domain: sdv sde

text:
Françoise Hennion
email: francoise.hennion@univ-rennes1.fr

Bastien Labarrere

Marine Renaudon

Andreas Prinzing

Plasticity in plant populations may be constrained

Keywords: Plant population plasticity constrained by costs, environments, integration Pringlea antiscorbutica, Ranunculus biternatus, Ranunculus pseudotrullifolius, abiotic treatment, costs, environments, performance, phenotypic integration, phenotypic plasticity, plant species, plant populations

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INTRODUCTION

Climate change alters the environments and plants, being sessile organisms, have to cope with these alterations (Sultan, 2000). Phenotypic plasticity is the capacity of a given genotype to result in different phenotypes under different environmental conditions, and plants are well known for having generally high plasticity [START_REF] Bradshaw | Evolutionary significance of phenotypic plasticity in plants[END_REF]Schlichting, 1986;Sultan, 2000). Phenotypic plasticity is one of the major means by which plants cope with environmental heterogeneity (Valladares et al., 2007;[START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF] and is now considered as part of a species' adaptive potential to environmental changes [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF][START_REF] Hoffmann | Climate change and evolutionary adaptation[END_REF][START_REF] Callahan | Phenotypic plasticity, costs of phenotypes, and costs of plasticity: toward an integrative view[END_REF]. However, the expression of a phenotype under a certain environment is an integrative result of all local responses of many modular traits and their interactions, so plasticity should be analysed within a frame of trait variation and co-variation to understand phenotypic response and evolution [START_REF] Pigliucci | Phenotypic integration: studying the ecology and evolution of complex phenotypes[END_REF][START_REF] Wang | Morphological canalization, integration, and plasticity in response to population density in Abutilon theophrasti: Influences of soil conditions and growth stages[END_REF]. For instance, plasticity is not always adaptive, only plasticity producing phenotypes in the same direction as natural selection, and thus with the ability to increase plant fitness, is considered adaptive (Bell and Galloway, 2008), a currently developing research question [START_REF] Murren | Constraints on the evolution of phenotypic plasticity: limits and costs of phenotype and plasticity[END_REF][START_REF] Molina-Montenegro | Adaptive phenotypic plasticity and competitive ability deployed under a climate change scenario may promote the invasion of Poa annua in Antarctica[END_REF][START_REF] Diamond | Buying time: plasticity and population persistence[END_REF][START_REF] Fox | Beyond buying time: the role of plasticity in phenotypic adaptation to rapid environmental change[END_REF]. If plasticity is too costly or too limited by other constraints, then plasticity may reduce rather than increase performance and finally fitness, but our understanding of the costs or limitations of plasticity is still rather low [START_REF] Pigliucci | Evolution of phenotypic plasticity: where are we going now?[END_REF]Snell-Rood et al., 2018;[START_REF] Wei | Elevation-dependent selection for plasticity in leaf and root traits of Polygonum hydropiper in response to flooding[END_REF]Auld et al., 2009).

A plant may be only little plastic because of energy-allocation constraints: plasticity is costly and may reduce performance (Auld et al., 2009;[START_REF] Bongers | Drought responses, phenotypic plasticity and survival of Mediterranean species in two different microclimatic sites[END_REF]. Plastic response is dependent upon physiological and morphological machinery that involves the acquisition of environmental information, transduction of the signal, and the expression of a phenotypic response [START_REF] Vinocur | Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations[END_REF][START_REF] Dewitt | Costs and limits of phenotypic plasticity[END_REF]. Such machinery induces costs in organisms, which may be classified in two kinds: constitutive and induced [START_REF] Callahan | Phenotypic plasticity, costs of phenotypes, and costs of plasticity: toward an integrative view[END_REF][START_REF] Murren | Constraints on the evolution of phenotypic plasticity: limits and costs of phenotype and plasticity[END_REF].

Constitutive costs include the costs of maintaining physiological machinery and signal acquisition, and exist independent of whether the plant performs a plastic response or not. Induced costs include the costs of the realized phenotypic changes and depend on the intensity of the phenotypic response (Sultan and Spencer, 2002;Auld et al., 2009). Costs of plasticity might reduce plant fitness, and even challenge plant survival [START_REF] Pigliucci | Evolution of phenotypic plasticity: where are we going now?[END_REF]Valladares et al., 2007;[START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF][START_REF] Bongers | Drought responses, phenotypic plasticity and survival of Mediterranean species in two different microclimatic sites[END_REF]. In view of the great role played by phenotypic plasticity in plant response to global change, trying to evaluate plasticity and performance together remains a key challenge [START_REF] Givnish | Ecological constraints on the evolution of plasticity in plants[END_REF]Valladares et al., 2007;Auld et al., 2009;[START_REF] Bongers | Drought responses, phenotypic plasticity and survival of Mediterranean species in two different microclimatic sites[END_REF].

Another limit to plasticity in plants may lay in the multifactorial aspect of environmental variation in nature (Valladares et al., 2007). Plastic response to such complex environmental changes is more difficult to decipher than responses to simple laboratory environments under which plasticity was often studied, with only one condition varying at a time (Valladares et al., 2007;[START_REF] Westneat | Causes and consequences of phenotypic plasticity in complex environments[END_REF][START_REF] Callahan | Phenotypic plasticity, costs of phenotypes, and costs of plasticity: toward an integrative view[END_REF]. In a complex environment, different conditions, for instance drought and shade, may lead to opposite plant responses (Sack and Grubb, 2002), hence hiding the overall response. Also, costs involved in the response to a first condition may limit responses to other conditions, due to constraints on energy allocation (Auld et al., 2009). How plant phenotype is changed in response to synchronous changes of multiple environmental conditions and the consequences on plant performance are topics that still require investigation [START_REF] Westneat | Causes and consequences of phenotypic plasticity in complex environments[END_REF].

Finally, plasticity may be limited by the integration among traits. Phenotypic integration is the pattern and magnitude of character correlations due to genetic, developmental, and/or functional connections among traits and allowing organism consistency [START_REF] Pigliucci | Phenotypic integration: studying the ecology and evolution of complex phenotypes[END_REF].

Integration may be strongly affected by a change in the environment [START_REF] Pigliucci | Reaction norms of Arabidopsis. 1. Plasticity of characters and correlations across water, nutrient and light gradients[END_REF][START_REF] Liu | The effects of mechanical stress and spectral shading on the growth and allocation of ten genotypes of a stoloniferous plant[END_REF][START_REF] Wood | Environmental effects on the structure of the G-matrix[END_REF][START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF]. Integration has been suggested to possibly constrain plasticity. Indeed, linkage with other traits was assumed to limit a trait's range of variation and so, expression of plasticity for a given trait was assumed to be limited by its overall integration (Valladares et al., 2007). However, this relationship is far from being solved [START_REF] Wang | Morphological canalization, integration, and plasticity in response to population density in Abutilon theophrasti: Influences of soil conditions and growth stages[END_REF][START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF]. We here suggest an alternative hypothesis: that in poorly integrated organisms, extreme plastic modification of one trait may not be consistent with the modification of other traits, limiting overall plasticity, whereas in highly integrated organisms, extreme modification of one trait value may be consistent with extreme modification of the value of other traits. Such consistency in highly integrated organisms might facilitate the plasticity of the first trait and increase the plasticity of other traits, increasing the overall degree of plasticity of the organism. Hence phenotypic integration might in theory both, limit and facilitate plasticity. So far only few studies considered the possible effect of integration of traits on their plasticity [START_REF] Gianoli | Phenotypic integration may constrain phenotypic plasticity in plants[END_REF][START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF]. The hypothesis of integration as a constraint to plasticity was supported by a study showing that plasticity in response to shading or drought was lowest in traits that were strongly integrated with other traits [START_REF] Gianoli | Phenotypic integration may constrain phenotypic plasticity in plants[END_REF].

The hypothesis of lack of integration as a constraint to plasticity was partly supported by [START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF] showing that phenotypic plasticity of a given trait to drought was positively associated with phenotypic integration of this trait with other traits (both in the optimum and the stressful environments). Inevitably, such studies remain correlative as integration cannot be manipulated without damaging an organism. One can only compare integration prior to treatment with plasticity in response to treatment. Importantly, these few existing studies tried to identify whether more integrated traits are more or less plastic than less integrated traits. However, plasticity varies also among populations or other intraspecific lineages, and to our knowledge no study has tried to identify whether more integrated lineages respond more or less plastically to the environment than less integrated lineages.

Investigating plant plasticity and integration in response to environmental variation is particularly relevant in areas with drastic ongoing climate change. The sub-Antarctic region is characterized by cool and windy climate all year round. However, this region is subject to a rapid and intense climate change (Le [START_REF] Roux | Changes in climate extremes, variability and signature on sub-Antarctic Marion Island[END_REF][START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]Verfaillie et al., 2021). Such is the case in the Kerguelen Islands located in the southern Indian Ocean (Fig. 1) and with typical sub-Antarctic climate (mean annual temperature of 4.6°C and mean wind speed of 9.7 m.s -1 at "Port-aux-Français" from 1951 to 2018; (Verfaillie et al., 2021)). Climate change there resulted in a significant drying trend associated with a marked shift in the storm track location [START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF][START_REF] Verfaillie | Regional modeling of surface mass balance on the Cook Ice Cap, Kerguelen Islands (49 degrees S, 69 degrees E)[END_REF], whereas a large increase in the mean temperature was also observed e.g. [START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]Verfaillie et al., 2021). Direct impacts of climate change are already visible in the Kerguelen Islands, with more frequent and more prolonged summer droughts, the rise of the snow line and the drastic reduction of the Cook ice cap [START_REF] Hennion | High flexibility in growth and polyamine composition of the crucifer Pringlea antiscorbutica in relation to environmental conditions[END_REF][START_REF] Lebouvier | The significance of the sub-Antarctic Kerguelen Islands for the assessment of the vulnerability of native communities to climate change, alien insect invasions and plant viruses[END_REF]Verfaillie et al., 2015). Plant species in Iles Kerguelen show signs of stress such as leaf wilting or increased shoot necrosis suggesting they are particularly sensitive to ongoing changes [START_REF] Chapuis | Recovery of native plant communities after eradication of rabbits from the subantarctic Kerguelen Islands, and influence of climate change[END_REF][START_REF] Frenot | Impact des changements climatiques et de la fréquentation humaine sur la biodiversité des îles subantarctiques françaises[END_REF][START_REF] Marchand | Morphological variability of cushion plant Lyallia kerguelensis (Caryophyllales) in relation to environmental conditions and geography in the Kerguelen Islands: implications for cushion necrosis and climate change[END_REF]. As the Iles Kerguelen are geographically very isolated, species can hardly migrate to track changing environments in space, and so plants have to respond mainly by plasticity or adaptive evolution [START_REF] Hoffmann | Climate change and evolutionary adaptation[END_REF]. We studied three indigenous species from Iles Kerguelen: two Ranunculus species: R. biternatus and R. pseudotrullifolius (Ranunculaceae) and the Brassicaceae Pringlea antiscorbutica. Ranunculus species are known to show high plastic responses [START_REF] Garbey | Morphological plasticity of a spreading aquatic macrophyte, Ranunculus peltatus, in response to environmental variables[END_REF], and especially heterophylly that is considered as adaptive plasticity [START_REF] Wells | Adaptive phenotypic plasticity: the case of heterophylly in aquatic plants[END_REF].

Ranunculus biternatus and pseudotrullifolius are known to show trait plasticity between plants from the field and such under controlled conditions [START_REF] Hennion | Morphological and phytochemical relationships between Ranunculus species from Iles Kerguelen[END_REF]. Pringlea antiscorbutica is known to show decreased root or shoot growth in response to salt or heat (Hummel et al., 2004 a, b) and shows plasticity in shoot growth in response to cultivation conditions [START_REF] Hennion | High flexibility in growth and polyamine composition of the crucifer Pringlea antiscorbutica in relation to environmental conditions[END_REF].

We studied lineages (populations, maternal lines) within each of these three subantarctic species in a phytotron, subjecting plants to variation of different abiotic conditions such as warming treatment, and (in P. antiscorbutica) shading treatment and combination of both warming and shading treatment. We investigated the variation of morphological traits and the morphological or physiological performance of individuals. We aimed at testing the predictions of the above hypotheses that plasticity of intraspecific lineages might be costly, limited by environmental complexity and by either high or low phenotypic integration: (i) Intraspecific lineages of high plasticity have low performance. (ii) Intraspecific lineages show a lower degree of plasticity in response to variations of two environmental conditions than in response to variation of one single condition; (iii) Intraspecific lineages that show high phenotypic integration prior to environmental treatment, respond either less plastically or more plastically to this treatment.

MATERIALS AND METHODS

Species under study

The Iles Kerguelen (49°20'00" S, 69°20'00" E) are situated in the southern Indian Ocean within the sub-Antarctic region [START_REF] Lebouvier | Conservation and management in the french sub-Antarctic islands and surrounding seas[END_REF] (Fig. 1). We studied R. biternatus Smith, R.

pseudotrullifolius Skottsberg, and Pringlea antiscorbutica. R.Br. ex Hook.f. All are perennial plants. R. biternatus and R. pseudotrullifolius are two species of austral distribution with a magellanic origin [START_REF] Lehnebach | Around the pole: evolution of sub-Antarctic Ranunculus[END_REF] while P. antiscorbutica is endemic from the southern Indian Ocean province (Bartish et al., 2012). On the Iles Kerguelen these species occupy different habitats (Hennion and Walton, 1997a). Ranunculus biternatus is widespread on the island occurring in habitats below 300m above sea level. In contrast, R. pseudotrullifolius being halophilous has more restricted distribution and occurs within a short distance of the coast, occupying peaty or sandy shores and ponds (Hennion and Walton, 1997a). On the Iles Kerguelen P. antiscorbutica occurs in habitats ranging from coastal meadows to montane fellfields [START_REF] Hennion | Accumulation of organic and inorganic solutes in the subantarctic cruciferous species Pringlea antiscorbutica in response to saline and cold stresses[END_REF].

Seed collection

To grow plants under controlled conditions, we used seeds from different natural populations in the three species. Populations were defined as continuous groups of plants living at a given site.

Seeds from P. antiscorbutica were collected in the field in 2013 in six populations spread across the region covering Ile Australia, Mont Crozier and Ile Mayes (Fig. 1). Ten individuals were randomly selected within each population. In each of the ten individuals, fifteen siliques were then randomly collected from the median part of the longest infructescence where seed size is maximum and least variable (Hennion, Schermann-Legionnet and Atlan, unpubl.). Seeds were also collected from P. antiscorbutica plants growing from four populations in a phytotron in 2013. In R. biternatus and R. pseudotrullifolius, seeds were collected in 2014 from 20 individuals across three populations in Isthme Bas, the same in which phenotypic measurements had been performed in 10 2012. All seeds were stored dry with silica gel at 4°C until their use according to an established protocol (Hennion and Walton, 1997b).

Controlled conditions

We grew plants in a phytotron at Rennes. Seeds were sterilized and germinated in Petri dishes at 25°C for P. antiscorbutica and 21°C for Ranunculus species, under low light (1kLux) following an established protocol (Hennion and Walton, 1997b). To render the overall design more homogeneous we kept a constant number of seedlings from each population. In each species, three hundred seedlings were planted in vermiculite substrate and fertilized with ½ Hoagland solution [START_REF] Hoagland | Growing plants without soil by the water-culture method[END_REF]. For the R. pseudotrullifolius, NaCl was added to Hoagland solution to reach a concentration of 1g.L -1 , being consistent with the salinity in the field [START_REF] Labarrere | How do plants respond and adapt to climate change? Study at cold margins[END_REF].

Plants were grown hydroponically in a phytotron under conditions (light exposure, photoperiod, temperature and air humidity) mimicking austral summer in Iles Kerguelen at best, according to a previous protocol [START_REF] Hennion | Accumulation of organic and inorganic solutes in the subantarctic cruciferous species Pringlea antiscorbutica in response to saline and cold stresses[END_REF][START_REF] Hennion | High flexibility in growth and polyamine composition of the crucifer Pringlea antiscorbutica in relation to environmental conditions[END_REF]. Specifically, plants were grown in a photoperiod of 14h with an irradiance of 15klux from a combination of Philips TLD 79 fluorescent and Philips Aquarelle TLD 89 tubes. Temperature was of 9°C during the day and 4°C during the night with a relative humidity of 75/85% day/night.

Experimental treatments

The experimental design is summarized in Fig. 2. Plants were grown under standard, "control" conditions for 5 months at 9°C; 15klux. We then subjected subsets of plants to different treatments during 3 months. In P. antiscorbutica, we subjected plants to four conditions: (i) warming (13°C / +4°C), (ii) shading (5klux), (iii) both warming and shading (13°C; 5klux) and (iv) control (9°C; 15klux). In R. biternatus and R. pseudotrullifolius, we subjected plants to warming (13°C). Due to a low number of available seedlings, R. biternatus and R. pseudotrullifolius were not subject to shading treatment or combined warming-shading treatment. In P. antiscorbutica, a total 320 plants (32 plants from each of 10 source populations) were used: per each of four treatments 8 plants from 10 source populations. In R. biternatus, a total 152 plants (8 plants from each of 19 mothers) were grown: per each of two treatments (warming treatment and control) 4 plants from each of 19 mothers for a total of 76 plants. In R. pseudotrullifolius, a total 112 plants (8 plants from each of 14 mothers) were grown: per each of two treatments (warming treatment and control) 4 plants from 14 mothers for a total of 56 plants. Placement of plants within a treatment was random with respect to levels of integration or of performance, thereby avoiding any confounding effect of possible variation within-treatment variation of microenvironmental conditions and how different lineages of plants responded to a given treatment. Plants were all 8-month old at the end of experiments.

Trait measurements

We conducted two sets of plant measurements. First, we measured plants after 5 months growth while all plants were subjected to control conditions to determine baseline differences between plants (T0). We measured plants again after 3 months growth in the different abiotic conditions (T1). Traits considered were height, diameter and number of leaves of the rosette, and of the 2 longest leaves: length and width of lamina and length of petiole. These traits were shown to respond to increased temperature in previous work [START_REF] Hennion | High flexibility in growth and polyamine composition of the crucifer Pringlea antiscorbutica in relation to environmental conditions[END_REF][START_REF] Hermant | Endemic species have highly integrated phenotypes, environmental distributions and phenotype-environment relationships[END_REF][START_REF] Labarrere | How do plants respond and adapt to climate change? Study at cold margins[END_REF]. In R. biternatus and R. pseudotrullifolius, diameter changed during plant handling and therefore was not measured. We also estimated physiological and morphological plant performances. To determine physiological performance, we used the maximum efficiency of photosystem II [START_REF] Maxwell | Chlorophyll fluorescence-a practical guide[END_REF][START_REF] Zhu | What is the maximum efficiency with which photosynthesis can convert solar energy into biomass?[END_REF], a useful performance parameter known to decrease with stress [START_REF] Maxwell | Chlorophyll fluorescence-a practical guide[END_REF][START_REF] Kalaji | Effects of salt stress on photosystem II efficiency and CO 2 assimilation of two Syrian barley landraces[END_REF]. Maximum PSII efficiency (Fv/Fm) was measured using a PAM chlorophyll fluorometer system (Heinz Walz, Effeltrich, Germany) in saturating pulse mode. After 30 minutes of dark adaptation, the maximum fluorescence of leaves was measured under dark conditions (Fm) and a subsequent saturating pulse of light was applied. Minimum fluorescence (F0) was determined under weak light before a pulse, and variable fluorescence (Fv=Fm-F0) was calculated. To determine morphological performance, we used leaf spread [START_REF] Zamora | Fitness responses of a carnivorous plant in contrasting ecological scenarios[END_REF]. Leaves that are not fully spread, but curled, indicated that the plant is not fully performant. Part of leaf that is curled is unexposed to light and therefore does not contribute to photosynthesis. We measured leaf spread as the ratio between length of leaf that is not curled and maximum flattened leaf length.

Data analyses

Trait means and plasticity

We investigated plant plasticity comparing plant trait values between the different experimental conditions, i.e. a trait change, with in whatever direction, with a change in environment. Prior to calculating plasticity of traits, we aimed to remove the intrinsic trait value differences among plants to correct for differences between plants already present prior to exposing them to controls or treatments during months 5 -8. Thus, we calculated trait value as standardized trait value Ts = (T1-T0)/T0. With T0 being the measurement of the trait done during the first set of measurements -when all plants were subjected to the same conditions -and T1 being the measurement of the trait done during the second set of measurements, when plants were subjected to different experimental conditions. In P. antiscorbutica, we calculated trait means of these standardized trait values across plants from the same population subjected to same condition. In R. biternatus and R. pseudotrullifolius, we calculated trait means across plants from the same mother subjected to same condition. So, to summarize, we had one data point per maternal line (or population) and treatment.

In each species, we calculated -for each population (or mother) and treatment -trait plasticity as the absolute value of the difference between trait mean under treatment and trait mean under control. In P. antiscorbutica we also calculated the mean degree of plasticity across the three treatments. Samples sizes did not permit the same calculus for Ranunculus species. These means across individuals per population or per mother could then be compared to phenotypic integration calculated across individuals per population or per mother. For consistency we used source populations or mothers as our level of analyses in all further comparisons.

Phenotypic integration

We calculated phenotypic integration across P. antiscorbutica individuals from the same source populations. We did so prior to treatments, thereby avoiding any effect of environment on integration [START_REF] Wang | Morphological canalization, integration, and plasticity in response to population density in Abutilon theophrasti: Influences of soil conditions and growth stages[END_REF]. Samples sizes did not permit the same calculus for Ranunculus species. We focused on the dominant axis of phenotypic integration, quantified as the percentage of variance explained by the first axis of a Principal Component Analysis (PCA) performed on traits (Tucic et al., 2013;[START_REF] Haber | Disintegrating the fly: A mutational perspective on phenotypic integration and covariation[END_REF][START_REF] Michaud | Phenotypic integration in feliform carnivores: Covariation patterns and disparity in hypercarnivores versus generalists[END_REF]. PCA was performed using correlation matrix. Traits were height and diameter of the rosette, and of the 2 longest leaves: length and width of lamina and length of petiole, leaf 1 older than leaf 2. Trait values were scaled and PCA conducted using the FactoMineR package of R 4.0 [START_REF] Lê | FactoMineR: an R package for multivariate analysis[END_REF][START_REF]R: A language and environment for statistical computing[END_REF]. Note that all PCAs are calculated across the same number of traits hence the same total number of axes, rendering % explained variance directly comparable among PCAs. We investigated whether degree of phenotypic integration differed before and after the exposure to treatment, but found no differences [ Supplementary Information Table S2a ]. Moreover, we found that degree of phenotypic integration did not differ among treatments [ Supplementary Information Table S2b ].

Hypothesis Testing

The relationships between trait means, plasticity or integration were tested using simple linear regression. We determined differences of trait values among treatments using ANOVA. We used MANOVA to determine whether plant phenotype (accounting for all traits) differed among treatments. The same traits were tested in MANOVA and ANOVA analyses. Analyses were conducted in R 4.0 (R, 2020). For multiple testing on the same data set, p-values were corrected using sequential Bonferroni's correction [START_REF] Cabin | To Bonferroni or not to Bonferroni: when and how are the questions[END_REF]. In all regression analyses we verified the assumptions of the analyses using residual plots.

RESULTS

Effects of treatments on plant traits

In P. antiscorbutica, plant phenotype of maternal lineages was significantly larger in warming treatment than in control (p=0.005; F=5.9; dferror=16; MANOVA). In contrast, plant phenotype was significantly smaller in response to shading or combined warming-shading treatments than in control (respectively: p=0.0043; F=6.8; dferror =16 and p=0.012; F=5.1; dferror =16; MANOVA).

We did not find any difference in plant phenotype between shading and combined warmingshading treatments (p=0.55; F=0.93: dferror =16; MANOVA). Differences of individual traits between treatments and the control are indicated Table 1.

In R. biternatus, number of leaves as well as petiole length and lamina width of leaf 2 of populations were significantly larger in warming treatment compared to control (Table 2). In R.

pseudotrullifolius, lamina length of leaf 1 was significantly larger in warming treatment than in control (Table 2). Overall, phenotype was larger (marginally significant) in warming in R. biternatus but not in R. pseudotrullifolius (respectively: p=0.058; F=2.2; dferror =34 and p=0.57; F=0.8; dferror =24; MANOVA).

Effects of treatments on plant performance

In P. antiscorbutica, we found that morphological performance (percentage of leaf spread) of maternal lineages was lower in combined warming-shading treatment than in the other treatments or in the control (Table 3). In R. biternatus we found that plant performances of populations did not differ significantly between control and warming treatment (Table 4). In R. pseudotrullifolius, we found that physiological performance of populations was higher in warming treatment. Yet, this result is only marginally significant after sequential Bonferroni's correction (Table 4).

Effect of treatments on the degree of plasticity

In P. antiscorbutica, we found that the degree of plasticity of maternal lineages was higher in response to warming treatment than in response to shading treatment or combined warmingshading treatment (respectively: p=0.05; F=2.19; dferror =16 and p=0.036; F=2.21; dferror =16; MANOVA). In contrast, the degree of plasticity did not differ between shading treatment and combined warming-shading treatment (p=0.45; F=0.46; dferror =16; MANOVA). Specifically, degrees of plasticity of plant height and lamina width of the tallest leaf were higher in response to warming treatment than to shading treatment (Table 5). Degrees of plasticity of lamina width of leaf 1 and leaf 2 were higher in response to warming treatment than in response to combined warming-shading treatments (Table 5).

Relationship between degree of plasticity and performance

In P. antiscorbutica, we found significant negative relationship between the degree of trait plasticity of maternal lineages and plant performance (percentage of leaf spread and marginally photosynthetic efficiency) in response to warming treatment (Fig. 3). Also, we found a significant negative relationship between trait plasticity and performance (percentage of leaf spread) in response to combined warming-shading treatment (Fig. 4). We did not find any significant relationships between the degree of trait plasticity and plant performances in response to shading treatment (p-value>0.05; all adj.r²<0.1; simple linear regressions). To explore constituent costs of being plastic we compared across-treatment means of plasticity per source population to performances in control condition, where no plasticity is expressed. We found no relationships (p-value>0.05; all adj.r²<0.1; simple linear regression).

In R. biternatus and R. pseudotrullifolius, we did not find any significant relationship between the degree of trait plasticity of populations and plant performance.

Relationship between phenotypic integration prior to treatment and the degree of plasticity in response to treatment In P. antiscorbutica, we found a significant positive relationship between phenotypic integration of maternal lineages and the degree of plasticity of leaf 1 length in shading treatment (Fig. 5). Also, we found marginally significant positive relationships between phenotypic integration and the degree of plasticity of leaf 2 length in shading treatment (Fig. 5). We found no significant relationship between integration and the degree of trait plasticity in other treatments.

DISCUSSION

As sessile organisms, plants have to face changes of their environmental conditions. Phenotypic plasticity is considered as a major mean for plants to face environmental change [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF][START_REF] Fox | Beyond buying time: the role of plasticity in phenotypic adaptation to rapid environmental change[END_REF]). Yet, plasticity has costs for plant performance and limits that have been recognized in theory but remain to be evidenced in practice [START_REF] Pigliucci | Evolution of phenotypic plasticity: where are we going now?[END_REF]Valladares et al., 2007;[START_REF] Molina-Montenegro | Adaptive phenotypic plasticity and competitive ability deployed under a climate change scenario may promote the invasion of Poa annua in Antarctica[END_REF][START_REF] Wei | Elevation-dependent selection for plasticity in leaf and root traits of Polygonum hydropiper in response to flooding[END_REF][START_REF] Callahan | Phenotypic plasticity, costs of phenotypes, and costs of plasticity: toward an integrative view[END_REF][START_REF] Murren | Constraints on the evolution of phenotypic plasticity: limits and costs of phenotype and plasticity[END_REF]. Also, plasticity was mainly studied analysing the independent response of traits to the variation of a single environmental condition (Valladares et al., 2007;[START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF]. Yet, in nature plants are subject to the variation of multiple conditions. Moreover, traits are correlated to each other (i.e. phenotypic integration) and cannot vary independently. We investigated the relationships between plasticity and performance in experimental treatments of plants. Moreover, we investigated the effects of combined abiotic conditions on plant plasticity, and examined the relationship between plant phenotypic plasticity and integration. We showed that plasticity may decrease performance suggesting strong induced costs of plasticity. Also, we showed that plasticity of plants may be lower when plants respond to two conditions combined than when plants respond to a single condition. Finally, some positive correlation between phenotypic plasticity and integration was evidenced.

Trait values under different treatments

Iles Kerguelen face rapid and intense climate change, characterized by an increase of temperature and a decrease of rainfall [START_REF] Lebouvier | The significance of the sub-Antarctic Kerguelen Islands for the assessment of the vulnerability of native communities to climate change, alien insect invasions and plant viruses[END_REF][START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]Verfaillie et al., 2021). Plants in Iles Kerguelen already exhibit signs of stress such as leaf wilting or increased shoot necrosis during dry summer periods [START_REF] Chapuis | Recovery of native plant communities after eradication of rabbits from the subantarctic Kerguelen Islands, and influence of climate change[END_REF][START_REF] Frenot | Impact des changements climatiques et de la fréquentation humaine sur la biodiversité des îles subantarctiques françaises[END_REF][START_REF] Marchand | Morphological variability of cushion plant Lyallia kerguelensis (Caryophyllales) in relation to environmental conditions and geography in the Kerguelen Islands: implications for cushion necrosis and climate change[END_REF]. Previous work evidenced plastic responses in the studied species (Hummel et al., 2004b;[START_REF] Hennion | Morphological and phytochemical relationships between Ranunculus species from Iles Kerguelen[END_REF]Hennion and Walton, 1997b). In this study, we showed that all species exhibit plastic morphological responses to abiotic changes. In P. antiscorbutica we found that traits (4 out of 9 traits) increased in response to warming. This is consistent with the response of plants at the 15-day seedling stage where shoot growth increased in response to a warm temperature treatment (22°C night/ 25°C day) compared to colder temperature regime mimicking Kerguelen summer conditions (5°C night/ 10°C day) (Hummel et al., 2004b). In R. biternatus, various traits increased in response to warming (3 out of 8 traits). In contrast, in R. pseudotrullifolius, only one out of 8 traits was plastic, i.e. increased in response to warming.

Degrees of plasticity are low under simultaneous change of multiple environmental conditions

In P. antiscorbutica, we investigated differences of degree of plasticity among treatments.

Particularly, we investigated to what extent simultaneous variation of multiple environmental conditions affect the degree of plant plasticity -compared to the variation of a single condition.

Firstly, we found that the degree of plasticity is higher in response to warming than in response to shading. We yet recognize that the two conditions do not have the same units and difference of plasticity might result from a difference of the intensity of changes, hardly comparable, between the two conditions. Secondly, we found that the degree of plasticity is lower in response to warming-shading than in response to warming. Warming and shading have opposite effects on plant growth. A first explanation for the decreased degree of plasticity in response to multiple conditions is hence that opposite effects of warming and shading may annihilate each otherresulting in a lower degree of plasticity than that resulting from one single factor. Further studies may investigate whether conditions having the same effect on plant growth have cumulative effects on the degree of plasticity. Another explanation for our result is that constraints of resource allocation might prevent traits to respond to multiple environmental conditions (Auld et al., 2009).

We showed that the degree of plasticity is higher in response to warming than in response to shading. We might therefore expect the plant phenotype in warming-shading to be closer to the plant phenotype in warming than to the plant phenotype in shading. Yet, we found that plant phenotypes in warming-shading are closer to plant phenotypes in shading than to plant phenotypes in warming. We hence suggest that, in addition to the opposite effects of shading and warming treatments, shading imposes a stronger constraint than warming. Resource constraints imposed by shading might prevent plants from responding to temperature and might possibly explain lower performance. This result is consistent with the current view that multidimensional phenotypic plasticity (i.e. plasticity of a trait with respect to variation in multiple environmental factors) cannot be explained solely by scaling up ideas from models of unidimensional plasticity [START_REF] Westneat | Causes and consequences of phenotypic plasticity in complex environments[END_REF]. Climate change alters simultaneously multiple environmental factors and plasticity might hence not suffice to compensate such changes.

Plasticity in face of climate-change-induced warming and shading by competitors

Species that show plasticity in response to abiotic conditions are believed better armed face to climate change (Valladares et al., 2007). A large increase in the mean temperature is observed in Kerguelen Islands (e.g. [START_REF] Favier | Atmospheric drying as the main driver of dramatic glacier wastage in the southern Indian Ocean[END_REF]Verfaillie et al., 2021). Plasticity shown in P. antiscorbutica and R. biternatus in response to warming might hence favour their capacity to face climate change, whereas R. pseudotrullifolius seems less plastic in relation to temperature. Notably, in the three species this plasticity did not decrease performance. Possibly, this maintained performance is because our treatment lasted for three months only and we acknowledge that a persistent increase of temperature might affect plant performance in the long term as suggested by former experiments. Indeed, under well-watered cultivation in a common garden under a temperate climate, warmer than the sub-Antarctic, at Brest (France), young plants of P. antiscorbutica, R. biternatus and R. pseudotrullifolius collected from Iles Kerguelen first showed increased growth but finally collapsed after several months (Hennion, pers. com.). In addition, in the field, plants are also exposed to the wind and different levels of soil water saturation or soil nutrients. Combined with these conditions, increased temperature might affect plant performance. Increased temperatures in Iles Kerguelen are suggested to favour expansion of invasive plant species [START_REF] Frenot | Impact des changements climatiques et de la fréquentation humaine sur la biodiversité des îles subantarctiques françaises[END_REF], which may compete with native species, for instance for light. In P. antiscorbutica, we found that the phenotypes were similar in response to shading or combined warming and shading, and that individual traits decreased. Yet, we showed that neither shading nor warming did affect plant performance but combined warming and shading reduced plant performance. P. antiscorbutica might thus be sensitive to the combination of climate change and shading due to colonization by invasive species in Kerguelen at low altitude. We suggest that competition for light (with native and introduced species) might impact the capacity of P. antiscorbutica plants to cope with warming. Importantly, by triggering plant growth, warming may itself be the cause of increased light competition.

Phenotypic plasticity is high in maternal lineages showing high phenotypic integration prior to treatment

The relationship between the degree of plasticity and phenotypic integration still requires investigation (e.g. [START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF][START_REF] Wang | Morphological canalization, integration, and plasticity in response to population density in Abutilon theophrasti: Influences of soil conditions and growth stages[END_REF]. Before determining the influence of phenotypic integration on the degree of plasticity in response to a given abiotic condition, we must verify whether phenotypic integration responds or not to that abiotic condition. Here, we did not find any response of degree of integration to abiotic conditions. Given that integration does not respond to treatments, integration might potentially influence trait plasticity to those treatments. Some authors suggest integration may decrease plasticity [START_REF] Gianoli | Phenotypic integration may constrain phenotypic plasticity in plants[END_REF].

Alternatively, however, we hypothesized that integration might also favour plastic response of a trait as in highly integrated organisms, extreme variation of one trait value matches extreme variation of other trait values, allowing extreme trait modification to maintain plant consistency.

Here, we found no relationships between integration of maternal lineages prior to treatment and plasticity in response to warming or warming-shading treatment. But we did find that maternal lineages of high integration prior to the treatment responded more plastically to shading treatment. This latter result may explain positive relationships between plasticity and integration found posttreatment (density, [START_REF] Wang | Morphological canalization, integration, and plasticity in response to population density in Abutilon theophrasti: Influences of soil conditions and growth stages[END_REF] by an effect of integration on plasticity. We note, however, that we cannot rule out more complex causalities where both integration and plasticity are driven by a third, unknown property of plants. To our knowledge our results are the first evidence that intraspecific lineages of higher integration react more plastically to certain environmental changes, suggesting that plastic change of a trait is easier when integrated with other traits.

Costs of plasticity: high degree of plasticity relates to decreased performance Plasticity helps plants facing environmental changes, yet, plasticity also has costs which may compromise plant fitness (Valladares et al., 2007;Bell and Galloway, 2008;Auld et al., 2009[START_REF] Murren | Constraints on the evolution of phenotypic plasticity: limits and costs of phenotype and plasticity[END_REF]. We investigated whether the degree of plasticity of populations/maternal lineages statistically increased or decreased performance in P. antiscorbutica. We showed that the degree of plasticity statistically decreased plant performance in high temperature treatment. Yet, we found no significant relationship between plasticity and performance in the other treatments.

Overall, we found evidence for costs of plasticity, and these costs appeared to depend on the environmental context. Note that our study is essentially correlative as we did not directly manipulate neither integration nor plasticity as imposing integration or plasticity risks to damage the organism and may not reflect levels of integration or plasticity actually achieved in nature.

Costs of plasticity may be dissociated in two kinds of costs, constitutive and induced.

Constitutive costs result from maintaining the basic physiological machinery of plasticity while induced costs depend on the amount of phenotypic changes (Sultan and Spencer, 2002;[START_REF] Callahan | Phenotypic plasticity, costs of phenotypes, and costs of plasticity: toward an integrative view[END_REF]. Constitutive costs have to be paid even without phenotypic change in a constant environment (Auld et al., 2009). However, we found that performance under stable conditions did not relate to the capacity to plastically respond to changing conditions. Plasticity might hence have only little constitutive costs in our study system. Induced costs, in contrast, reflect the costs of morphological modification in response to environmental change. High induced costs might impose resource allocation constraints (Auld et al., 2009), which does not allow plants to maintain the same degree of performance. Here we found that the degree of plasticity is indeed negatively related to plant performance post-treatment, notably in warming treatment. Above, we discussed that warming induced a higher degree of plasticity than other treatments. We suggest that a high degree of plasticity generates important induced costs that impact plant performance. We also observed a negative plasticity-performance relationship in response to a combined warmingshading treatment. We suggest that the most stressful environments, such as combined warming and shading, reduce energy available to plants. In our systems, the complete absence of positive relationship between the degree of plasticity and performance tends to suggest that more plastic organisms may not inevitably better sustain environmental change. On the opposite, plants that show high degree of plasticity may perform poorly. Performance is poor once the environmental change occurs, not before, i.e. reflecting induced rather than constitutive costs.

CONCLUSION

Although the concept of plastic response to environmental change has long been studied, our understanding has to be improved about costs and limits of such plastic response in individuals [START_REF] Pigliucci | Evolution of phenotypic plasticity: where are we going now?[END_REF][START_REF] Matesanz | Phenotypic integration does not constrain phenotypic plasticity: differential plasticity of traits is associated to their integration across environments[END_REF][START_REF] Murren | Constraints on the evolution of phenotypic plasticity: limits and costs of phenotype and plasticity[END_REF]. Here we showed that simultaneous shift of two environmental factors may limit plasticity of individuals from different lineages. This may reduce the capacity of populations to respond to climate warming under increased light competition. Also, we found some evidence for a positive relationship between phenotypic plasticity in response to treatment and phenotypic integration prior to treatment suggesting that a trait can respond to treatment more plastically if changes are well integrated into a body plan. Moreover, we found that highly plastic individuals performed poorly. Due to induced costs, plant plastic response to environmental change does not guarantee plant success in the new environment.

Hence, little plastic individuals may be as, or even more, performant in the new environment than Degree of plasticity of leaf 2 length in response to shading treatment (difference between shading treatment and control).

Phenotypic integration (%)

Figure 1 .

 1 Figure 1. a. Location of Iles Kerguelen in the southern Indian Ocean; b. sampled sites in Iles

Figure 2 .

 2 Figure 2. Illustration of experimental design. The numbers of plants used per treatment, per species

Figure 3 .

 3 Figure 3. Negative relationship between the degree of plasticity and performance in the warming

Figure 4 .

 4 Figure 4. Negative relationship between the degree of plasticity and performance in the warming-

Figure 5 .

 5 Figure 5. Positive relationship between phenotypic integration and the degree of plasticity of leaf

Fig

  Fig. 2.

Fig

  Fig. 3.

Table 1 .

 1 ANOVA analyses indicating trait differences between different abiotic treatments in P. antiscorbutica. P-values (P) after sequential Bonferroni's correction and F values (F) are indicated. "</>": trait is lower/larger in the first treatment indicated. dferror =18 source populations.

	Plant

Table 2

 2

				Plant						Leaf number 1								Leaf number 2
		Plant height		Number of leaves	Petiol length		Lamina length	Lamina width		Petiol length	Lamina length	Lamina width
	Control vs warming	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>
	R. biternatus	0.14	3.62	0.0091 10.94	<	0.085 4.12		0.12	2.44		0.11	4.67		0.03 7.19	<	0.066 1.07		0.0003	5.8	<
	R. pseudotrullifolius	0.54	0.66		1	1.36		0.43	0.18		0.033	3.1	<	0.077	2.6		0.85	1		0.70	0.39		0.90	0.6

. ANOVA analyses indicating trait differences between warming and control in R. biternatus and R. pseudotrullifolius. P-values (P) after sequential Bonferoni's correction and F values (F) are indicated. "</>": trait is lower/larger in the control. In R. biternatus, dferror = 36 mothers; in R. pseudotrullifolius, dferror =26 mothers.

Table 3 .

 3 ANOVA analyses indicating differences of performance (photosynthetic efficiency and leaf spread) between treatments in P.antiscorbutica. P-values (P) after sequential Bonferroni's correction and F values (F) are indicated. "</>": performance is lower/higher in the first treatment indicated. dferror =18 source populations.

	Photosynthetic efficiency		Leaf spread
	P	F	</>	P	F	</>

Table 4 .

 4 ANOVA analyses indicating differences of performance (percentage of survival, photosynthetic efficiency and leaf spread) between warming and control in R. biternatus and R. pseudotrullifolius. P-values (P) after sequential Bonferroni's correction and F values (F) are indicated. "</>": performance is lower/higher in the control. In R. biternatus, dferror = 36 mothers; in R. pseudotrullifolius, dferror =26 mothers.

	Percentage of		Photosynthetic
		survival			efficiency			Leaf spread
	P	F	</>	P	F	</>	P	F	</>

Table 5 .

 5 ANOVA analyses indicating differences of the degree of plasticity between treatments in P. antiscorbutica. "</>": trait is less/more plastic in the first treatment indicated. P-values (P) and F values (F) are indicated. dferror =16 source populations.

				Plant					Leaf number 1							Leaf number 2
		Plant height		Number of leaves	Petiole length	Lamina length	Lamina width	Petiole length	Lamina length	Lamina width
		P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>	P	F	</>
	Warming vs shading 0.054	4.31	>	0.13	2.53		0.28	1.23		0.19	1.87		0.036 5.19	>	0.35	0.94		0.38 0.83		0.30	1.18
	Warming vs warming-shading	0.13	2.55		0.54	0.38		0.87	0.03		0.35	0.91		0.012 7.97	>	0.72	0.13		0.06 3.97		0.017 6.99	>
	Shading vs warming-shading	0.64	0.22		0.43	0.64		0.23	1.54		0.71	0.15		0.41	0.73		0.25	1.45		0.34 0.95		0.31	1.09

ACKNOWLEDGEMENTS

We thank IPEV programs 136 Ecobio (M. Lebouvier), 1116 PlantEvol (F. Hennion) and Réserve Naturelle TAF for contribution to seed collection in Iles Kerguelen. We thank personnel of common facility ECOLEX, ECOBIO Rennes (Valérie Gouesbet, Jean-Luc Foulon, Thierry Fontaine-Breton and †Fouad Nassur) for help in plant cultivation. This research is linked to CNRS Zone-Atelier Antarctique. FH, BL and AP conceived the ideas. FH and BL performed the fieldwork. BL and MR performed the laboratory work. BL, MR and AP analysed the data. BL, FH and AP led the writing. All authors contributed to discussions.

FUNDING Field work was supported by IPEV (programs 1116 PlantEvol -F. Hennion and 136 Ecobio -M. Lebouvier). Laboratory work was supported by CNRS Zone-Atelier Antarctique, CNRS INEE grant (PICS "AntarctBiodiv" to FH) and University Rennes 1 (Direction de la Recherche et de l'Innovation). B.L. was supported by a PhD grant from Ministry of Research and Education (France). LITERATURE CITED Auld JR, Agrawal AA, Relyea RA. 2009. Re-evaluating the costs and limits of adaptive phenotypic plasticity. Proceedings of the Royal Society of London B: Biological Sciences: rspb20091355. Bartish IV, Ainouche A, Jia D, Bergstrom D, Chown SL, Winkworth RC, Hennion F. 2012. Phylogeny and colonization history of Pringlea antiscorbutica (Brassicaceae), an emblematic endemic from the South Indian Ocean Province. Molecular Phylogenetics and Evolution, 65: 748-756. Bell DL, Galloway LF. 2008. Population differentiation for plasticity to light in an annual herb: Adaptation and cost. American Journal of Botany, 95: 59-65. Benavides R, Carvalho B, Matesanz S, Bastias CC, Cavers S, Escudero A, Fonti P, Martinez-Sancho E, Valladares F. 2021. Phenotypes of Pinus sylvestris are more coordinated under local harsher conditions across Europe. Journal of Ecology, 109: 2580-2596.

highly plastic individuals. Future studies might aim to verify whether the relationships we found also hold for traits other than vegetative morphology.

SUPPLEMENTARY DATA

Supplementary data are available online in the Supporting Information section and consist of the following. Table S1: MANOVA analyses indicating trait differences among source mothers (R. biternatus and R. pseudotrullifolius) or source populations (P. antiscorbutica) in the control condition. Table S2: ANOVA analyses indicating in P. antiscorbutica whether a) degree of phenotypic integration differs before and after application of treatments, b) phenotypic integration differs among treatments.

The datasets generated during the current study will be available online on Osuris geonetwork: https://www.osuris.fr/geonetwork /srv/fre/catalog.search#/.