M.D Christine Lebrun-Frenay
email: lebrun-frenay.c@chu-nice.fr

MD Aksel Siva

PhD Maria Pia Sormani

MSc Cassandre Landes-Chateau

MD Lydiane Mondot

PhD Francesca Bovis

MD Patrick Vermersch

MD Caroline Papeix

MD Eric Thouvenot

MD Pierre Labauge

MD Françoise Durand-Dubief

MD Husnu Efendi

MD Emmanuelle Le Page

MD Murat Terzi

MD Nathalie Derache

MD Bertrand Bourre

MD Robert Hoepner

MD Rana Karabudak

MD Jérôme De Seze

MD Jonathan Ciron

MD Pierre Clavelou

MD Sandrine Wiertlewski

MD Omer Faruk Turan

MD Nur Yucear

MD Mikael Cohen

MD Christina Azevedo

MD Orhun H Kantarci

MD Darin T Okuda

MD Daniel Pelletier

Teriflunomide and Time to Clinical Multiple Sclerosis in Patients With Radiologically Isolated Syndrome

Keywords: Disease prevention, multiple sclerosis, radiologically isolated syndrome, presymptomatic, teriflunomide, MRI, prevention Clinically Isolated Syndrome, CNS: Central Nervous System, CSF: Cerebrospinal fluid, DMT: Disease-modifying treatment, MRI: Magnetic resonance imaging MS: Multiple sclerosis, OCB: Oligoclonal bands, PP: Primary Progressive, RIS: Radiologically isolated syndrome, RR: Relapsing-remitting, HR: hazard ratio, DIS: Dissemination in space, DIT Paced Auditory Serial Addition Test, CSCT: Computerized Speed Cognitive Test, MFIS: Modified Fatigue Impact Scale

Question: Is it possible to prevent multiple sclerosis with a disease-modifying treatment in preclinical demyelinating disease? Findings: In this 96-week randomized clinical trial, a significant reduction in the risk of clinical multiple sclerosis was demonstrated in those treated with disease-modifying treatment, compared to placebo. The observed safety profile was consistent with known outcomes reported in prior pivotal MS studies.

Meaning:

The findings from this randomized clinical trial provide the first evidence of early treatment intervention with teriflunomide in preventing multiple sclerosis in radiologically isolated syndrome subjects.

Accepted manuscript

Introduction

Multiple sclerosis (MS) is a common cause of severe neurological disability in young adults resulting from an inflammatory immune-mediated insult of myelin, axons, and their supporting cells within the central nervous system (CNS). The diagnosis can only be made following clinical symptoms consistent with acute or progressive CNS injury disseminated in time and space by further clinical or MRI criteria. [START_REF] Thompson | Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria[END_REF] When these MRIs reveal unexpected anomalies highly suggestive of demyelinating plaques given their size, location, and shape in the absence of symptomatology explained by these lesions, they can fulfill formally validated criteria for the radiologically isolated syndrome (RIS). [START_REF] Lebrun | Association between clinical conversion to multiple sclerosis in radiologically isolated syndrome and magnetic resonance imaging, cerebrospinal fluid, and visual evoked potential: follow-up of 70 patients[END_REF][START_REF] Okuda | Incidental MRI anomalies suggestive of multiple sclerosis: the radiologically isolated syndrome[END_REF] This preclinical stage, defined objectively by MRI, expands the spectrum of at-risk individuals for future clinical demyelinating events.

The dissemination in space (DIS) requirement for RIS incorporates the 2005 McDonald criteria [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] , requiring 3 or 4 of four imaging criteria to be met. The RIS criteria [START_REF] Okuda | Incidental MRI anomalies suggestive of multiple sclerosis: the radiologically isolated syndrome[END_REF] , when applied to individuals with incidental demyelinating-appearing MRI lesions without defining clinical features of MS, predict conversion to a first clinical event at a rate of 34% at five years and 51% at ten years. 5,6, Conversion to primary progressive MS (PPMS) [START_REF] Kantarci | on behalf of RISC and CFSEP. Primary Progressive Multiple Sclerosis Evolving From Radiologically Isolated Syndrome[END_REF] or pediatric MS [START_REF] Makhani | Oligoclonal bands increase the specificity of MRI criteria to predict multiple sclerosis in children with the radiologically isolated syndrome[END_REF] has also been described, further validating a pre-clinical MS phase.

The early use of disease-modifying treatments (DMTs) is typically recommended in established MS. [START_REF] Montalban | ECTRIMS/EAN Guideline on the pharmacological treatment of people with multiple sclerosis[END_REF] Despite advancements in the characterization of RIS subjects [START_REF] Lebrun | Association between clinical conversion to multiple sclerosis in radiologically isolated syndrome and magnetic resonance imaging, cerebrospinal fluid, and visual evoked potential: follow-up of 70 patients[END_REF][START_REF] Okuda | Incidental MRI anomalies suggestive of multiple sclerosis: the radiologically isolated syndrome[END_REF][START_REF] Kantarci | on behalf of RISC and CFSEP. Primary Progressive Multiple Sclerosis Evolving From Radiologically Isolated Syndrome[END_REF][START_REF] Makhani | Oligoclonal bands increase the specificity of MRI criteria to predict multiple sclerosis in children with the radiologically isolated syndrome[END_REF] and our understanding of risk factors for initial symptom development [START_REF] Okuda | Radiologically Isolated Syndrome: 5-Year Risk for an Initial Clinical Event[END_REF][START_REF] Lebrun | the ten years RISC Group on behalf of SFSEP and OFSEP. Radiologically Isolated Syndrome: 10-Year Risk estimate of a clinical event[END_REF][START_REF] Lebrun-Frénay | Risk factors and time to clinical symptoms of multiple sclerosis among patients with a radiologically isolated syndrome[END_REF] , the support for DMTs in RIS was only recently demonstrated in the ARISE study [START_REF] Okuda | Multi-center, Randomized, Double-blinded Assessment of Dimethyl Fumarate in Delaying the Time to a First Attack in Radiologically Isolated Syndrome (ARISE)[END_REF] with oral dimethyl fumarate, in extending the time to the first clinical event in RIS.

Teriflunomide (HMR1726; Aubagio ® ) is an oral immunomodulator, taken once daily and approved in more than 80 countries for treating relapsing-remitting MS. [START_REF] Comi | Pooled safety and tolerability data from four placebo-controlled teriflunomide studies and extensions[END_REF][START_REF] O'connor | Long-term safety and efficacy of teriflunomide[END_REF] This study Accepted manuscript investigated the efficacy of teriflunomide in extending the time to a first clinical event related to MS. The rationale for this proposed early treatment intervention trial was to test another DMT with a different mode of action and confirm the impact of an immune intervention at the preclinical stage of MS.

Methods

Study oversight and ethics statement

The RIS Consortium (RISC) designed the trial and named adjudication referees for clinical and MRI application of RIS criteria in screened subjects. Referees were also asked to adjudicate neurological events when reported by participants. An independent safety monitoring committee reviewed all safety data (See online only the trial oversight section, eFigure 1 and eFigure 2). All RISC core group members independently authored and developed the study protocol and participated in writing the manuscript. All authors approved the draft sent to Sanofi, which was only involved in funding support. The trial was conducted according to the provisions of the International Conference on Harmonization Guidelines for Good Clinical Practice. [START_REF]ICH harmonised tripartite guideline for good clinical practice: E6(R1)[END_REF] The Declaration of Helsinki was followed during the TERIS trial. [START_REF]WMA Declaration of Helsinki -ethical principles for medical research involving human subjects[END_REF] The study protocol, patient information, and consent form were accepted by the relevant Ethics Committee (IEC) and registered as EUDRA-CT 2016-002428-97. The Study Sponsor was the University Hospital of Nice and registered at ClinicalTrials.gov as NCT03122652 (TERIS). Data were secured by clinical research organizations and remained confidential throughout the study. Data analysis was performed solely by the investigators independent of the funding source.

Participants

Written informed consent from participants was required. Inclusion criteria were i) age >18 years, ii) fulfillment of 2009 RIS criteria [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] (eFigure 1) with structural neuroimaging abnormalities not Accepted manuscript explained by another disease process, and no historical accounts of remitting clinical symptoms consistent with neurological dysfunction.

Exclusion criteria comprised severe hepatic, renal, and immune system impairments, lactating or pregnant women, or previous exposure to immunosuppressive or disease-modifying therapy.

Subjects of reproductive potential were eligible only if the teriflunomide use guidelines were met (online only). [START_REF]Summary of safety product characteristics for teriflunomide[END_REF]

Study design

TERIS was planned as a 96-week, multi-center, randomized (1:1), double-blinded study of teriflunomide 14mg daily or placebo in RIS subjects fulfilling 2009 criteria 3 (eFigure 1), with the option to continue in the randomization arm up to week 144. Medication, placebo capsules, and packaging were identical to those used in the pivotal teriflunomide studies. [START_REF] Lebrun | the ten years RISC Group on behalf of SFSEP and OFSEP. Radiologically Isolated Syndrome: 10-Year Risk estimate of a clinical event[END_REF][START_REF] Kantarci | on behalf of RISC and CFSEP. Primary Progressive Multiple Sclerosis Evolving From Radiologically Isolated Syndrome[END_REF][START_REF] Makhani | Oligoclonal bands increase the specificity of MRI criteria to predict multiple sclerosis in children with the radiologically isolated syndrome[END_REF] An independent clinical adjudication committee (AS, MC, DTO) verified study entry criteria before randomization. MRI scans were analyzed through adjudicated consensus by the Advanced Imaging in Multiple Sclerosis (AIMS) Laboratory (LM, CJA, DP) in Los Angeles, California.

Each clinical center was responsible for recruitment, treatment, evaluation of participants, and collection of study data according to the protocol. Randomization was stratified according to geographic region and was performed centrally by an independent interactive web-response system. Expanded Disability Status Scale (EDSS) scores, functional scores, Paced Auditory Serial Addition Test (PASAT-3), Computerized Speed Cognitive Test (CSCT) assessments, Modified Fatigue Impact Scale (MFIS), and quality of life (EQ-5D-5L and MUSICOL) were performed at baseline, every six months, and any unscheduled visit following a neurological event.

RIS subjects who completed the 96-week study without symptoms suggestive of MS or side effects to the allocated drug were offered to enter the third year in the same arm. RIS subjects who Accepted manuscript converted to MS or did not wish to continue in the assigned treatment were included in the safety follow-up. Patients diagnosed with MS were offered to start or continue oral teriflunomide.

The wash-out procedure was applicable for women in the event of pregnancy while on study medication, for women who planned to become pregnant, for premature discontinuation before or at the end of the study, or in specific situations according to the investigator's decision (online only material).

Brain and Spinal Cord MRI protocols:

Imaging studies were conducted on 1.5 Tesla (T) or 3T MRI units from different manufacturers using a standardized protocol (online only material). Baseline MRI anomalies were verified as consistent with CNS demyelinating disease and meeting spatial dissemination criteria by a central reading unit. MRI scans were performed at baseline, week 48, week 96, week 144, and any time during the study if warranted by the investigator.

Outcomes

The primary outcome was the time to a first acute or progressive neurological event resulting from CNS demyelination, expressed as a rate of conversion to clinical MS. An acute neurological event was defined by a clinical symptom localized to the optic nerve, brainstem, cerebellum, spinal cord, or long sensory or motor tracts, lasting >24 hours and followed by symptom improvement. A progressive event was defined by the onset of a clinical symptom with the temporal profile revealing at least a 12-month progression.

Secondary outcomes included the following brain MRI metrics; the cumulative number of new and/or enlarging T2-weighted hyperintense lesions, the cumulative number of new gadoliniumenhancing (Gd+) lesions, changes in T2-weighted hyperintense lesion volumes, and the proportion Accepted manuscript of participants with new or newly enlarging T2-weighted hyperintense lesions and new Gd+ lesions. The additional clinical metrics (PASAT-3, CSCT, MFIS, EQ-5D-5L, and MUSICOL) were assessed at baseline and yearly.

Statistical Analysis

Sample size calculations using Cox proportional hazards regression indicated that 80 patients per arm would have been needed to have 80% power to detect a 50% reduction in the risk of the first clinical event if 25% of participants experienced a first clinical event at two years having a trial duration of 5 years. The prior distribution for the effect of teriflunomide versus placebo was derived from the TOPIC 17 (NCT 000622700), TEMSO [START_REF] O'connor | Randomized trial of oral teriflunomide for relapsing multiple sclerosis[END_REF] (NCT 00134563), and TOWER [START_REF] Confavreux | Oral teriflunomide for patients with relapsing multiple sclerosis (TOWER): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF] (NCT 00751881) trials in RRMS.

Survival curves were computed using the Kaplan-Meier method to display the treatment effect.

Both adjusted and unadjusted Cox proportional-hazards regression models were used to assess the impact of treatment from randomization to the first acute or initial progressive neurological event.

The adjusted model used key baseline variables to adjust for potential confounding effects, such as sex, age at the time of RIS diagnosis, MS family history, brain T2-weighted hyperintense lesion volume (log-transformed), and presence of Gd+/-lesions. The statistical analyses for the secondary and the exploratory endpoints are detailed in the online-only material. All statistical analyses (MPS, FB) were performed using SAS software, version 9.4 (SAS Institute), or R, version 4.1.3.

Results

Participants

From September 25, 2017, to October 31, 2022, 124 RIS patients were screened. Thirty-Three subjects were excluded due to RIS diagnostic criteria not being fulfilled (18), study consent withdrawals (16), and loss of follow-up (1). Of the 89 randomized RIS subjects from 20 centers

Accepted manuscript

in France, Switzerland, and Turkey, 63 (70.8%) were female. The mean (±SD) age at study enrolment was 39.8 (±12.1) years, and the mean (±SD) age at index MRI was 37.9 (±12) years.

The baseline demographic and clinical data were well-balanced between groups (Table 1). The most common reasons for obtaining the index MRIs were headache (46.1%), follow-up for other neurological diseases (28.1%), dizziness/vertigo (16.9%), and visual symptoms (6.7%) (Table 1).

Adverse reactions and reasons for study discontinuation are provided in the appendix (eTable 1, and eTable2). The retention rate for the study was 82%. Follow-up MRI studies performed at week 96 were acquired in 62 (69.7%) of subjects, 32 within the placebo group and 30 in the teriflunomide group (eFigure2).

Efficacy

Primary outcome

After randomization, 28 primary clinical endpoints were detected; four (14.3%) were consistent with a progressive clinical syndrome qualifying as primary progressive MS, and 24 (85.7%) were consistent with acute relapses. Syndromic localization of the acute events was optic nerve (4 (14.3%)), spinal cord (8 (28.5%)), brainstem/cerebellum (3 (10.7%)), and subcortical white matter (9 (32.1%)). Progressive events involved cognitive impairment (2 (7.2%)) and progressive paraparesis (2 (7.2%)).

The mean (±SD) time to the first clinical event was 119.0 (±5.2) weeks.

The risk of a first clinical event was significantly reduced in the teriflunomide arm (mean time to event=128.2 (±7.25) weeks, eight clinical events; six acute, two progressive) versus the placebo arm (mean time to event=109.6 (±7.44) weeks, 20 clinical events; 18 acute, two progressive), with an unadjusted HR=0.37, 95% confidence interval (CI)=0.16-0.84, p=0.018 (Figure 1 and Table 2).

Accepted manuscript

This result corresponded to a 63% reduction in the risk of a first MS-related clinical event favoring teriflunomide.

Results from the adjusted Cox proportional-hazards regression model were consistent with those of the unadjusted analysis, with a significant effect favoring teriflunomide on reducing the risk of the first event after adjusting for sex, age at the time of diagnosis, MS family history, EDSS, T2weighted hyperintense volume (log-transformed) and the presence of gadolinium-enhancing lesions (HR=0.28, 95% CI=0.11-0.71, p=0.007; Table 2).

Secondary outcomes

Compared to placebo, the cumulative number of new and/or newly-enlarging T2 lesions (RR=0.57, 95% CI=0.27-1.20, p=0.139) and the cumulative number of Gd+ lesions (RR=0.33, 95%CI:0.09-1.17, p=0.086) were reduced in the teriflunomide arm in the adjusted analysis, although statistical significance was not achieved. The proportions of subjects with new and/or newly-enlarging T2 lesions (OR=0.72 95% CI=0.25-2.06), p=0.537) and with Gd+ activity (OR=0.28 95% CI=0.07-1.13), p=0.075) were also reduced in the teriflunomide arm, but not statistically significant.

Like imaging metrics, the results in other secondary clinical outcomes favored the teriflunomide arm but without statistical significance (Table 3). The largest difference between teriflunomide and placebo was observed for the EQ-5D-5L (mean difference in the annualized change +3.69 (95%CI=-0.56; 7.95), p=0.089).

Safety

Adverse events were closely monitored during the follow-up. The treatment arm reported one severe COVID infection declared as possibly related to the drug. Only one patient stopped the Accepted manuscript study due to side effects (eTable 2). The most common adverse events observed more often in patients treated with teriflunomide were gastrointestinal disorders (11.4%), dysmenorrhea (9.1%), benign respiratory infections (6.8%), general disorders conditions (6.8%), and transient increase of transaminases (4.5%) (eTable 1). Reasons for study discontinuation after randomization were loss of follow-up (27.8%), consent withdrawal (22.2%), patient's decision (22.2%), adverse event (16.7%), pregnancy (5.5%), and sponsor decision (5.5%).

Discussion

Following the recent ARISE study [START_REF] Okuda | Multi-center, Randomized, Double-blinded Assessment of Dimethyl Fumarate in Delaying the Time to a First Attack in Radiologically Isolated Syndrome (ARISE)[END_REF] , TERIS is the second randomized multi-center study to demonstrate the benefit of a DMT previously approved for use in established MS in delaying or preventing the development of clinical MS in subjects with RIS with a risk reduction of over 60% relative to placebo. An overwhelming number of natural history studies and clinical trials in clinically isolated syndrome (CIS) and early MS support the paradigm that earlier treatment is beneficial in delaying long-term disability accumulation. [START_REF] Montalban | ECTRIMS/EAN Guideline on the pharmacological treatment of people with multiple sclerosis[END_REF][START_REF] Miller | Clinically isolated syndromes[END_REF] The TERIS study and ARISE 11 demonstrate that different DMTs with different immune mechanisms of action, studied in different populations derived from diverse healthcare systems, yield comparable results, supporting that early intervention in RIS may improve clinical outcomes.

Even with demonstrated efficacy and reassuring teriflunomide long-term safety data [START_REF] Comi | Pooled safety and tolerability data from four placebo-controlled teriflunomide studies and extensions[END_REF][START_REF] O'connor | Long-term safety and efficacy of teriflunomide[END_REF][START_REF]Summary of safety product characteristics for teriflunomide[END_REF][START_REF] Papeix | French Group for Recommendations in Multiple Sclerosis (France4MS), the Société Francophone de la Sclérose En Plaques (SFSEP). Infections and multiple sclerosis: Recommendations from the French Multiple Sclerosis Society[END_REF] , the indication of starting a DMT needs to be thoroughly discussed in a presymptomatic situation, as not all RIS subjects might benefit from it. The cornerstone of the risk-benefit balance is the absence of an induced severe adverse event for a drug prescribed at the preclinical stage of the disease. Several retrospective and prospective data-driven RISC cohorts have identified risk factors for developing a first clinical event and have demonstrated a risk stratification with an increased risk for developing MS at a younger age, the presence of spinal cord or infratentorial lesions, the identification of new MRI lesions over time, and the presence of gadolinium-

Accepted manuscript

enhancing lesions or OCBs in the CSF. [START_REF] Okuda | Radiologically Isolated Syndrome: 5-Year Risk for an Initial Clinical Event[END_REF][START_REF] Lebrun | the ten years RISC Group on behalf of SFSEP and OFSEP. Radiologically Isolated Syndrome: 10-Year Risk estimate of a clinical event[END_REF][START_REF] Lebrun-Frénay | Risk factors and time to clinical symptoms of multiple sclerosis among patients with a radiologically isolated syndrome[END_REF][START_REF] Lebrun-Frénay | The radiologically isolated syndrome: revised diagnostic criteria[END_REF] Whether these observed risk factors should be used to guide the decision to initiate treatment in RIS patients remains to be seen. The risk-benefit calculation should favor patient safety at the preclinical stage of the disease. This is the first limitation of our study as we could not stratify at-risk subgroups according to the aforementioned risk factors for developing MS, mainly because our proof-of-concept study was powered to detect the overall impact of active treatment versus placebo arms. Any such stratification would require a much larger clinical trial with a different research question and preselection of high and low-risk groups rather than post-hoc analyses, which are inappropriate for the current study. Now that two studies (ARISE and TERIS) have proven the efficacy of DMTs in RIS, future study designs can focus on identifying the ideal subjects who would benefit most from treatment. As expected, TERIS shares additional limitations similar to the preceding ARISE study. [START_REF] Okuda | Multi-center, Randomized, Double-blinded Assessment of Dimethyl Fumarate in Delaying the Time to a First Attack in Radiologically Isolated Syndrome (ARISE)[END_REF] TERIS was also prematurely discontinued by the study sponsor, mainly due to the reduced pace of study enrollment. When the study started, the sample size was calculated from published pivotal teriflunomide CIS and RRMS studies. [START_REF] Miller | TOPIC Study Group. Oral teriflunomide for patients with a first clinical episode suggestive of multiple sclerosis (TOPIC): a randomised, doubleblind, placebo-controlled, phase 3 trial[END_REF][START_REF] O'connor | Randomized trial of oral teriflunomide for relapsing multiple sclerosis[END_REF][START_REF] Confavreux | Oral teriflunomide for patients with relapsing multiple sclerosis (TOWER): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF] A sample size calculation based on data-driven cohorts was proposed for RIS studies more recently. [START_REF] Lebrun-Frénay | Risk factors and time to clinical symptoms of multiple sclerosis among patients with a radiologically isolated syndrome[END_REF] The onset of PPMS in the current study is in keeping with the overall expected incidence of PPMS in MS and RIS populations. [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF][START_REF] Tutuncu | Onset of progressive phase is an age-dependent clinical milestone in multiple sclerosis[END_REF] In the ARISE study, primary progressive clinical courses were not observed during the two-year study period. [START_REF] Okuda | Multi-center, Randomized, Double-blinded Assessment of Dimethyl Fumarate in Delaying the Time to a First Attack in Radiologically Isolated Syndrome (ARISE)[END_REF] In TERIS, PPMS onset was observed in 4 (14%) individuals diagnosed during the third year of the study. Taken together, these results suggest that we may, for the first time, have an opportunity to better identify those at risk for a primary progressive clinical course at this preclinical stage and prevent or delay clinical progression from the onset, a clear unmet need in MS clinical practice.

MRI is now the most helpful tool to predict conversion to MS in patients with CIS [START_REF] Tintore | Baseline MRI predicts future attacks and disability in clinically isolated syndromes[END_REF] . Particularly gadolinium-enhancing and spinal cord lesions at baseline and enhancing lesions at one year and Accepted manuscript three years best predict future physical and cognitive disability, including conversion to secondary progressive MS at 15 years. [START_REF] Brownlee | Early imaging predictors of long-term outcomes in relapse-onset multiple sclerosis. Early imaging predictors of long-term outcomes in relapseonset multiple sclerosis[END_REF] RISC cohorts demonstrated a similar risk profile for the presymptomatic phase. [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF][START_REF] Okuda | Radiologically Isolated Syndrome: 5-Year Risk for an Initial Clinical Event[END_REF][START_REF] Lebrun | the ten years RISC Group on behalf of SFSEP and OFSEP. Radiologically Isolated Syndrome: 10-Year Risk estimate of a clinical event[END_REF][START_REF] Lebrun-Frénay | Risk factors and time to clinical symptoms of multiple sclerosis among patients with a radiologically isolated syndrome[END_REF] In TERIS, although all the secondary MRI measures favored teriflunomide over placebo, none achieved statistical significance. MRI activity in RIS is less known than those in established MS. Consequently, the individual variance may have led to reduced statistical power. Similarly, statistical trends toward benefit in the present study were also observed on fatigue, quality of life, and cognitive metrics. Since all secondary outcome measures consistently trended in the same direction, such results support the primary clinical endpoint and provide an opportunity to properly power future clinical trials in RIS to detect DMT impact on secondary endpoints.

The MS community now has two randomized, multi-center, placebo-controlled, double-blind studies showing that an active treatment extends the time to symptomatic MS in RIS individuals meeting a previously unmet need. Earlier treatment during the presymptomatic phase of MS can delay disability even further after symptomatic MS onset. However, before considering any immune intervention, it is essential to highlight the risk of misdiagnosing individuals with nonspecific MRI anomalies as having RIS, which underlines the role of MS tertiary centers in managing the diagnosis and intervention in RIS. [START_REF] Solomon | The contemporary spectrum of multiple sclerosis misdiagnosis. A multicenter study[END_REF][START_REF] Calabrese | Better explanations" in multiple sclerosis diagnostic workup[END_REF] EDSS, median (IQR) 0 (0-0) 0 (0-0) 0 (0-0)

Follow-up

Analyzed ITT (n=45)

♦ Excluded from analysis (give reasons) (n=0)

Analyzed ITT (n=44)

♦ Excluded from analysis (give reasons) (n=0) Analysis

Figure 1 :Figure S2 .

 1S2 Figure 1: Kaplan-Meier estimates of time from randomization to the first demyelinating event (unadjusted analysis)

Table 1 :

 1 Baseline characteristics of the study population

	Total	Teriflunomide	Placebo
	(n=89)	(n=44)	(n=45)

Acknowledgments

The research investigators sincerely thank all study participants, research coordinators, and support staff for their dedication and commitment to expanding knowledge within the field of MS. We would like to thank Dr. Isabel Firmino (Alexion, then Genzyme), Dr. Aymeric de Chastenier (Sanofi Genzyme, then La Roche Posay), and Dr. Amel Gheribenblidia (Sanofi) who believe in our project from the beginning and supported it to Sanofi executing committee. We also thank Mrs.

PL received honoria and grants from Biogen, Sanofi Genzyme, Merck, Novartis, Alexion and Janssen.

FDD none HE has received speaking honoraria, personal compensation for participating on advisory boards , Biogen Idec/Gen Pharma of Turkey, Merck-Serono; Novartis; ELP none MT Received a research scholarship from Ondokuz Mayıs University Research Support Funds Received honoraria or consultancy fees from Novartis, Teva, Merck-Serono, Biogen Idec/Gen Pharma, Sanovel, Ali Raif, Abdi İbrahim in exchange for travel and registration guarantee for participation in advisory boards, teaching training courses and/or participation in scientific congresses or symposiums, or consulting fees ND none BB serves on scientific advisory board, has received funding for travel and honoraria from Alexion, Biogen, BMS, Merck, Novartis, Sanofi and Roche RH received speaker/advisor honorary from Merck, Novartis, Roche, Biogen, Alexion, Sanofi, Janssen, Bristol-Myers Squibb, Teva/Mepha and Almirall. He received research support within the last 5 years from Roche, Merck, Sanofi, Biogen, Chiesi, and Bristol-Myers Squibb. He also Accepted manuscript received research grants from the Swiss MS Society and is a member of the Advisory Board of the Swiss and International MS Society. He also serves as Rana Karabudak has received honoraria for giving educational lectures, consultancy fees for participating advisory boards, and travel grants for attending scientific congresses or symposia from Roche, Sanofi-Genzyme, Merck-Serono, Novartis, Teva, Biogen Idec/Gen Pharma of Turkey , consulting, serving on a scientific advisory board, speaking, or other activities with Biogen, Novartis, Merck, Sanofi-Genzyme, Roche, BMS, Alexion, Horizon Therapeutics, none related to this study. PC none SW received consultancy fees, speaker fees, honoraria and clinical research grants from Alexion, Biogen, Janssen-Cilag, Merck, Novartis, Roche and Sanofi. OFT Received honoraria or consultancy fees for participating to advisory boards giving lectures and/or Received research grants from The Scientific and Technological Research Council Of Turkey and Ege University Research Support Funds Received honoraria or consultancy fees for participating to advisory boards, giving educational lectures and/or travel and registration coverage for attending scientific congresses or symposia from F. Hoffmann-La Roche Ltd, Sanofi-Genzyme, Merck-Serono Novartis, Biogen Idec/Gen Pharma of Turkey, TEVA , Alexion, ARİS, Sanovel and Abdi Ibrahim Turkey MC has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Ad Scientiam, Biogen, Merck, Sanofi-Genzyme, Roche, Celgene-BMS, Alexion, Horizon Therapeutics, none related to this study. CJA receives grant support from the National Multiple Sclerosis Society and the National Institutes of Health (NIH). In the last three years, she has received honoraria or consulting fees from Biogen, Sanofi-Genzyme, Novartis, Genentech, Alexion, EMD Serono, and Horizon Therapeutics for participation on advisory boards and data safety monitoring committees. Honoraria for lectures from American Academy of Neurology, Spire Learning, Department of Defense, Catamount Education, Projects in Knowledge, Oregon Health and Science University. Serono, Genentech, Genzyme, Janssen Pharmaceuticals, and research support from Biogen and EMD Serono/Merck. He was also a study protocol author and served as the lead investigator of the ARISE study.

Accepted manuscript

Yanica Mathieu for her support as an FCRIN4MS organizer and Mrs. Cynthia Caillon from the Research Clinic Direction of Nice University Hospital for her constant support.

Data availability

Anonymized data collected for the study, including individual participant data and a data dictionary defining each field in the set, will be made available to others for publication six months later. Data not published within this article will be made available by request from any qualified investigator.

The full study protocol, statistical analysis plan, and informed consent form will also be supplementary data. The corresponding author had final responsibility for the decision to submit the manuscript for publication. All named authors met the International Committee of Medical Journal Editors criteria for authorship for this manuscript and take responsibility for the integrity of the work as a whole.

Contributions

Conflicts of interest

CLF is the principal investigator of the TERIS Study, with no personal fees.

Accepted manuscript

Data and Safety Monitoring Committee

Cassandre Landes-Chateau, CCS, and Samir Yamani, Nice University Hospital, Nice France. *Mean annualized changes were calculated as the difference between baseline and the last follow-up available All the adjusted analyses were corrected for sex, age at RIS diagnosis, MS family history, EDSS, T2-lesion volume (log-transformed), and presence of gadolinium-enhancing lesions at baseline. Means, differences, and p-values were calculated using a generalized regression model, with the follow-up value as the dependent variable after controlling for the corresponding baseline value and with an offset term to account for follow-up duration.

Accepted manuscript

Accepted manuscript


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Aridity and cold temperatures drive divergent adjustments of European beech xylem anatomy, hydraulics, and leaf physiological traits

Keywords: Intraspecific trait variation, trait coordination, trait variances, water use, climatic stress, xylem hydraulics, leaf physiology

Understanding plant trait coordination and variance across climatic gradients is critical for assessing forests' adaptive potential to climate change. We measured eleven hydraulic, anatomical and leaf-level physiological traits in European beech (Fagus sylvatica L.) along a moisture and temperature gradient in the French Alps. We assessed how traits covaried, and how their population-level variances shifted along the gradient. The intrapopulation variances of vessel size and xylem-specific conductivity reduced in colder locations as narrow vessels were observed in response to low temperature. This decreased individual-level water transport capacity compared to the warmer and more xeric sites. Conversely, the maximum stomatal conductance and Huber value variances were constrained in the arid and warm locations, where trees showed restricted gas exchange and higher xylem-specific conductivity.

The populations growing under drier and warmer conditions presented wide variance for the xylem anatomical and hydraulic traits. Our results suggest that short-term physiological acclimation to raising aridity and heat in southern beech populations may occur mainly at the leaf level. Furthermore, the wide variance of the xylem anatomical and hydraulic traits at these sites may be advantageous since more heterogeneous hydraulic conductivity could imply populations' greater tree-tree complementarity and resilience against climatic variability. Our study highlights that both intrapopulation trait variance and trait network analysis are key

 [START_REF] Mcdowell | Mechanisms of plant survival and mortality during drought: Why do some plants survive while others succumb to drought?[END_REF][START_REF] Klein | Drought-induced tree mortality: From discrete observations to comprehensive research[END_REF][START_REF] Anderegg | Metaanalysis reveals that hydraulic traits explain cross-species patterns of drought-induced tree mortality across the globe[END_REF]). Thus, forest health very much depends on trees' capacity to modulate their water-use strategies to a changing environment [START_REF] Messier | Similarities and differences in intrapopulation trait correlations of co-occurring tree species: consistent water-use relationships amid widely different correlation patterns[END_REF]. In this context, functional traits related to xylem anatomy, hydraulic conductivity and leaf-level water use emerge as key proxies of tree water-use economy [START_REF] Gleason | Shifts in leaf and stem hydraulic traits across aridity gradients in eastern Australia[END_REF][START_REF] Anderegg | Spatial and temporal variation in plant hydraulic traits and their relevance for climate change impacts on vegetation[END_REF].

Variation in the functional traits that regulate water use has been widely assessed along environmental gradients to identify adaptive patterns. As climate goes from wetter to drier conditions, trees tend to shift from an acquisitive to a conservative water-use strategy [START_REF] Reich | The world-wide "fast-slow" plant economics spectrum: A traits manifesto[END_REF]. For instance, xeric environments promote the formation of narrower xylem vessels (e.g. [START_REF] García-Cervigón | Xylem adjusts to maintain efficiency across a steep precipitation gradient in two coexisting generalist species[END_REF][START_REF] López | Coordination of stem and leaf traits define different strategies to regulate water loss and tolerance ranges to aridity[END_REF]. Trees from dry environments also tend to optimise water supply to functional leaves, which is consistently observed across species as an increment in Huber value (i.e. the ratio of stem section per leaf area unit) [START_REF] Martínez-Vilalta | Hydraulic adjustment of Scots pine across Europe[END_REF][START_REF] Gleason | Shifts in leaf and stem hydraulic traits across aridity gradients in eastern Australia[END_REF][START_REF] Pritzkow | Phenotypic plasticity and genetic adaptation of functional traits influences intra-specific variation in hydraulic efficiency and safety[END_REF], Rosas et al. 2019). Additionally, the gas exchange rates of trees growing in more arid conditions are usually lower (i.e. stomatal conductance and assimilation capacity), and their water-use efficiency increases to prevent

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5 leaf hydraulic damage [START_REF] Aranda | Variation in photosynthetic performance and hydraulic architecture across European beech (Fagus sylvatica L.) populations supports the case for local adaptation to water stress[END_REF][START_REF] Lübbe | Acclimation of leaf water status and stem hydraulics to drought and tree neighbourhood: Alternative strategies among the saplings of five temperate deciduous tree species[END_REF][START_REF] Torres-Ruiz | Genetic differentiation in functional traits among European sessile oak populations[END_REF][START_REF] Henry | A stomatal safetyefficiency trade-off constrains responses to leaf dehydration[END_REF].

Temperature is also a primary driver of variation in water-use-related functional traits. Rising temperatures lead to high vapour pressure deficit (VPD), which increases the dehydration risk [START_REF] Kurz | Mountain pine beetle and forest carbon feedback to climate change[END_REF], Will et al. 2013). Plants tend to mitigate heat impacts by performing stronger stomatal control and reducing their whole-tree hydraulic conductance [START_REF] Nathalie | Temperate forest trees and stands under severe drought: a review of ecophysiological responses, adaptation processes and long term consequences[END_REF][START_REF] Grossiord | Plant responses to rising vapor pressure deficit[END_REF]. Low temperatures can trigger frost-induced cavitation in temperate and cold ecosystems [START_REF] Mayr | Repeated freeze-thaw cycles induce embolism in drought stressed conifers (Norway spruce, stone pine)[END_REF][START_REF] Charrier | Monitoring of freezing dynamics in trees: A simple phase shift causes complexity[END_REF]. As an adaptation mechanism to this disturbance in cold environments, trees promote narrower vessels [START_REF] Castagneri | Divergent climate response on hydraulic-related xylem anatomical traits of Picea abies along a 900-m altitudinal gradient[END_REF][START_REF] Schreiber | Variation of xylem vessel diameters across a climate gradient: Insight from a reciprocal transplant experiment with a widespread boreal tree[END_REF][START_REF] García-Cervigón | Xylem anatomy needs to change, so that conductivity can stay the same: Xylem adjustments across elevation and latitude in Nothofagus pumilio[END_REF], similarly to when they are subjected to dry conditions. Trees exposed to frequent frosts and longer winters have higher maximum assimilation rates compared to those growing under milder temperature conditions [START_REF] Huxman | Photosynthetic resource-use efficiency and demographic variability in desert winter annual plants[END_REF][START_REF] Bresson | To what extent is altitudinal variation of functional traits driven by genetic adaptation in European oak and beech?[END_REF].

However, there are still many uncertainties about the intraspecific trait variation dimension in response to environmental variability in trees because current knowledge is based mainly on interspecific comparisons. Yet, intraspecific trait variability can be wider than interspecific variability (e.g. [START_REF] Jung | Intraspecific trait variability mediates the response of subalpine grassland communities to extreme drought events[END_REF], Siefert et al. 2015). Furthermore, interspecific adaptive patterns to shifting environments can vastly vary within species [START_REF] Lübbe | Acclimation of leaf water status and stem hydraulics to drought and tree neighbourhood: Alternative strategies among the saplings of five temperate deciduous tree species[END_REF][START_REF] Fuchs | Hydraulic variability of three temperate broadleaf tree species along a water availability gradient in central Europe[END_REF], which indicates the need for more studies to assess intraspecific trait variability.

Moreover, most existing studies have addressed trait variation by a univariate approach, although functional traits involved in water use normally show consistent correlations (i.e. trait coordination). Indeed vessel anatomy is closely coupled with hydraulic conductivity [START_REF] Zimmermann | Xylem Structure and the Ascent of Sap[END_REF], Sperry et al. 2008). The latter has been positively related to greater stomatal conductance and leaf-level water use [START_REF] Brodribb | Hydraulic and photosynthetic co-ordination in seasonally dry tropical forest trees[END_REF][START_REF] Brodribb | Stomatal closure during leaf dehydration, correlation with other leaf physiological traits[END_REF]. In this context, studying trait covariation using coordination networks has been

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suggested to gain more mechanistic insights into adaptive processes [START_REF] Poorter | Trait correlation networks: a whole-plant perspective on the recently criticized leaf economic spectrum[END_REF][START_REF] Messier | Interspecific integration of trait dimensions at local scales: the plant phenotype as an integrated network[END_REF]. The emergence of covariation structures would depict the selection of specific trait combinations in response to climate shifts. However, the trait coordination structures observed across species do not necessarily hold within species [START_REF] Umaña | Does trait variation within broadly distributed species mirror patterns across species? A case study in Puerto Rico[END_REF]Swenson 2019, Rosas et al. 2019). Furthermore, recent studies conducted on an intraspecific scale suggest that trait coordination may vastly vary depending on climate conditions [START_REF] Carvalho | Intraspecific perspective of phenotypic coordination of functional traits in Scots pine[END_REF][START_REF] Benavides | Phenotypes of Pinus sylvestris are more coordinated under local harsher conditions across Europe[END_REF]. So it is necessary to better understand how traits are coordinated within species and along environmental gradients.

In addition to adjustments to trait values and coordination, understanding how trait variance patterns are distributed across a given species' populations might be essential for predicting its adaptive potential [START_REF] Ahrens | Intra-specific trait variation remains hidden in the environment[END_REF]. Indeed population-level heritable trait plasticity can be modified due to local adaptations [START_REF] Linhart | Evolutionary significance of local genetic differentiation in plants[END_REF][START_REF] Valladares | Ecological limits to plant phenotypic plasticity[END_REF][START_REF] Reich | The world-wide "fast-slow" plant economics spectrum: A traits manifesto[END_REF]. For instance, stressful climate conditions can filter out unfavourable trait values (i.e. climatic filtering effect) which, in turn, reduces community-level trait variance (Weiher et al. 1995, Swenson and[START_REF] Swenson | Ecological and evolutionary determinants of a key plant functional trait: Wood density and its community-wide variation across latitude and elevation[END_REF]. Such reductions in pools of available trait variances can limit population adjustment capacity (Molina-Montenegro and Naya 2012, [START_REF] Anderegg | Aridity drives coordinated trait shifts but not decreased trait variance across the geographic range of eight Australian trees[END_REF]). In this context, studies performed on an interspecific scale report narrow variances for woody traits (i.e. wood density and bark thickness) in communities growing under cold conditions (Swenson et al. 2012), and the same applies to leaf anatomical traits in water-limited environments (Swenson et al. 2012(Swenson et al. , Šímová et al. 2017). Within species, [START_REF] Anderegg | Aridity drives coordinated trait shifts but not decreased trait variance across the geographic range of eight Australian trees[END_REF] observed constrained variance patterns for leaf dry mass content in more xeric populations.

Nonetheless, very few studies have addressed intraspecific trait variance patterns along broad climatic gradients, and current knowledge on their climatic drivers is still weak [START_REF] Šímová | Stress from cold and drought as drivers of functional trait spectra in North American angiosperm tree assemblages[END_REF](Šímová et al. , Anderegg et al. 2020)).

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In order to improve our understanding of intraspecific water-use strategy variation, our study focuses on European beech (Fagus sylvatica L.). Beech is a widely distributed species in European forests. It is quite sensitive to both drought [START_REF] Geßler | Potential risks for European beech (Fagus sylvatica L.) in a changing climate[END_REF][START_REF] Friedrichs | Speciesspecific climate sensitivity of tree growth in Central-West Germany[END_REF]) and temperature extremes [START_REF] Weigel | Winter matters: Sensitivity to winter climate and cold events increases towards the cold distribution margin of European beech (Fagus sylvatica L.)[END_REF][START_REF] Gazol | Summer drought and spring frost, but not their interaction, constrain European beech and Silver fir growth in their southern distribution limits[END_REF], which makes it a key species for understanding trait adjustments along climatic gradients. Specifically, we centre on xylem anatomy (i.e. vessel size and density), hydraulics (i.e. maximum xylem-and leaf-specific conductivity, loss of conductivity, Huber value) and leaf physiology (i.e. stomatal conductance, assimilation rate, water-use efficiency) traits. Our study area lies along a 300 kilometre-long precipitation and temperature gradient in the southern French Alps. The gradient covers an extensive geographical range from the southern Mediterranean edge to the wetter alpine limit of beech, where heat+aridity and low temperature are, respectively, its main limiting factors.

Our objectives are to assess: (i) how the functional traits that modulate beech water use vary in different climates along the gradient; (ii) how coordination among these functional traits varies along the climatic gradient; (iii)

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Materials and Methods

Study area

This study was carried out at six sites scattered along a climatic gradient in the French Alps (Fig. 1) [START_REF] Jourdan | How neighbourhood interactions control the temporal stability and resilience to drought of trees in mountain forests[END_REF][START_REF] Jourdan | The effect of tree diversity on the resistance and recovery of forest stands in the French Alps may depend on species differences in hydraulic features[END_REF], Jourdan et al. 2021).

The mean annual precipitation and temperature among the sites along the south to north gradient range from 877 mm to 1,292 mm and from 11ºC to 6.7ºC, respectively. At the three most southern and driest sites (SB, LU, LA), forest type is composed mostly of beech and downy oak (Quercus pubescens Willd.), and of beech and silver fir (Abies alba Mill.) at the three northern sites (VT, VC, BG). A recent study along the same gradient depicts that there are no discrete genetic differences between sites, which suggests that they belong to the same genetic population [START_REF] Capblancq | Climate-associated genetic variation in Fagus sylvatica and potential responses to climate change in the French Alps[END_REF]. To reduce the impact of interspecific interactions, two monospecific plots were selected at each site consisting of a 17.5 metre-radius circle where at least 90% of the basal area is represented by beech. In each plot, five dominant trees were selected for sampling (10 trees per site, n=60 trees in all). All the plots along the gradient are north-or north-west oriented, and of a limestone bedrock type. The selected stands have not been managed in at least the last decade, and their structure is high forest in all the plots, except LU, where forest is coppice. The main attributes of each site are depicted in Table 1, and more details about site selection and characteristics can be found in [START_REF] Jourdan | The effect of tree diversity on the resistance and recovery of forest stands in the French Alps may depend on species differences in hydraulic features[END_REF] and [START_REF] Jourdan | Tree diversity and the temporal stability of mountain forest productivity: testing the effect of species composition, through asynchrony and overyielding[END_REF].

Field sampling and laboratory measurements

One field campaign per site was carried out between July and August 2019. Two 3-4 metrelong healthy top-of-canopy branches were harvested at predawn and midday from all the selected trees. Canopy was accessed with tree-climbing equipment in all the plots, except LU, where a telescopic pole pruner was used. Directly after branch cutting at predawn, leaf water

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9 potential (Ѱpd) was measured on one terminal leaf per branch by a Scholander pressure chamber (PMS Instrument Co., Corvallis, Oregon, USA).

From the branches harvested at midday, a terminal section of at least 1.5 m was immediately removed and placed in water, where it was recut at least twice to remove potential xylem cavitation effects. Afterwards, maximum stomatal conductance and assimilation rates (gsmax and Amax, respectively) were measured on one terminal leaf using an infrared gas-exchange analyzer (LI-COR 6400, LI-COR, Lincoln, USA). Measuring the gas exchange rates by this method had no impact on Amax and gsmax [START_REF] Verryckt | Coping with branch excision when measuring leaf net photosynthetic rates in a lowland tropical forest[END_REF]. Measurements were taken at the following settings in a 2×3 LED chamber: 400 ppm of reference CO2 concentration, 1,500 μmol m -2 s -1 light-saturating photosynthetic photon flux density, 50% relative chamber humidity, and block temperature adjusted to 20°C or 30°C to match the ambient conditions at each site while measurements were taken (i.e. BG, VC, VT = 20°C, LA, LU, SB = 30°C). As the measured leaves covered the whole LI-COR 6400 cuvette, no correction for leaf area was conducted. Leaf temperature was measured using the energy balance. Data were recorded after the steadystate gas exchange rates had remained for at least 4 min. Intrinsic water-use efficiency (WUEi)

was obtained by dividing Amax by gsmax.

Foliar carbon isotopic composition (δ 13 C, ‰), a proxy for water-use efficiency integrated over the entire period since leaves formed [START_REF] Farquhar | Models of integrated photosynthesis of cells and leaves[END_REF], Ehleringer et al. 1993), was measured on the sunlight leaves taken from the same branches used for the gas exchange measurements. Leaf material was dried at 65°C for 48 h, Dried samples were finely ground.

About 1 mg of the powdered material was placed inside tin capsules (Säntis, Teufen, Switzerland) for the δ 13 C analysis. Measurements were taken on an elemental analyzer interfaced to a DeltaPlusXP isotope ratio mass spectrometer (EA-IRMS; Thermo EA 1100

Deltaplus XL; 0.1‰ precision) in the stable isotope research centre (SIRC) of WSL (Birmensdorf, Switzerland).

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From every branch taken at midday, 20 centimetre-long shoots (approx. 70 cm from the apex, diameter between 2-5 mm) were taken and placed inside vials with 50% ethanol solution to determine wood anatomy. In the laboratory, 12 micrometre-width cross-sections were obtained from each sample using a Reichert Om E based microtome [START_REF] Gärtner | New perspectives for wood anatomical analysis in dendrosciences: The GSL1-microtome[END_REF].

Cross-sections were stained with safranine-astrablue (1% and 0.5% in distilled water, respectively), mounted on glass slides with Canada balsam, and dried in an oven at 60ºC for 24 h. Slides were photographed by a microscope Olympus BX41 at 40x magnification. Vessel diameter (D), vessel density (Vd) and relative vessel lumen area (i.e. vessel lumen to sapwood area ratio) (VA) were calculated by the ImageJ software. Xylem anatomical measurements were taken on the last two rings because this wood section is responsible for most twig water transport (Domec andGartner 2002, Bouche et al. 2014). Vessel hydraulic diameter (i.e. the effective vessel diameter that contributes to hydraulic conductivity) was calculated according to [START_REF] Sperry | Analysis of circular bordered pit function I. Angiosperm vessels with homogenous pit membranes[END_REF] as follows:

𝐷ℎ = 2 ( ∑ 𝑟 5 ∑ 𝑟 4 )
where r is the vessel radius in microns (m).

A 1-metre terminal section was cut from every branch for the hydraulic traits measurements and immediately placed inside sealed dark plastic bags with wet tissue to maintain high moisture conditions. Samples were sent to the laboratory over the next 24 h, stored in a cold chamber at 5ºC, and processed within the next 2 or 3 days. The analysis was performed using a Xyl´em device (Bronkhorst, France) according to [START_REF] Cochard | Evaluation of a new centrifuge technique for rapid generation of xylem vulnerability curves[END_REF]. Deionised, filtered and degassed water, with 1M and 10M concentrations of CaCl2 and KCl, respectively, was used for the analysis. Eight small shoots were cut underwater from each branch and placed in a manifold. Then a low pressure (45kPa) flow was applied to measure the initial hydraulic conductivity of each shoot. Samples were flushed at 0.1 MPa for 3 minutes to remove any air bubbles that obstructed vessels. Previous tests determined that a maximum of 3 minutes

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11 allowed all air bubbles to be removed, and the maximum hydraulic conductivity (Khf) could be recorded. The percent loss of conductivity (PLC) was calculated using the ratio of the initial hydraulic conductivity (Khi) recorded before flushing samples were divided by Khf, and expressed as percentage units as follows:

𝑃𝐿𝐶 = 100 (1 - 𝐾ℎ 𝑖 𝐾ℎ 𝑓 )
Xylem-specific conductivity (Ks) was calculated for every shoot by dividing Khf by the sapwood area, estimated as the cross-section without bark. Simultaneously, leaf-specific conductivity (LSC) was calculated by dividing Khf by the leaf area associated with every shoot, measured using a LICOR scanner (LI-3100 Area Meter, LI-COR, USA). Huber value (HV) was estimated as the sapwood area/leaf area ratio.

All the measured traits and their units are summarised in Table 2.

Climate data

Climate data were obtained for each site from the current climate WorldClim database at ~1km 2 resolution grid from https://www.worldclim.org/data/index.html [START_REF] Fick | WorldClim 2: new 1-km spatial resolution climate surfaces for global land areas[END_REF]. A set of the potential explanatory variables was selected for trait variability, which accounted for water availability and temperature effects (i.e. averages and extremes): mean annual precipitation (pp); mean minimum temperature of the coldest month (MTCM); mean maximum temperature of the warmest month (MTWM). For each site, the annual P/PET (precipitation/potential evapotranspiration) index from the layer available at http://www.cgiar-csi.org/data/global-aridity-and-pet-database was downloaded, which derives from the same WorldClim database.

U N C O R R E C T E D M A N U S C R I P T 12 Statistical analysis
We tested the correlation between the explanatory climate variables (pp, MTCM, MTWM, P/PET) and found that they were highly correlated (i.e. Pearson's correlation coefficient higher than 0.7 in all cases). We thus summarised the climate variables in a single variable by performing a principal component analysis (PCA). The first obtained component, PC1, explained 95% of variance. It was chosen as the best representative of the moisture and temperature gradient. We performed Pearson's correlation analysis between PC1 and every measured trait to assess the relations between climatic gradient and trait variability. The normality for all the traits was previously tested by the Shapiro-Wilk test and was logtransformed whenever necessary. Gaussian kernel density estimations were used to explore the vessel diameter distribution among populations, following [START_REF] Schreiber | Variation of xylem vessel diameters across a climate gradient: Insight from a reciprocal transplant experiment with a widespread boreal tree[END_REF] and [START_REF] García-Cervigón | Xylem anatomy needs to change, so that conductivity can stay the same: Xylem adjustments across elevation and latitude in Nothofagus pumilio[END_REF]. Kernel density estimation is a non-parametric procedure that calculates the probability density function of a variable from a finite number of samples [START_REF] Silverman | Density Estimation for Statistics and Data Analysis[END_REF]). R function density was used for this purpose.

Coordination between traits was evaluated with path analyses using R-packages lavaan v.06-7 [START_REF] Roseel | lavaan: An R Package for Structural Equation Modeling[END_REF]) and semplot v. 1.1.2 [START_REF] Epskamp | Path Diagrams and Visual Analysis of Various SEM Packages[END_REF]). A first model was performed for all trees and sites (i.e. general model) to characterise the trait coordination along the whole gradient.

To better assess the climate effect on the coordination network, and the gradual differentiation between northern and southern populations [START_REF] Capblancq | Climate-associated genetic variation in Fagus sylvatica and potential responses to climate change in the French Alps[END_REF], two more models were carried out: one for the trees at our three southern, drier and warmer sites (i.e.

arid-warm model, PC1<0: SB, LU, and LA), and another for the trees at our three northern, wetter and colder sites (i.e. wet-cold model, PC1>0: VT, VC, and BG). Path structures were built by assuming direct relations among vessel hydraulic diameter (Dh), hydraulic conductivity (Ks and LSC) and maximum stomatal conductance (gsmax), and by accounting for empirical interactions between the traits of each group of traits (i.e. wood anatomy, xylem hydraulics,

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13 leaf physiology). We eliminated the less significant paths stepwise until the best fitted and predicted models were found (based on χ 2 , RMSEA and CFI indices).

We also analysed the population-level trait variance for every site using the TIC/IR statistic proposed by [START_REF] Violle | The return of the variance: Intraspecific variability in community ecology[END_REF] by taking each site as a different population. TIC/IR is calculated as the ratio of a trait's community variance (population-level variance in our case) in relation to its variance observed on the regional scale (the variance observed all along the climatic gradient in our case) assessed at the individual level. This index represents trait values' dispersion and assesses the strength of the environmental filtering in every trait's expression [START_REF] Violle | The return of the variance: Intraspecific variability in community ecology[END_REF]). Values close to or higher than 1 depict no environmental filtering in the expression of traits, with population-level variances being relatively equal to regional ones.

When values are below 1 for a given population, this index denotes that trait expression is less scattered than its heterogeneity on the regional scale. For instance, lower values indicate environmental filtering, which implies that the population-level trait expression is less heterogeneous than the species regional pool of variance. To assess if our climatic gradient was related to trait variances, we established relations with Pearson's correlation in each trait's TIC/IR and PC1.

All the statistical analyses were carried out with version 4.1 of the R software (R Core Team 2020).

Results

Trait adjustments along the gradient

We found a positive correlation (i.e. the mean trait values rising towards the gradient's colder and wetter end) between climate (PC1) and HV, the maximum assimilation rate (Amax), maximum stomatal conductance (gsmax), δ 13 C and the predawn leaf water potential (Ѱpd) (Fig. S1). On the contrary, we found that PC1 was negatively correlated (i.e. trait values lowering towards the colder and wetter end of the gradient) with xylem-specific conductivity (Ks), WUEi, hydraulically weighted vessel diameter (Dh) and percent loss of conductivity (PLC).

U N C O R R E C T E D M A N U S C R I P T 14 2, Table
We also observed marginally significant negative correlations between PC1 and vessel area per sapwood unit (VA) (Fig. 2, Table S1). LSC and Vd did not significantly correlate with climate.

The density curves for the vessel diameters (Fig. 3) revealed that they ranged from 7 to 55 µm.

A first peak on the curves for all the populations was observed at 10 µm, with a second one at around 20 µm, which corresponded to their average lumen diameter. The frequency of larger diameters notably decreased in the populations located on the cold-wet extreme of the gradient (i.e. kernel density estimations for the 30 µm diameter-width vessels was around 0.02 in all the populations, except in Vercors and Bauges, with drops to 0.012 and 0.011, respectively).

Trait coordination

Path analyses with the best adjustment included all the traits, except Ѱpd and WUEi, which were rejected by the model (Table 3, Fig. 4). Wood anatomical traits were closely correlated, with Dh positively influencing VA and lowering Vd. Dh was a central trait that significantly increased Ks and LSC. Dh also indirectly influenced HV (Table 3) by promoting a reduction in the leaf area when vessels were narrower. Ks and LSC strongly correlated with HV. PLC significantly increased LSC and positively correlated with HV (Table 3). Of the stem hydraulic traits, only Ks showed a negative relation with gsmax. Leaf physiological traits correlated with higher stomatal conductance to promote greater assimilation.

The path analysis for the arid and warm sites (Fig. 4B) depicted a weaker relation among trait groups. A lesser predictive response of Dh was found on Ks, with only a tendency for LSC and of Ks on gsmax. The relation between Dh and Vd was not significant in the arid-warm model.

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However, only this model depicted a significant direct influence of gsmax on δ 13 C. The path analysis for the wet-cold sites (Fig. 4C) maintained the correlation strength between the wood anatomical and hydraulic traits observed in the analysis for all the traits. However, PLC showed no significant correlation with any trait in this model.

Trait variance patterns

The trait variance analysis performed at the community-level showed that climate significantly correlated with the community-level trait variances in relation to their regional pool (TIC/IR) for HV and gsmax, and both reduced at the drier and warmer sites (Fig. 5, Table S2). A tendency was noted with strong correlation coefficients for Ks, Dh, and VA (Fig. 5), whose variances lowered at the wetter and colder sites. No significant filtering pattern was found in the communitylevel variances for the other traits in response to the climatic gradient (Fig. 5, Table S2).

Discussion

Our trait adjustments and coordination results evidence that beech trees follow two different strategies along the gradient to cope with cold temperature or aridity and heat. Both these two strategies are based on the acclimation of xylem hydraulic traits and leaf physiological ones. Interestingly, our outcomes also suggest that climatic extremes modulate the intrapopulation variances of these trait groups, which may affect their plasticity and acclimation capacity to a shifting environment.

Trait adjustments along the gradient

Our results show that as conditions became colder and moister, beech develop more conservative traits related to hydraulic efficiency (i.e. small vessel hydraulic diameter, Dh; and xylem specific conductivity, Ks), while leaf physiology traits adjust to more acquisitive

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16 behaviour (i.e. high gsmax, Amax, and low water-use efficiency). The drop in Dh is consistent with results from studies showing vessel diameter reductions in cold ecosystems as a prevention mechanism against freeze-thaw cavitation [START_REF] Castagneri | Divergent climate response on hydraulic-related xylem anatomical traits of Picea abies along a 900-m altitudinal gradient[END_REF][START_REF] Schreiber | Variation of xylem vessel diameters across a climate gradient: Insight from a reciprocal transplant experiment with a widespread boreal tree[END_REF][START_REF] García-Cervigón | Xylem anatomy needs to change, so that conductivity can stay the same: Xylem adjustments across elevation and latitude in Nothofagus pumilio[END_REF]. In line with this, several works observe that vessel diameters larger than 30 µm are highly vulnerable to cavitation due to freeze-thaw cycles [START_REF] Davis | The relationship between xylem conduit diameter and cavitation caused by freezing[END_REF][START_REF] Schreiber | Sixteen years of winter stress: An assessment of cold hardiness, growth performance and survival of hybrid poplar clones at a boreal planting site[END_REF][START_REF] Medeiros | Freezing regime and trade-offs with water transport efficiency generate variation in xylem structure across diploid populations of Larrea sp. (Zygophyllaceae)[END_REF], and the vessel size of trees adapted to cold conditions tends to go below that threshold [START_REF] Schreiber | Variation of xylem vessel diameters across a climate gradient: Insight from a reciprocal transplant experiment with a widespread boreal tree[END_REF]. Our results agree with these outcomes because the trees in the coldest populations show a substantially lower frequency of vessels above 30 µm (Fig. 3). These results demonstrate the important selective pressure of low temperatures in northern populations. Nevertheless, the high gas exchange rates (i.e. gsmax and Amax) at these sites agree with beech's high assimilation capacity in mountainous environments and at high latitudes to compensate for shorter growing seasons [START_REF] Bresson | To what extent is altitudinal variation of functional traits driven by genetic adaptation in European oak and beech?[END_REF]. This strategy also allows high carbon acquisition and water-use efficiency during early spring when late frosts occur [START_REF] Huxman | Photosynthetic resource-use efficiency and demographic variability in desert winter annual plants[END_REF][START_REF] Hernández-Calderón | Altitudinal changes in tree leaf and stem functional diversity in a semi-tropical mountain[END_REF]. Along these lines, physiological adjustments that enhance photosynthetic efficiency in colder environments and higher elevations have been reported to promote more positive leaf δ 13 C values [START_REF] Hultine | International Association for Ecology Altitude Trends in Conifer Leaf Morphology and Stable Carbon Isotope Composition[END_REF]. This agrees with our observed trend of more positive δ 13 C towards the cold-wet sites.

Our results unexpectedly show higher HVs (i.e. smaller leaf area per sapwood unit) as climate becomes wetter and colder, which contradicts consistent intraspecific evidence [START_REF] Pritzkow | Phenotypic plasticity and genetic adaptation of functional traits influences intra-specific variation in hydraulic efficiency and safety[END_REF], Rosas et al. 2019[START_REF] López | Coordination of stem and leaf traits define different strategies to regulate water loss and tolerance ranges to aridity[END_REF]). However, our results are similar to those reported by [START_REF] Fuchs | Hydraulic variability of three temperate broadleaf tree species along a water availability gradient in central Europe[END_REF], who reported higher HV in Norway maple trees growing in moister climates, but were limited by low hydraulic conductivity. Indeed HV is also known to increase in response to hydraulic efficiency losses to maintain homeostatic water uptake to leaves without risking excessive reductions in water potential [START_REF] Mencuccini | Leaf economics and plant hydraulics drive leaf : wood area ratios[END_REF][START_REF] López | Coordination of stem and leaf traits define different strategies to regulate water loss and tolerance ranges to aridity[END_REF]). Thus, our results show that HV is considerably mediated by Ks and Dh (Fig. 4, Table 3),

U N C O R R E C T E D M A N U S C R I P T 17
and its unexpected response is likely triggered by the low Ks values at the colder sites. This result is also supported by the non-significant response of leaf-specific hydraulic conductivity (i.e. LSC) along the gradient.

Towards more arid and warmer conditions, beech shifts its leaf physiology to a more conservative strategy (i.e. reductions in gsmax and Amax, and better water-use efficiency). This potentially prevents excessive dehydration and reduces the cavitation risk at low water potentials. This result agrees with previous works showing high plasticity and acclimation capacity of gas exchange traits in beech [START_REF] Aranda | Variation in photosynthetic performance and hydraulic architecture across European beech (Fagus sylvatica L.) populations supports the case for local adaptation to water stress[END_REF][START_REF] Lübbe | Acclimation of leaf water status and stem hydraulics to drought and tree neighbourhood: Alternative strategies among the saplings of five temperate deciduous tree species[END_REF][START_REF] Didion-Gency | Interactive effects of tree species mixture and climate on foliar and woody trait variation in a widely distributed deciduous tree[END_REF][START_REF] Martin-Blangy | Mixing beech with fir or pubescent oak does not help mitigate drought exposure at the limit of its climatic range[END_REF]) and other species [START_REF] Torres-Ruiz | Genetic differentiation in functional traits among European sessile oak populations[END_REF][START_REF] López | Coordination of stem and leaf traits define different strategies to regulate water loss and tolerance ranges to aridity[END_REF]). However, one surprising finding is that the trees on the arid-warm edge of the gradient have the highest Dh and Ks, which could mean higher dehydration and hydraulic failure risks during drought [START_REF] Hajek | Trade-offs between xylem hydraulic properties, wood anatomy and yield in Populus[END_REF]). This situation contradicts previous studies, which show reductions in vessel diameter and Ks in beech populations with increasing aridity [START_REF] Schuldt | How adaptable is the hydraulic system of European beech in the face of climate change-related precipitation reduction?[END_REF][START_REF] Stojnić | Variation in xylem vulnerability to embolism in European beech from geographically marginal populations[END_REF]. Nonetheless, our results agree with a recent work showing that the hydraulic conductivity and lumen diameters of top P. sylvestris branches increase in arid populations with slight carbon input [START_REF] Kiorapostolou | Scots pine trees react to drought by increasing xylem and phloem conductivities[END_REF]. This adjustment may allow low-productive trees to compensate for reduced whole-tree hydraulic efficiency caused by narrower rings forming at their stem base [START_REF] Pellizzari | Wood anatomy and carbon-isotope discrimination support long-term hydraulic deterioration as a major cause of drought-induced dieback[END_REF][START_REF] Prendin | Xylem anatomical adjustments prioritize hydraulic efficiency over safety as Norway spruce trees grow taller[END_REF], and to relocate carbon to other organs to cope with drought (e.g. developing deeper roots) [START_REF] Eilmann | Drought-induced adaptation of the xylem in Scots pine and pubescent oak[END_REF][START_REF] Matisons | Plasticity and climatic sensitivity of wood anatomy contribute to performance of eastern Baltic provenances of Scots pine[END_REF]. Maintaining a certain hydraulic conductivity level can also optimise water use and carbon assimilation during high water availability periods, despite the higher water stress risk during summer drought [START_REF] Hajek | Trade-offs between xylem hydraulic properties, wood anatomy and yield in Populus[END_REF][START_REF] Kiorapostolou | Scots pine trees react to drought by increasing xylem and phloem conductivities[END_REF].

Indeed the trees growing at the arid-warm sites display more stress (i.e. more negative predawn water potentials, Ѱpd; a higher percent loss of conductivity, PLC). Nevertheless, their PLC values are low (i.e. mainly < 30%) which suggests that the observed leaf-physiological adjustments successfully buffer the impacts of higher VPD and aridity.

U N C O R R E C T E D M A N U S C R I P T 18
However, maintaining high Ks and Dh can be a risky strategy in the southern populations of our gradient as regards climate change predictions. Previous studies conducted with beech and other species demonstrated that these traits show low plasticity [START_REF] Eilmann | Wood structural differences between northern and southern beech provenances growing at a moderate site[END_REF][START_REF] Hajek | Intraspecific variation in wood anatomical, hydraulic, and foliar traits in ten European beech provenances differing in growth yield[END_REF][START_REF] Pritzkow | Phenotypic plasticity and genetic adaptation of functional traits influences intra-specific variation in hydraulic efficiency and safety[END_REF][START_REF] Torres-Ruiz | Genetic differentiation in functional traits among European sessile oak populations[END_REF]) and slow responses to environmental changes [START_REF] Tomasella | Acclimation of branch and leaf hydraulics in adult Fagus sylvatica and Picea abies in a forest through-fall exclusion experiment[END_REF][START_REF] Pritzkow | Phenotypic plasticity and genetic adaptation of functional traits influences intra-specific variation in hydraulic efficiency and safety[END_REF]. Such limited plasticity in the short term can increase these populations' vulnerability to sudden heat waves and extreme droughts in forthcoming decades. Their short-term fitness might rely on trait adjustments in other organs, such as shifts in leaf physiology, leaf shedding or deeper roots developing. We suggest that future works address the acclimation of water-use strategies and also include root traits [START_REF] Hertel | Fine root biomass and dynamics in beech forests across a precipitation gradient -Is optimal resource partitioning theory applicable to water-limited mature trees?[END_REF] to, therefore, provide a more complete understanding of how trees modulate their water economy in response to climate variability.

Trait coordination

Interestingly, our results demonstrate that environmental conditions modulate the correlation between traits, which agrees with the results of [START_REF] Carvalho | Intraspecific perspective of phenotypic coordination of functional traits in Scots pine[END_REF] and [START_REF] Benavides | Phenotypes of Pinus sylvestris are more coordinated under local harsher conditions across Europe[END_REF]. Our results also suggest that the relative importance of adjustments at xylem and leaf levels depends on the acting climatic constraint. Trees that grow at sites limited by low temperature show a closer coupling between the xylem anatomical and hydraulic traits. This agrees with the outcomes of [START_REF] Carvalho | Intraspecific perspective of phenotypic coordination of functional traits in Scots pine[END_REF], who report that P. sylvestris trees display more coordination between traits in harsher environments. This reinforces our results about the important selective pressure of the low temperatures at our northern sites, which promotes a specific combination of hydraulic traits to avoid freeze-thaw cavitation and harmful water potentials (i.e. smaller vessel diameter, lower hydraulic conductivity and higher HV).

U N C O R R E C T E D M A N U S C R I P T 19
Conversely, trait coordination depicted by the arid-warm model suggests that the acclimation strategy to increasing aridity and heat involves mainly leaf traits through selecting trait combinations to increase water-use efficiency and to minimise water losses. Specifically, dry origin trees show significant increases in leaf-specific conductivity and HV in response to incremented percent loss of conductivity. This coordination denotes a smaller leaf area triggered by PLC, which suggests that they are prone to early leaf shedding under summer conditions. This adjustment reduces trees' transpiration surface and prevents cavitation at high VPD and water depletion [START_REF] Pivovaroff | Multiple strategies for drought survival among woody plant species[END_REF][START_REF] Leuschner | Drought response of European beech (Fagus sylvatica L.)-A review[END_REF]. Indeed, pre-senescent leaf shedding is reported in beech in July or August if drought conditions are intense enough [START_REF] Leuschner | Drought response of European beech (Fagus sylvatica L.)-A review[END_REF][START_REF] Schuldt | A first assessment of the impact of the extreme 2018 summer drought on Central European forests[END_REF]. Moreover, the negative correlation between gsmax and leaf δ 13 C depicts marked stomatal control in trees, which limits their gas exchange capacity.

This allows them to optimise leaf-level water use upon maximum stomatal conductance, which helps to prevent hydraulic failure [START_REF] Grossiord | Plant responses to rising vapor pressure deficit[END_REF]. This response falls in line with studies that have observed consistent links between lower δ 13 C discrimination and leaf morphological adjustments to limit water loss (large leaf mass per area) [START_REF] Wright | Modulation of leaf economic traits and trait relationships by climate[END_REF], Rosas et al. 2019).

It is worth noticing that Dh comes over as a key trait in beech water-use strategies to adapt to climate variability as our results depict that it mediates the response of many other traits (i.e.

Vd, VA, Ks, Kl, and HV). The significant negative correlation between Ks and gsmax observed in all the path analyses is also noteworthy. This contrasts with the assumptions made that higher xylem water transport capacity promotes incremented leaf-level water use [START_REF] Brodribb | Hydraulic and photosynthetic co-ordination in seasonally dry tropical forest trees[END_REF][START_REF] Brodribb | Stomatal closure during leaf dehydration, correlation with other leaf physiological traits[END_REF][START_REF] Reich | The world-wide "fast-slow" plant economics spectrum: A traits manifesto[END_REF]. However, the low leaf-level water use and high water transport capacity of the xylem can also prevent dehydration and maintain a wellhydrated tree status. In this context, stomata at the leaf-level act as a bottleneck for water movement and reduce water depletion during maximum gas exchange periods (Zimmermann 2002, Sack and[START_REF] Sack | Leaf hydraulics[END_REF]. This strategy may be particularly relevant for trees growing in

U N C O R R E C T E D M A N U S C R I P T 20
drier environments or those exposed to high seasonality because it can also enhance xylem protection from hydraulic failure caused by low water potentials [START_REF] Pivovaroff | Coordination of stem and leaf hydraulic conductance in southern California shrubs: a test of the hydraulic segmentation hypothesis[END_REF][START_REF] Pivovaroff | Multiple strategies for drought survival among woody plant species[END_REF].

For instance, similar adjustments have been reported as an adaptation mechanism to drought in Mediterranean semiarid shrubs, especially in species that operate on low safety margins [START_REF] Pivovaroff | Coordination of stem and leaf hydraulic conductance in southern California shrubs: a test of the hydraulic segmentation hypothesis[END_REF].

Overall, these results highlight the variability of the trait combinations that beech can display in response to different environmental conditions, and indicate that the vessel hydraulic diameter is a key trait for modulating the acclimation strategies of beech's water economy.

Further research is needed to test whether the observed coordination structures are sustained in other populations limited by high aridity and VPD, and also by low temperatures.

Trait variance patterns.

Our results show that the climatic extremes in our gradient reduce the variances of gsmax, HV, Dh, VA and Ks. These results support previous observations showing that the variances of some traits reduce when climatic conditions become harsher, i.e., climatic filtering (Swenson et al. 2012[START_REF] Anderegg | Aridity drives coordinated trait shifts but not decreased trait variance across the geographic range of eight Australian trees[END_REF]. We note two response patterns associated with low temperature and aridity that align with the observed individual trait adjustments and coordination along the gradient.

The variances of the traits related to water transport efficiency (Dh, VA, Ks) show signs of climatic filtering in the populations growing under moister and colder conditions (i.e. TIC/IR <1).

This observation reinforces the notion that such conditions filter out wide vessels to reduce vulnerability to frost-induced cavitation, especially those above the 30 µm threshold. Indeed, this disturbance can be particularly harmful if it takes place early in spring when leaves are already transpiring [START_REF] Magnani | Interpretation of seasonal changes of xylem embolism and plant hydraulic resistance in Fagus sylvatica[END_REF]Borghetti 1995, Lemoine 2001). For instance, frost-induced cavitation in spring has been reported to cause conductivity losses up to 60% in beech [START_REF] Magnani | Interpretation of seasonal changes of xylem embolism and plant hydraulic resistance in Fagus sylvatica[END_REF], which can significantly increase decay and the mortality risk.

U N C O R R E C T E D M A N U S C R I P T 21
These results are consistent with reported patterns of reduced community variances of wood anatomy traits in response to low temperatures (Swenson andEnquist 2007, Swenson et al. 2012). On the contrary, the gsmax and HV variances display an opposite pattern by lowering in the populations growing under drier and hotter conditions. This suggests that the high temperatures, VPD and aridity along our gradient select trait values that promote stomatal regulation; i.e. lower leaf-level water use, and maintain water status. It should be noted that although Ks and gsmax are closely related according to the path analyses, their divergent variance patterns suggest that the adjustment of gsmax in response to higher aridity is uncoupled from hydraulics. Hence selecting a lower gsmax may rely on other mechanisms linked with water-use dynamics at the leaf-level, such as stomatal sensitivity, osmotic adjustments or hormonal regulation mechanisms, including aquaporins [START_REF] Schroeder | Guard cell signal transduction[END_REF][START_REF] Kapilan | Regulation of aquaporins in plants under stress[END_REF].

Interestingly, although our previous high Ks and Dh outcomes at drier sites suggest more vulnerability to drought, their wide variance at these sites could be advantageous for the overall population. Firstly because it may depict greater acclimation capacity as these populations would have a wider pool of heritable trait plasticity available [START_REF] Valladares | The effects of phenotypic plasticity and local adaptation on forecasts of species range shifts under climate change[END_REF][START_REF] Anderegg | Aridity drives coordinated trait shifts but not decreased trait variance across the geographic range of eight Australian trees[END_REF]. This would promote better tolerance to disturbances for some trees, while others would succumb, which would increase populations' chances to remain functional. Secondly, more water transport efficiency diversity could imply higher functional diversity and enhanced resilience against drought or heat waves. For instance, beech has higher growth and growth recovery rates after extreme droughts in more functionally diverse stands (Pretzsch et al. 2013, Mölder andLeuschner 2014). The same positive effect of stand functional diversity on resilience has been confirmed for co-existing Abies alba [START_REF] Gazol | Functional diversity enhances silver fir growth resilience to an extreme drought[END_REF] and tropical forests [START_REF] Schmitt | Functional diversity improves tropical forest resilience: Insights from a long-term virtual experiment[END_REF]. One probable mechanism behind this positive effect is tree complementarity in water use based on root and/or stem hydraulic traits [START_REF] Gazol | Functional diversity enhances silver fir growth resilience to an extreme drought[END_REF]. Tree complementarity can buffer or slow down soil water

  in trees is crucial for predicting forest dynamics under novel stressful conditions linked with climate change. Dysfunctions in tree water relations and hydraulic efficiency are globally correlated with climate-associated forest decline and mortality

  reducing resource use overlapping and competition[START_REF] Morin | Tree species richness promotes productivity in temperate forests through strong complementarity between species[END_REF][START_REF] De Andrés | Increased complementarity in water-limited environments in Scots pine and European beech mixtures under climate change[END_REF]. However, these studies addressed functional diversity based on species identity in mixed stands. To what extent hydraulic traits' heterogeneity in both monospecific and mixed stands can significantly enhance beech resilience when facing extreme droughts remains unclear. Future studies are needed to further explore this topic and to better understand the influence of population-level trait variances on species acclimation.ConclusionsThis study highlights two main beech responses to climate variability along a temperature and aridity gradient. Colder and moister conditions promote narrower vessels, and greater stomatal and assimilation capacity, which are likely to prevent frost-induced cavitation and to compensate for shorter growing seasons. As climate becomes drier and warmer, the adjustment and coordination of traits are based mainly on leaves to promote reductions in stomatal capacity, increments in water-use efficiency and early leaf shedding. However, the trees at the arid and warm sites still possess high water transport capacity along the xylem and wide vessel diameters. This suggests that acclimation to sudden rises in aridity and temperature relies mainly on leaves to minimise water depletion and to protect the xylem from negative water potentials. At the population-level, the trait variances controlling water transport capacity increase with more arid and hotter conditions. This could be advantageous for the overall populations on the arid-warm end of the gradient. Higher heterogeneity (i.e. plasticity) can increase their acclimation capacity and the chances of some trees better tolerating the impact of extreme droughts. High water-use diversity could also favour tree complementarity and populations' resilience. Intrapopulation variance is, thus, highlighted as a key parameter for gaining a better understanding of species responses to climate change.Future studies are needed to better assess the role of intrapopulation variances in acclimation influence on stands' resilience. We also advocate the inclusion of root traits in future research. Assessing traits from all the organs involved in water use will provide a more complete understanding of how tree species acclimate their water-use strategies.

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Acknowledgements

EV was supported by a predoctoral fellowship funded by the Spanish Ministry of Economy and Competitiveness as part of Project HYDROMED (PID2019-111332RB-C21). AV and LM were supported by Projects INERTIA (PID2019-111332RB-C22), and IMAGINA (Prometeu program/2019/110, GVA). L.M. was additionally supported by the Spanish MICINN (PTA2019-018094). AV was also supported by a WSL Visiting Fellowship. MD and CG were supported by the Swiss National Science Foundation (SNF) (PZ00P3_174068) and the Sandoz Family Foundation. We are grateful to the two tree climbers, J. Baudry and E. Norois, for their help during fieldwork. We thank C. Donzé for support in the field and laboratory. The plot design used in this study forms part of the GMAP plot network (https://oreme.org/observation/foret/gmap/), partly funded by the OSU OREME in Montpellier, and by the ANR project BioProFor (contract no. 11-PDOC-030-01). The CEAM foundation is supported by the Generalitat Valenciana.

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Conflict of interest

The authors declare no conflict of interest.

Authors' contributions