial grass

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract

• Background and Aims More intense droughts under climate change threaten species resilience. Hydraulic strategies determine drought survival in woody plants but have been hardly studied in herbaceous species. We explored the intra-specific variability of hydraulic and morphological traits as indicators of dehydration tolerance in a perennial grass, cocksfoot (Dactylis glomerata) which has a large biogeographical distribution in Europe.

• Methods Twelve populations of cocksfoot originating from Mediterranean, Temperate and Northern European areas were grown in a controlled environment in pots. Dehydration tolerance, leaf and stem anatomical traits and xylem pressure associated with 88% or 50% loss of xylem conductance (P 88, P 50 ) were measured.

• Key Results Across the twelve populations of cocksfoot, P 50 ranged from -3.06 to -6.36 MPa, while P 88 ranged from -5.06 to -11.6 MPa. This large intraspecific variability of embolism thresholds corresponded with the biogeographical distribution and some key traits of the populations. In particular, P 88 was correlated with dehydration tolerance (r=-0.79). The dehydration-sensitive temperate populations exhibited the highest P 88 . The most dehydration-tolerant Mediterranean populations had the greatest leaf dry matter content and leaf fracture toughness, and the lowest P 88 . The Northern populations displayed intermediate trait values, potentially attributable to frost resistance. The thickness of metaxylem vessel walls in stems was highly correlated with P 50 (r=-0.92) but no trade-off with stem lignification was observed. The relevance of the linkage between hydraulic and stomatal traits is discussed for drought survival in perennial grasses.

• Conclusions Compared to woody species, the large intra-specific variability in dehydration tolerance and embolism resistance within cocksfoot has consequences for its sensitivity to climate change. To better understand adaptive strategies of herbaceous species to increasing drought and frost requires exploring further the role of hydraulic and mechanical traits using a larger inter-and intra-specific range of species.

INTRODUCTION

Higher temperatures due to climate change have led to more frequent extreme climate events around the world (I.P.C.C., 2014, [START_REF] Orlowsky | Global changes in extreme events: regional and seasonal dimension[END_REF]Seneviratne, 2012, Seneviratne et al., 2014).

Extreme events, including intense droughts and heat waves, will become important drivers of future ecosystem dynamics and function [START_REF] Smith | The ecological role of climate extremes: current understanding and future prospects[END_REF]. Resilience, defined as the capacity of a population or community to recover following disturbance, may therefore be the key to the persistence of ecosystems (Hodgson et al., 2015;Nimmo et al., 2015).

Forests and grasslands are major terrestrial ecosystems, covering one-quarter and onethird of the total land area, respectively. They provide many important ecosystem services including timber and forage supply, carbon storage, and biodiversity preservation, but their persistence is threatened under more frequent extreme climate events [START_REF] Anderegg | Consequences of widespread tree Mortality triggered by drought and temperature stress[END_REF][START_REF] Pilgrim | Interactions among agricultural production and other ecosystem services delivered from European temperate grassland systems[END_REF]. Climate-induced forest dieback is an increasing global concern (Allen et al., 2010) and major research efforts have been carried out to identify strategies for drought survival among woody plant species [START_REF] Pivovaroff | Multiple strategies for drought survival among woody plant species[END_REF]. In parallel, declines in grassland productivity [START_REF] Brookshire | Long-term decline in grassland productivity driven by increasing dryness[END_REF] and long-term degradation from drought may gradually become more common in the future [START_REF] Ciais | Europe-wide reduction in primary productivity caused by the heat and drought in 2003[END_REF]. Drought reduces plant productivity and induces plant mortality in grasslands under extreme events [START_REF] Griffin | Mortality of Australian alpine grasses (Poa spp.) after drought: species differences and ecological patterns[END_REF][START_REF] Hodgkinson | Death model for tussock perennial grasses: a rainfall threshold for survival and evidence for landscape control of death in drought[END_REF][START_REF] Moran | Functional response of U.S. grasslands to the early 21st-century drought[END_REF][START_REF] Poirier | Persistence and production of perennial grasses under water deficits and extreme temperatures: importance of intraspecific vs. interspecific variability[END_REF][START_REF] Van Peer | Survival, gap formation, and recovery dynamics, in grassland ecosystems exposed to heat extremes: The role of species richness[END_REF]. Although the ability of woody species to survive intense drought was strongly related to their embolism resistance i.e. the vulnerability to embolism [START_REF] Mcdowell | Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF], the traits and processes involved in the survival and recovery of herbaceous species after extreme stress are still poorly characterized [START_REF] Craine | Global diversity of drought tolerance and grassland climate-change resilience[END_REF][START_REF] Hoover | Resistance and resilience of a grassland ecosystem to climate extremes[END_REF].

Recent methodological developments have enabled inter-specific study of the vulnerability to embolism in stems of herbaceous plants [START_REF] Lens | Embolism resistance as a key mechanism to understand adaptive plant strategies[END_REF], Tixier et al., 2013), opening the door for explorations of the role of hydraulic traits in stress survival of non-woody species. Building on these recent results showing large interspecific variability in embolism resistance in grasses distributed along an aridity gradient [START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF], the present study aimed to explore the extent of intraspecific variability in the perennial grass Dactylis glomerata L. This model species was chosen since it has a large biogeographical distribution over Europe and Asia [START_REF] Borrill | Evolution and genetic resources in cocksfoot[END_REF] and a high genetic variation within both populations and geographical origins [START_REF] Lumaret | Cytology, genetics, and evolution in the genus Dactylis[END_REF][START_REF] Xie | Diversity comparison and phylogenetic relationships of cocksfoot (Dactylis glomerata L.) germplasm as revealed by SSR markers[END_REF].

Intraspecific variation with respect to embolism resistance is still poorly understood [START_REF] Martinez-Vilalta | Hydraulic adjustment of Scots pine across Europe[END_REF]. In woody trees, phenotypic plasticity in resistance to embolism was found to be low and buffered against environmental variation in a large range of Pinus pinaster [START_REF] Lamy | Uniform Selection as a Primary Force Reducing Population Genetic Differentiation of Cavitation Resistance across a Species Range[END_REF][START_REF] Lamy | Limited genetic variability and phenotypic plasticity detected for cavitation resistance in a Mediterranean pine[END_REF] but was not different across populations of Fagus sylvatica [START_REF] Herbette | Insights into xylem vulnerability to cavitation in Fagus sylvatica L.: phenotypic and environmental sources of variability[END_REF][START_REF] Wortemann | Genotypic variability and phenotypic plasticity of cavitation resistance in Fagus sylvatica L. across Europe[END_REF]; however, not a single study has assessed the intraspecific variation within an herbaceous grass species. Perennial species exhibit various functional strategies to cope with moderate to severe drought [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF][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] Zwicke | What functional strategies drive drought survival and recovery of perennial species from upland grassland?[END_REF], since they undergo many successive periods of stress and recovery during their life cycle. Under severe drought, the basal aerial meristems and roots are the key organs for plant survival and recovery, while most leaves of perennial grasses senesce to reduce water loss (Volaire et al., 2009). As a result, no significant correlation was found between vulnerability of leaf hydraulic conductance and whole-plant survival of drought in grasses [START_REF] Ocheltree | A safety vs efficiency trade-off identified in the hydraulic pathway of grass leaves is decoupled from photosynthesis, stomatal conductance and precipitation[END_REF]. The role of hydraulic traits in herbaceous species should therefore be analysed through a whole-plant perspective.

Survival in increasingly drought prone environments depends on a range of traits which include sufficient mechanical strengthening to avoid irreversible effects of tissue dehydration. Embolism resistant tree species are often characterized by a high wood density (Hacke et al., 2001[START_REF] Lens | Embolism resistance as a key mechanism to understand adaptive plant strategies[END_REF][START_REF] Ogasa | Recovery performance in xylem hydraulic conductivity is correlated with cavitation resistance for temperate deciduous tree species[END_REF] although no strong evidence was that denser woods conferred higher survival, or that the risk of embolism caused by wide conduits increased mortality [START_REF] Russo | Interspecific relationships among growth, mortality and xylem traits of woody species from New Zealand[END_REF]. In addition, increased tolerance to hydraulic dysfunction implies increased carbon costs for leaf construction and water use [START_REF] Nardini | Trade-offs between leaf hydraulic capacity and drought vulnerability: morpho-anatomical bases, carbon costs and ecological consequences[END_REF]. Low embolism threshold values were associated with narrower and denser vein conduits, increased thickness of conduit wall and vein density and also with reduced leaf area [START_REF] Nardini | Trade-offs between leaf hydraulic capacity and drought vulnerability: morpho-anatomical bases, carbon costs and ecological consequences[END_REF]. In herbaceous species too, Arabidopsis plants with increased wood formation in their stems were shown to be significantly more embolism-resistant [START_REF] Lens | Embolism resistance as a key mechanism to understand adaptive plant strategies[END_REF], Tixier et al., 2013) and grass species from the most arid environments had more lignified stems compared to the vulnerable species native to wetter habitats [START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF].

The main objective of this study is to identify relationships between embolism resistance, whole plant dehydration survival, leaf traits, and stem anatomy amongst twelve populations of Dactylis glomerata L originating from Scandinavia to Morocco. Given the contrasted biogeographic origins of the studied populations, we expected that they differ strongly for hydraulic and anatomical traits which would subsequently contribute to their dehydration tolerance. To determine ecological responses of grassland species under climate extremes (Smith, 2011a), this study therefore aims to (a) identify the role of embolism thresholds and associated traits for drought survival and mechanical resistance in a perennial herbaceous species and (b) determine the intra-specific variability of these traits as indicators of potential drought survival.

MATERIAL AND METHODS

Plant Material

Twelve populations of Dactylis glomerata L. (cocksfoot), including native populations and cultivars, were selected from germplasm banks to be representative from three climatic regions: four Mediterranean, four Temperate, and four Northern populations (Table 1). Cocksfoot (D. glomerata L.) is a tetraploid (2n=4x=28) species from the genus Dactylis whose taxonomic constitution is debated, but broadly consists of a complex of diploids and tetraploids [START_REF] Borrill | Evolution and genetic resources in cocksfoot[END_REF]. The twelve populations tested were all verified to be tetraploid (Barre, pers. comm).

Experimental sites and design

Three pot-experiments were carried out to determine dehydration tolerance, embolism resistance as well as anatomy and leaf traits, at the CEFE-CNRS research center in Montpellier, France (43°6'N, 3°8'E). Seeds from each D. glomerata population were sown in seedling trays in February 2013 and the adult plants (over one year old) were used for all subsequent experiment.

-Experiment 1 was carried out to determine plant dehydration tolerance measured as plant survival rate in plants growing in short pots, after rewatering following an intense drought [START_REF] Volaire | A unified framework for plant drought adaptive strategies: across scales and disciplines[END_REF]. Plants were transplanted into 4L, 18cm-diameter pots in May 2013. As roots were equally limited in depth for all the populations in pots, plant drought survival mirrored plants dehydration tolerance under a similar soil water availability [START_REF] Volaire | Plant traits and functional types to characterise drought survival of pluri-specific perennial herbaceous swards in Mediterranean areas[END_REF]. It allowed us to discount the effect of rooting depth associated mainly with dehydration avoidance [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF][START_REF] Volaire | Drought survival in Dactylis glomerata and Festuca arundinacea under similar rooting conditions in tubes[END_REF]. For this experiment, the quantity of dry substrate was measured to be identical for each of the 4L pot so that the time dynamics of soil moisture could be accurately assessed and compared simply by pot weight during the period of intense water deficit in summer 2014.

-Experiment 2 was carried out to measure embolism threshold and stem anatomy.

Individuals started from tillers taken from the adult plants sown in 2013 were transplanted in September 2014 into 10L (24 cm-diameter) pots in order to obtain reproductive tillers from spring 2015.

-Experiment 3 was carried out to measure biomechanical and leaf traits. Individuals taken from the initial adult plants were transplanted into new 4L pots in September 2015.

Each 4L and 10L pot contained four clones from a single population, to ensure soil cover close to field conditions, with 10 and 4 replicates for 4L pots and 10L pots respectively per population.

Pots were filled with a substrate composed of 65% local loamy-clay soil and 35% compost.

For all experiments, the plants were grown under an open-ended hoop-house and they were kept fully irrigated and fertilized (40 U N after each cut) except during the imposition of the severe drought in Experiment 1 during summer 2014. Pesticides were applied to prevent the development of diseases caused by insects (aphids) or fungi (rust). For all experiments, pots were distributed on a bench according to a full randomization design. In addition, the position of pots was modified multiple times during the experiment to minimize placement effects.

In addition, plants of one of the Mediterranean populations (Kasbah), that was grown in a nearby field in a 1.30 m deep loamy soil since 2011 [START_REF] Barkaoui | Mean root trait more than root trait diversity determines drought resilience in native and cultivated Mediterranean grass mixtures[END_REF] and without irrigation nor fertilization for two years, were used for stem sampling in order to assess the plasticity of embolism resistance in one population. Twenty stems of this field-grown 'Kasbah' population were collected in spring 2015 and processed for the measurement of stem embolism (see below).

No other measurement was carried out on these plants.

Mean temperatures were 9.5°C in winter, 16.6°C in spring and 24.2°C in summer, with 63% mean air relative humidity and mean radiation of 800 µmol m -2 s -1 from spring to summer.

Measurements

Dehydration tolerance under severe summer drought (Experiment 1)

Plants of Experiment 1 were kept fully irrigated and fertilized until June 2014 when the severe summer drought started to be imposed as follows. In June 2014, the soil water content was raised to field capacity (60% soil water content for the substrate used) and irrigation was ceased thereafter. Pots were weighed at frequent intervals (every 2-5 days) throughout the experiment to monitor the decrease in soil water content. Once full leaf senescence was reached for all populations, and when soil water content in the pots decreased to near 12% (27 to 35 days after irrigation cessation), plants were harvested at 3cm from the soil surface and the pot was put back under full irrigation (field capacity), thus ending the severe drought. All pots were re-watered after they reached the same soil water content to ensure that plant survival corresponded to an identical soil final dehydration for all populations irrespective of their growth potential. The plants were irrigated for 15 days after which dehydration survival was measured. Plants that did not produce new shoots after 15 days were counted as dead. The survival rate was measured as the number of living plants divided by the total number of plants (plant survival; SURV %).

Embolism Resistance (Experiment 2)

In late spring 2015, three pots for each population were maintained under irrigated conditions and allowed to flower. Stems were then collected and processed using the static centrifuge technique [START_REF] Alder | Use of centrifugal force in the study of xylem cavitation[END_REF]. A negative pressure was applied to stem pieces in a standard centrifuge with a custom-built, 26 cm rotor. Each stem segment was only spun once in the centrifuge. After centrifugation, a 2 cm long segment containing a node was excised under water from the middle of the stem segment. The segment was connected to the XYL'EM apparatus (Bronkhorst, Montigny-les-Cormeilles, France) to measure its hydraulic conductance using a solution of 10 mM KCl and 1mM CaCl2 in deionized ultrapure water. The sample was then resaturated with pressurised water to determine the percentage of loss of conductance caused by embolised conduits. For each pressure point in the vulnerability curve (VC), one to seven grass stems were used with 14 to 36 pieces per curve (min-max), and one S-shaped curve per population was fitted (Fig. S1) according to a sigmoid function [START_REF] Pammenter | A mathematical and statistical analysis of the curves illustrating vulnerability of xylem to cavitation[END_REF]. Embolism resistance was expressed by the 50% or 88% percentage loss of hydraulic conductance (P 50 , P 88 ). P 50 is the metrics most used to compare hydraulic thresholds between species and populations while P 88 was shown to be the embolism threshold leading to irreversible drought damage in angiosperm trees [START_REF] Urli | Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees[END_REF]

Stem anatomy

To evaluate which stem characters contribute to the variation in embolism resistance observed among the D. glomerata populations, light microscopy observations were performed on cross sections of stemspreviously used for the measurement of embolism resistanceat the level of internodes (from three stems per population). After vulnerability curve determination, samples were frozen until processed for anatomy. Samples were defrost in water, stored in 50% ethanol and then were embedded with LR White (hard grade, London Resin, UK) following [START_REF] Hamann | A comparison of paraffin and resin-based techniques used in bark anatomy[END_REF], sectioned with a rotary microtome (Leica RM 2265) equipped with disposable blades.

Transverse sections of 4 µm were made, heat-attached to the slides, stained with toluidine blue, and mounted in Entellan®. The slides were observed with a Leica DM2500 light microscope equipped with a Leica DFC-425C digital camera (Leica Microscopes, Wetzlar, Germany). A range of stem anatomical characters [START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF], such as total stem area (A S ), area of lignified outer stem tissue (A LIG ), surface area of fibre wall (A FW ) and fibre lumen (A FL ), thickness of the metaxylem vessel wall (T VW ), hydraulically weighted diameter of vessels (D HV ), and traits derived from these measurements, such as the proportion of lignified tissue per total stem area (P LIG ), the proportion of cell wall per fiber (P CW F), the proportion of total fiber wall area in lignified area (P FW F*A LIG ), the proportion of total fiber wall in lignified area per stem area ((P FW F*A LIG ):A S ). Observations were made based on three individuals per population and at least 30 measurements per feature using ImageJ v 1.43 (National Institutes of Health, Bethesda, Maryland, USA).

The diameter of the vessels (D V ) was calculated based on the lumen area that was considered to be a circle following the equation:

𝐷𝑣 = √ 4𝐴 𝜋 Eqn (1)
where D is the vessel diameter and A is the area of the vessel. The hydraulically weighted vessel diameter (D Hv ; weights diameters of vessels according to their hydraulic conductance) was calculated following the equation [START_REF] Sperry | Xylem embolism in ring-porous, diffuse-porous, and coniferous trees of northern Utah and interior Alaska[END_REF]:

𝐷ℎ𝑣 = ∑ 𝐷 5 ∑ 𝐷 4 Eqn (2)
where D is the vessel diameter as measured in eqn. (1).

Leaf traits under full optimum conditions (Experiment 3)

In grasses, leaf tissue density can be assessed both by leaf dry matter content (Cornelissen et al., 2003) which is a reasonable indirect measure of dry matter concentration [START_REF] Shipley | Dry matter content as a measure of dry matter concentration in plants and their parts[END_REF] and resistance of the leaf to mechanical fracture, that can be measured in a variety of ways [START_REF] Ang | Novel way of measuring the fracture toughness of leaves and other thin films using a single inclined razor blade[END_REF][START_REF] Aranwela | Methods of assessing leaf fracture properties[END_REF][START_REF] Onoda | Global patterns of leaf mechanical properties[END_REF]. For experiment 3, in early spring (2016), at the time before reproductive stage, one fully extended leaf was collected from one plant per pot (four replicates). Fracture toughness was measured with a portable mechanical measuring device (Instron® Inspec 2200) adapted for cutting with a single razor cut, following Ang et al [START_REF] Ang | Novel way of measuring the fracture toughness of leaves and other thin films using a single inclined razor blade[END_REF]. Leaf blades were placed on the cutting platform across a gap 2 mm wide permitting the passage of the diagonally angled blade in the middle of each leaf blade (single edged blade, "Personna GEM, 0.23 mm thickness, Cat.# 71962). Leaf toughness, the energy required to shear the leaf cross section in J m -² was calculated after subtracting initial elastic loading and final elastic unloading, as well as friction energy from the force distance curves. As in [START_REF] Ang | Novel way of measuring the fracture toughness of leaves and other thin films using a single inclined razor blade[END_REF], the initial cut was followed by a second pass that measured just the friction force generated by the blade against the cut surfaces of the leaf. Leaves were cut with a razor angle of 20° and at a crosshead speed of 0.1 mm s -1 . Leaf thickness and width were measured with a binocular at the central vein of the leaf and at the widest part of the leaf respectively. The leaf was then immediately placed into a tube with the cut end submerged in deionized water and stored in the dark at 4 °C for 24 h. The fresh and dry leaf biomass (g) were weighed to obtain leaf dry matter content (LDMC; Leaf dry matter content is the oven-dry mass (mg) of a leaf divided by its water-saturated fresh mass (g), expressed in mg g -1 ) following [START_REF] Perez-Harguindeguy | New handbook for standardised measurement of plant functional traits worldwide[END_REF]. This trait is used as a proxy for leaf density which is a trait more difficult to measure [START_REF] Shipley | Dry matter content as a measure of dry matter concentration in plants and their parts[END_REF].

Statistical analysis

All statistical analyses were performed with Rstudio (version 0.98.501). The effect of biogeographical origin was tested with anova ('lmtest' package) followed with Tukey's post hoc tests. A principal component analysis (PCA; 'ade4' package) was performed on the twelve variables showing significant differences between origins, scaled and centered, to visualize their covariations. The four bioclimatic variables (www.worldclim.org) chosen to characterize the drought and frost occurrence in the sites of origins of the populations (annual precipitations, precipitations of the driest quarter, aridity index [START_REF] Trabucco | Climate change mitigation through afforestation/reforestation: A global analysis of hydrologic impacts with four case studies[END_REF] and minimum temperature of the coldest months) were used as supplementary variables in the PCA. Pairwise correlations between all measured traits for each season were evaluated using Pearson's method ('Hmisc' package). We tested the relationships between key-variables using simple linear regressions.

RESULTS

Across the twelve populations of D. glomerata studied, P 50 ranged from -3.06 to -6.36 MPa (Fig. 1a), while P 88 ranged from -5.06 to -11.6 MPa (Fig. 1b). The Mediterranean populations had significantly lower P 50 (-5.8 and -10.6 MPa respectively) than the Temperate populations (-3.3 and -6.1 MPa respectively), while the Northern populations showed intermediate embolism thresholds (Table 2). In addition, P 50 of the Mediterranean population Kasbah significantly differed according to the growing environment (Fig. 2), either in pots (P 50 = -6.05±0.31) or in the field (P 50 = -5.21±0.29). P 50 and P 88 measured on potted irrigated plants were both highly and more correlated (r=0.88 and 0.89, P<0.001, Table 3) with the mean precipitation of the driest quarter of the year than with the annual precipitation of the origin site. Both embolism thresholds were, however, less strongly correlated with the aridity index of the site of origin (r=0.60, P=0.039). The mean aridity index was 1.20 for Northern populations (less arid) compared to 0.82 (more arid) for Temperate populations despite of the greater rainfall in Temperate areas. In addition, no correlation was detected between cavitation resistance and minimum temperatures of the coldest months of the sites studied.

Plant dehydration survival in pots was significantly greater (Table 2) for the Mediterranean populations (58%) than for the group of Northern and Temperate ones (7 to 13%).

Although leaf thickness did not discriminate the populations according to their origin, leaf width almost doubled with increasing latitude from on average 4.3 mm in Mediterranean populations to 8 mm in Northern ones. Mediterranean populations also had a higher LDMC (20% greater) and a very significantly greater leaf fracture toughness (2.5 fold greater) than the Northern and Temperate populations.

The Mediterranean populations were distinguished from the others by having thinner stems with a higher fibre wall proportion in lignified area per stem (Table 2). The traits for Northern and Temperate populations were mostly not significantly different although the proportion of total fibre wall area in lignified area was greater in Northern populations. The thickness of metaxylem vessel wall highly discriminated all origins and spanned from 1.4, 1.2 and 1.1 μm in Mediterranean Northern and Temperate populations respectively (Table 2). P 88 was highly and better correlated to dehydration survival (r=-0.79, Table 3, Fig. 4a) and leaf traits such as fracture toughness (r=-0.78, Table 3, Fig. 4b) and LDMC (r=-0.82, Table 3, Fig. 3c), than P 50 . A highly significant correlation was found between both embolism thresholds (Table 3, Fig. 5,6) and the thickness of metaxylem vessel wall in stems (T VW : r=-0.92 for P 50 ).

Notably no other stem anatomical trait was correlated with either embolism thresholds. In contrast, plant dehydration survival was correlated to the total stem area (A S : r=-0.66) and therefore with the area of lignified outer stem tissue (A LIG : r=-0.61) as well as with the proportion of fibre wall in lignified area over the entire stem area ((P FW F*A LIG ):A s r=0.59).

Overall, the covariation between LDMC, leaf fracture toughness, embolism thresholds and dehydration tolerance was very significant (Table 3). Unexpectedly, leaf toughness was not correlated with any trait associated with stem lignification over the entire data set. However, a PCA (Fig. 7a,b) clearly illustrates that high embolism resistance and dehydration survival associated with small and more lignified lamina and lignified stems discriminate the group of Mediterranean populations. In contrast, Temperate and Northern populations were characterised by low dehydration survival and embolism resistance, bigger leaves and stems with thickerwalled fibres especially for Northern populations.

DISCUSSION

This study is the first to explore intraspecific variability of embolism resistance, whole plant dehydration tolerance and associated traits in an herbaceous grass species. Our results show a huge range of embolism thresholds amongst the twelve D. glomerata populations studied, reflecting its biogeographical distribution and corresponding with some key traits.

Large intraspecific variability in cocksfoot

If we compare the interspecific range for P 50 [START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF] observed within 18 Poaceae species native to similar mesic and xeric environments (-3.2 to -7.5 MPa) with the intraspecific variability in D. glomerata found here (-3.06 to -6.36 MPa), the overlap is striking. Only Stipa pennata, a grass native to arid rangelands exhibited a lower P 50 (-7.5 MPa) compared to the Mediterranean populations of D. glomerata. In addition, this intra-specific variation is much greater than that reported for a dicot herbaceous perennial Solidago Canadensis sampled from dry to humid sites and showing P 50 ranging from -2.37 to -3.08 MPa [START_REF] Nolf | Physiological acclimation to drought stress in Solidago canadensis[END_REF]. The intraspecific range in P 50 variation within cocksfoot matches with most of the woody species studied in angiosperms (-2 to -4 MPa) and gymnosperms (-4 to -8 MPa), although far from the extreme embolism threshold (-18.8 MPa) found so far in the gymnosperm Callitris tuberculata [START_REF] Larter | Extreme Aridity Pushes Trees to Their Physical Limits[END_REF]. This intraspecific variability is strikingly large in comparison with former results on woody species. For instance, across 17 populations of Fagus sylvatica originating from sites in a large European latitudinal gradient with annual precipitations spanning from 563 to 1362 mm, P 50 was relatively constant from -2.8 to -3.2 MPa [START_REF] Wortemann | Genotypic variability and phenotypic plasticity of cavitation resistance in Fagus sylvatica L. across Europe[END_REF]. No significant differences in embolism resistance (-3.9 to -4.1 MPa) was found either across 24 populations of Pinus pinaster from Northern African and French sites [START_REF] Lamy | Uniform Selection as a Primary Force Reducing Population Genetic Differentiation of Cavitation Resistance across a Species Range[END_REF].

Nevertheless, plasticity in vulnerability of embolism (from -3 to -6 MPa) was shown in the very dry limits of Pinus canariensis under a strong aridity gradient [START_REF] Lopez | Plasticity in Vulnerability to Cavitation of Pinus canariensis Occurs Only at the Driest End of an Aridity Gradient[END_REF]. It could be interesting to explore whether the high embolism resistance of D. glomerata from the most arid sites (P 50 < -5 MPa) could be ascribed to a shallower rooting system than woody species by analogy with shrubs that were found to be more embolism tolerant than trees in the same habitat, probably reflecting differences in rooting depth and drought tolerance (Brendel & Cochard, 2011). The remarkable range in embolism resistance could be added to the list of factors contributing to explain why cocksfoot covers a greater span of biomes that that of most woody species.

Our intraspecific comparison is based on plants grown in similar optimum pot conditions for two years, underlining that the large differences within populations are genetically determined. Yet, our results also show a high plasticity of embolism threshold since for the tested Kasbah population, P 50 significantly varied between potted and field-grown plants of the same population. Many factors differ such as plant age at sampling date as well as many biotic and abiotic factors (soil depth, plant density…) between these two growing conditions for the same population. However, it may be hypothesized that a less negative P 50 of field-growing plants may be ascribed in particular to a deeper rooting system (130 cm in the field versus 20 cm in pots) which enhances dehydration avoidance versus dehydration tolerance and may have impacted hydraulic adaptations [START_REF] Scholz | Osmotic and elastic adjustments in cold desert shrubs differing in rooting depth: coping with drought and subzero temperatures[END_REF]. It could be interesting to measure embolism resistance in the same populations of cocksfoot growing all in a similar deep soil in order to analyse the impact of the expression of full rooting depth on embolism thresholds. However, it can therefore be assumed that if sampling was done using populations growing in their natural environment, our results would have probably generated stronger genotype x environments interactions, and even further increased the range of intra-specific variability in embolism thresholds. Significant site x population interactions were for instance found in beech [START_REF] Herbette | Insights into xylem vulnerability to cavitation in Fagus sylvatica L.: phenotypic and environmental sources of variability[END_REF][START_REF] Wortemann | Genotypic variability and phenotypic plasticity of cavitation resistance in Fagus sylvatica L. across Europe[END_REF] underlining the high phenotypic plasticity of embolism resistance. Further work is necessary to assess intra-specific P 50 and P 88 plasticity as a response to environments (e.g. soil depth, soil nutrients and water availability).

Relationships between embolism resistance and environmental limiting factors

Our results show a correlation between the annual and driest quarter precipitations of the sites studied with embolism thresholds measured on plants grown in a common garden under optimum irrigation. As a general pattern, dehydration survival in pots was greater for the populations coming from the driest sites with P 50 and P 88 more negative than -4.50 and -9.3 MPa respectively.

Plant mortality under severe drought when the resource is limited (shallow soils) can be then predicted by the hydraulic failure model assuming that a significant loss of conductivity can trigger plant mortality [START_REF] Mcdowell | Evaluating theories of drought-induced vegetation mortality using a multimodel-experiment framework[END_REF]. Our results are in line with a meta-analysis of tree species showing that hydraulic traits capture key mechanisms determining tree death [START_REF] Anderegg | Meta-analysis reveals that hydraulic traits explain cross-species patterns of drought-induced tree mortality across the globe[END_REF]. However, embolism resistance was less strongly correlated with the aridity index of the site of origin in contradiction to a former study dealing with a range of Temperate to semiarid grass species that showed a strong correlation between P 50 and aridity [START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF]. It would be interesting to test whether this interspecific correlation would hold when including herbaceous species from the highest latitudes. In D. glomerata, Northern populations showed a trend to more negative P 50 values than Temperate populations and are more frost tolerant, suggesting that the Northern populations could have also developed greater embolism resistance as a mechanism of frost tolerance [START_REF] Lens | Embolism resistance as a key mechanism to understand adaptive plant strategies[END_REF]. Both drought and freezing-thawing of stems induce a loss of hydraulic conductivity in woody plants [START_REF] Feng | Investigations Concerning Cavitation and Frost Fatigue in Clonal 84K Poplar Using High-Resolution Cavitron Measurements[END_REF]. Frost 'drought' had a large effect on plant water transport, adaptations in hydraulic safety and related anatomical parameters [START_REF] Mayr | Frost drought in conifers at the alpine timberline: Xylem dysfunction and adaptations[END_REF] and the propensity for freeze induced embolism increases as conduit diameter increases [START_REF] Zanne | Three keys to the radiation of angiosperms into freezing environments[END_REF]. The relationships between recovery from winter embolism and anatomical vessel traits also varied according to functional groups of tree species [START_REF] Niu | Divergence in strategies for coping with winter embolism among co-occurring temperate tree species: the role of positive xylem pressure, wood type and tree stature[END_REF]. The role of hydraulic dysfunction under frost remains to be explored in herbaceous species.

Tissue lignification and embolism resistance

In pseudostems, partly constituted with leaf sheaths, we found a strong positive correlation between the thickness of the metaxylem vessel wall and embolism resistance while lignification characters are not correlated with P 50 nor P 88 . Despite a significant positive correlation [START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF] between embolism resistance and stem lignification amongst 19 grass species (in terms of the thickness of the fibre walls and the relative proportion of lignified tissue per stem area), this trade-off is lacking within populations of D. glomerata. This lack of correlation is noteworthy given the huge range in P 50 variation within cocksfoot. There are slight indications that stem lignification may contribute to differences in embolism resistance. For instance, Figure 6ab shows that the proportion of lignified area per stem area clusters with other key traits for the embolism resistant Mediterranean populations. Furthermore, the two most resistant Mediterranean populations have more lignified fibres inside and surrounding the vascular bundles (Fig. S2) compared to the other cocksfoot populations studied. However, this character is difficult to quantify and seemingly shows at random variation amongst the remaining populations. Since we have sectioned internodes, we had to restrict ourselves to measurements of solitary vessels. It is plausible that our observations also reflect a correlation between more negative P 50 /P 88 values and increased inter-vessel wall thickness compared to vessel diameter (so called thickness-to span ratio) in case of vessel multiples that are much more present in the nodal parts of the stem.

Vessel wall reinforcement was suggested to be required for embolism resistance in order to prevent wall implosion when xylem pressure is very negative (Hacke et al., 2001). However, vessels have never been observed to implode in stems, probably because embolism events occur before the critical vessel implosion threshold is reached. Whatever the exact mechanism(s) operating during xylem embolism dysfunction in D. glomerata, it appears that plants with more fracture resistant leaves have stems that are more resistant to embolism than plants with less resistant leaves. In addition to possibly reducing the probability of lethal embolism (Hacke and Sperry, 2001), mechanically robust leaf architecture will also play a role in mechanically supporting water-stressed leaves and protecting leaf meristems as discussed below. This perhaps at least partly explains the tendency towards narrower, more compact, denser, tougher leaves in the Mediterranean ecotypes that are more likely exposed to more drought conditions. Of course leaf lamina traits are anatomically and functionally grade into the more proximal leaf sheath [START_REF] Bell | Plant Form[END_REF] which is also important for mechanical robustness and resistance to dehydration of the self-supporting meristems of D. glomerata.

Drought survival in perennial herbaceous species: hydraulic leaf traits may not be crucial

A general tenet of ecophysiology of drought adaptation is based on the assumption of a strong linkage between hydraulic and stomatal traits especially in woody species [START_REF] Skelton | Predicting plant vulnerability to drought in biodiverse regions using functional traits[END_REF], Bartlett et al., 2016) and also in herbaceous species [START_REF] Holloway-Phillips | Minimum hydraulic safety leads to maximum water-use efficiency in a forage grass[END_REF][START_REF] Lens | Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees[END_REF][START_REF] Nardini | Trade-offs between leaf hydraulic capacity and drought vulnerability: morpho-anatomical bases, carbon costs and ecological consequences[END_REF]. Regarding the extreme case of drought plant mortality in both annual and perennial grasses, basal leaf meristems are the tissues surviving longer and able to regenerate when the mature blades are dead [START_REF] Van Peer | Species richness and susceptibility to heat and drought extremes in synthesized grassland ecosystems: compositional vs physiological effects[END_REF]. These leaf meristems enclosed in mature leaf sheaths, are protected from intense evaporation and can survive more intense water deficits than older tissues [START_REF] Barlow | Drought responses of apical meristems[END_REF][START_REF] Munns | Solute accumulation in the apex and leaves of wheat during water-stress[END_REF]. As they accumulate carbohydrates such as fructans [START_REF] Schnyder | Growth rates and assimilate partitioning in the elongation zone of tall fescuue leaf blades at high and low irradiance[END_REF]Nelson, 1989, Volaire et al., 1998), apices exhibit the greatest osmotic adjustment relative to other tissues during drought [START_REF] Matsuda | Stress-induced osmotic adjustment in growing regions of barley leaves[END_REF]Riazi, 1981, West et al., 1990). In addition, the strategy of summer dormancy [START_REF] Gillepsie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF][START_REF] Gillepsie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF][START_REF] Volaire | Summer dormancy in perennial temperate grasses[END_REF] found in some species and populations of perennial species, triggers leaf senescence and meristem dehydration to reduce plant metabolism and consequently enhance drought survival. Our results show that the Mediterranean populations of D. glomerata which are the most summer dormant, 'sacrifice' their lamina in summer (irrespectively of the water supply) but also have the highest embolism resistance and dehydration tolerance. In frost-prone areas too, perennial herbaceous species avoid freezing by senescing aboveground tissues and overwintering as underground storage organs [START_REF] Zanne | Three keys to the radiation of angiosperms into freezing environments[END_REF]. For dehydration tolerance under frost or drought [START_REF] Gillepsie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF], these model species can then raise the relevance of the paradigm of hydraulic safety margins in leaves for stress survival. The leaf (lamina)-level drought tolerance (Nardini andLuglio, 2014, Ocheltree et al., 2016) may not be the key-level to explore drought survival in all species.

However, studies linking hydraulic conductivity and lamina anatomy [START_REF] Scoffoni | Leaf vein xylem conduit diameter influences susceptibility to embolism and hydraulic decline[END_REF] could be fruitful provided they take into account the continuum between expanded blades, sheaths and meristematic tissues. The co-variation between hydraulic traits and anatomical traits in meristems but also in root tissues should be explored further in grasses.

In conclusion, this study investigated dehydration survival amongst twelve populations of D. glomerata from a latitudinal gradient from Mediterranean to Scandinavian areas, representing only a restricted range of its much larger biogeographical distribution. D. glomerata represents an 'ideal' species for analyzing the interactions genotype x environment because of its biogeographical distribution is huge and spans over Europe and Asia [START_REF] Borrill | Evolution and genetic resources in cocksfoot[END_REF] and its phylogenetic relationships between populations are being investigated and show high genetic variation within both populations and geographical origins [START_REF] Lumaret | Cytology, genetics, and evolution in the genus Dactylis[END_REF][START_REF] Xie | Diversity comparison and phylogenetic relationships of cocksfoot (Dactylis glomerata L.) germplasm as revealed by SSR markers[END_REF].

Compared to woody species, the large degree of intra-specific variability in dehydration tolerance and embolism resistance within cocksfoot has consequences for its sensitivity to climate shifts, possible migrations and future biogeographical distribution [START_REF] Anderegg | Spatial and temporal variation in plant hydraulic traits and their relevance for climate change impacts on vegetation[END_REF]. In addition, given that the measurement of plant drought survival is experimentally difficult to standardize, our study demonstrating the key role of embolism threshold and tissue lignification as proxy for dehydration tolerance provide highly relevant knowledge to screen genetic resources and new breeding material.

Finally, this study calls for extending the numerous studies on mortality factors and lethal embolism thresholds in woody species to a large range of herbaceous species, both at inter and intra-specific levels, under contrasting environmental constraints. 1 0,00 0,00 0,10 0,19 0,00 0,00 0,13 0,18 0,89 0,45 0,67 0,38 0,37 0,88 0,14 0,09 0,00 0,00 0,00 0,76 0,04 P 50 0,94 1 0,02 0,21 0,25 0,02 0,03 0,24 0,45 0,58 0,51 0,47 0,38 0,27 0,73 0,18 0,33 0,00 0,00 0,00 0,92 0,04 SURV -0,79 -0,65 1 0,01 0,02 0,00 0,00 0,02 0,03 0,85 0,09 0,73 0,06 0,13 0,80 0,04 0,07 0,11 0,00 0,01 0,25 0,01 Lwidth 0,50 0,39 -0,71 1 0,01 0,00 0,00 0,00 0,02 0,40 0,14 0,87 0,16 0,35 0,15 0,09 0,71 0,36 0,10 0,21 0,00 0,00 Lthick 0,40 0,36 -0,66 0,72 1 0,02 0,02 0,20 0,42 0,53 0,08 0,48 0,04 0,07 0,30 0,73 0,81 0,78 0,06 0,12 0,03 0,01 LDMC -0,82 -0,68 0,87 -0,85 -0,65 1 0,00 0,01 0,07 0,50 0,23 0,66 0,18 0,22 0,64 0,07 0,13 0,07 0,00 0,04 0,09 0,01 LFrac -0,78 -0,61 0,92 -0,84 -0,68 0,97 1 0,02 0,03 0,81 0,21 0,62 0,16 0,18 0,62 0,08 0,10 0,11 0,01 0,05 0,08 0,00 A S 0,46 0,37 -0,66 0,83 0,40 -0,70 -0,67 1 0,00 0,24 0,15 0,22 0,30 0,99 0,14 0,00 0,90 0,30 0,11 0,19 0,08 0,13 A LIG 0,41 0,24 -0,61 0,65 0,26 -0,54 -0,62 0,78 1 0,36 0,24 0,24 0,42 0,88 0,06 0,08 0,71 0,34 0,30 0,49 0,17 0,13 P LIG -0,04 -0,18 0,06 -0,27 -0,20 0,22 0,08 -0,37 0,29 1 0,81 0,80 0,87 0,96 0,67 0,18 0,37 0,92 0,53 0,42 0,52 0,99 A F 0,24 0,21 -0,52 0,45 0,53 -0,37 -0,39 0,44 0,37 -0,08 1 0,66 0,00 0,06 0,09 0,99 0,87 0,96 0,19 0,23 0,41 0,32 A FL -0,14 -0,23 0,11 0,05 -0,22 0,14 0,16 0,38 0,37 -0,08 0,14 1 0,64 0,01 0,05 0,76 0,21 0,64 0,26 0,17 0,92 0,10 A FW 0,28 0,28 -0,55 0,43 0,59 -0,41 -0,44 0,33 0,26 -0,05 0,96 -0,15 1 0,00 0,28 0,92 0,60 0,93 0,09 0,10 0,43 0,13 P CW F 0,28 0,35 -0,46 0,29 0,53 -0,38 -0,41 0,00 -0,05 -0,02 0,56 -0,73 0,77 1 0,60 0,90 0,21 0,65 0,06 0,03 0,49 0,02 P FW F*A LIG -0,05 -0,11 -0,08 0,44 0,33 -0,15 -0,16 0,45 0,56 0,14 0,52 0,59 0,34 -0,17 1 0,60 0,11 0,68 0,69 0,53 0,20 0,78 (P FW F*A LIG ):A s -0,45 -0,42 0,59 -0,50 -0,11 0,53 0,53 -0,76 -0,52 0,41 0,00 -0,10 0,03 0,04 0,17 1 0,59 0,16 0,08 0,08 0,47 0,24 D HV -0,51 -0,31 0,53 -0,12 -0,08 0,46 0,50 0,04 -0,12 -0,28 -0,05 0,39 -0,17 -0,39 0,49 0,17 1 0,44 0,19 0,37 0,97 0,36 T VW -0,85 -0,92 0,48 -0,29 -0,09 0,53 0,49 -0,33 -0,30 0,03 0,02 0,15 -0,03 -0,14 0,13 0,44 0,25 1 0,01 0,01 0,96 0,09 P SUM 0,88 0,89 -0,84 0,50 0,55 -0,77 -0,74 0,48 0,32 -0,20 0,40 -0,35 0,51 0,56 -0,13 -0,53 -0,40 -0,71 1 0,00 0,83 0,01 P YEAR 0,75 0,86 -0,71 0,39 0,48 -0,61 -0,57 0,40 0,22 -0,26 0,38 -0,42 0,50 0,61 -0,20 -0,52 -0,29 -0,70 0,94 1 0,83 0,01 T MIN 0,10 -0,03 0,36 -0,82 -0,62 0,51 0,53 -0,53 -0,42 0,21 -0,26 -0,03 -0,25 -0,22 -0,40 0,23 0,01 -0,02 -0,07 -0,07 1 0,03 Aridity 0,60 0,60 -0,71 0,76 0,73 -0,74 -0,77 0,47 0,46 0,01 0,32 -0,49 0,46 0,65 0,09 -0,37 -0,29 -0,51 0,70 0,70 -0,62 1

ACKNOWLEDGMENTS

Legend of figures

  Thank to Pascal Chapon and Mathieu Ogier for excellent technical assistance, the 'Terrain d'expérience platform' and the 'Plateforme d'Analyses Chimiques en Ecologie' both at CEFE-CNRS (Labex CEMEB). Thanks to Mark Norton, Jeffrey Clary, Rajae Kallida, N. Shaimi, A. Palmé, S. Fourtier, B. Reid, G. Testa, and V. Copani, for providing seeds. This study was supported by the European Research Council (ERC) Starting Grant Project 'Ecophysiological and 25 753

Fig. 1 .Fig. 2 .

 12 Fig. 1. Thresholds of embolism resistance in cocksfoot. Means and standard errors of water potentials at 50 % embolism (a, P 50 ) and at 88% embolism (b, P 88 ) in stems of twelve populations of Dactylis glomerata of contrasting origins (red = Mediterranean; blue = Northern; green = Temperate)

Fig. 3 .

 3 Fig. 3. Embolism resistance and aridity of origin site in cocksfoot. Linear relationships between mean precipitation of the driest quarter at origin site and water potential at 50% embolism (P 50 ) in twelve populations of Dactylis glomerata of contrasting origins (red = Mediterranean; blue = Northern; green = Temperate). Bars correspond to standard errors.

Fig. 4 .

 4 Fig. 4. Embolism resistance and (a) dehydration tolerance, (b) leaf fracture toughness and (c) leaf dry matter content in cocksfoot. Linear relationships between water potential at 88% embolism (P 88 ) and (a) the proportion of surviving plants at soil wilting point in short pots (dehydration tolerance), (b) leaf fracture toughness of irrigated plants and (c) leaf dry matter content (LDMC) of irrigated plants in twelve populations of Dactylis glomerata of contrasting origins (red = Mediterranean; blue = Northern; green = Temperate). Bars correspond to standard errors.

Fig. 5

 5 Fig. 5 Embolism resistance and stem anatomy in cocksfoot. Linear relationships between water potential at 50% embolism (P 50 ) with thickness of metaxylem vessel walls in stem in twelve populations of Dactylis glomerata of contrasting origins (red= Mediterranean; blue= Northern; green= Temperate). Bars correspond to standard errors.

29 29Fig. 6

 296 Fig. 6 Thickness of metaxylem vessel wall in stems of cocksfoot: three populations of Dactylis glomerata of contrasting origins: a = 'car' (Temperate); b = 'swe' (Northern); c ='ma1' (Mediterranean). The arrows point to wall of the metaxylem vessels.

Fig. 7

 7 Fig. 7 PCA of 12 plant traits and 12 populations of cocksfoot. Principal component analysis (factorial axes 1 and 2) showing (a) the distribution of the twelve traits significantly discriminating origins of populations (only P 50 was included since it is highly correlated to P 88 ), see table 2 and (b) twelve populations of Dactylis glomerata of contrasting origins (red= Mediterranean; blue= Northern; green= Temperate). Climatic data of the origin or breeding site: Annual precipitations (Pyear), precipitations of the driest quarter (Pdry), aridity index (aridity) and minimum temperature of the coldest months (Tmin) were included as supplementary variables (bold and italics characters).

  Fig.3

  Fig. 5

Table 1 .

 1 List of the twelve populations of Dactylis glomerata. Climatic data associated either with their origin sites of collection or 754 sites of breeding (for cultivars cv in Temperate and Northern areas) from the WorldClim data set (http://www.worldclim.org; (Hijmans 755 et al., 2005). Seeds from Northern populations were provided by the Nordic Genetic Resource Center (NordGen). Temperate 756 populations were provided by the plant genetic resources information system of the National Institute for Agronomical Research 757 (INRA, France, Lusignan). The Sicilian ecotype came from University of Catania, Italy. The Moroccan ecotypes came from INRA

	758
	759	Rabat, Morocco

Table 2 :

 2 Results of variance analysis and Tukey test between means of variables for the three origins of Dactylis glomerata populations. ***: p<0.001; **: p<0.01; *: p<0.05; ns: nonsignificant. For each variable, different subscript letters indicate significant differences between

	origins
			Biogeographic origin
			of D. glomerata populations
		P value	Medit.	Northern	Temperate
	SURV = Plant dehydration survival (%)	<0.001***	58.2 a	13.1 b	6.9 b
	P 50 = Embolism pressure threshold for 50%	0.0016**	-5.8 a	-4.3 ab	-3.3 b
	embolism (MPa)
	P 88 = Embolism pressure threshold for 88%	<0.001***	-10.4 a	-7.6 ab	-6.1 b
	embolism (MPa)
	Lwidth = Leaf width (mm)	<0.001***	4.28 a	7.97 c	6.22 b
	Lthick = Leaf thickness (mm)	0.13ns	0.30	0.36	0.35
	LDMC = Leaf Dry Matter content (mg g DM -1 )	<0.001***	232.72 a	185.75 b	188.41 b
	LFrac = Leaf fracture resistance (J m-²)	<0.001***	478.6 a	188.47 b	225.55 b
	A S = Total stem area (mm²)	0.006**	2.07 a	3.09 b	2.9 b
	A LIG = lignified area (m 2 )	0.02*	0.27 a	0.38 b	0.33 ab
	P LIG = Proportion of lignified area per total stem	0.02*	0.1308 b	0.1258 ab	0.115 a
	area
	A F = fiber cell area (m 2 )	0.34ns	70.61	77.21	77.56
	A FL = fiber lumen area (m 2 )	0.50ns	15.95	14.16	14.76
	A FW = fiber wall area (m 2 )	0.12ns	54.66	63.04	62.79
	P CW F = Proportion of cell wall per fiber	0.029*	0.77 a	0.82 b	0.81 ab
	P FW F*A LIG = Proportion of total fiber wall area in	0.037*	0.30 ab	0.33 b	0.29 a
	lignified area
	(P FW F*A LIG ):A S = Proportion of total fiber wall in	0.002**	0.156 a	0.117 b	0.102 b
	lignified area per stem area
	D HV = hydraulically weighted diameter of vessels	0.17ns	23.04	21.77	21.18
	(m)
	T VW = Thickness of metaxylem vessel wall (m)	<0.001***	1.426 c	1.226 b	1.109 a

Table 3 :

 3 Pearson coefficients of correlations (lower part of the table) and P values (top part of the table) between traits measured on twelve populations of Dactylis glomerata. For abbreviations and units see table 1 and 2. Climatic variables of the origin sites (or breeding sites) of the populations: P DRY = Mean precipitations of the driest three months; P YEAR = Mean annual precipitations, T MIN = mean minimum temperatures of the coldest months, Aridity = aridity index. P<0.05 (italics,

	bold), P<0.01 (bold), P<0.001 (bold underlined)
							(P FW F*
						P FW F*	A LIG )
	P 88	P 50	SURV Lwidth Lthick LDMC LFrac A S	A LIG P LIG A F	A FL A FW P CW F	A LIG	:A s	D HV T VW P SUM P YEAR T MIN Aridity
	P 88

biophysical constraints on domestication in crop plants' [Grant ERC-StG-2014-639706-CONSTRAINTS], and by the Agence National de la Recherche [PRAISE project, ANR-13-BIOADAPT-0015]. Pauline Bristiel was financially supported by INRA (EA department).