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Tree Physiology

AN INTERNATIONAL BOTANICAL JOURNAL VOLUME 31 NUMBER 11 NOVEMBER 2011 www.treephys.oxfordjournals.org VOLUME 31 NUMBER 11 NOVEMBER 2011

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Tree Physiology

An International Botanical Journal

Cover image: Fagus sylvatica L. has a very widespread distribution in Europe. Wortemann et al. (pages 1175–1182) have analyzed the genetic variability of xylem vulnerability to cavitation across this whole range. The results indicate that cavitation resistance varies substantially within each population, but very little genetic differentiation was found across populations. A high phenotypic plasticity has also been identified for this trait. Photo: Hervé Cochard.

Tree Physiology 31, 1175–1182 doi:10.1093/treephys/tpr101

Research paper

Genotypic variability and phenotypic plasticity of cavitation resistance in Fagus sylvatica L. across Europe Rémi Wortemann1,2, Stéphane Herbette1,2, Têtè Sévérien Barigah1,2, Boris Fumanal1,2, Ricardo Alia3, Alexis Ducousso4, Dusan Gomory5, Patricia Roeckel-Drevet1,2 and Hervé Cochard1,2,6 1INRA,

UMR 547 PIAF, F-63100 Clermont-Ferrand, France; 2Clermont Université, Université Blaise-Pascal, UMR 547 PIAF, BP 10448, F-63000 Clermont-Ferrand, France; de Sistemas y Recursos Forestales, CIFOR, Instituto Nacional de Investigación y Tecnología Agraria, Carretera de La Coruña, Km 7.5, 28040 Madrid, Spain; 4INRA, UMR BIOGECO, 69 route d’Arcachon, 33612 Cestas, France; 5Technical University in Zvolen, T.G. Masaryka 24, 960 53 Zvolen, Slovakia; 6Corresponding author (herve. [email protected]) 3Departamento

Received July 28, 2011; accepted August 30, 2011; published online October 10, 2011; handling Editor Marc Abrams

Xylem cavitation resistance is a key physiological trait correlated with species tolerance to extreme drought stresses. Little is known about the genetic variability and phenotypic plasticity of this trait in natural tree populations. Here we measured the cavitation resistance of 17 Fagus sylvatica populations representative of the full range of the species in Europe. The trees were grown in three field trials under contrasting climatic conditions. Our findings suggest that the genotypic variability of cavitation resistance is high between genotypes of a given population. By contrast, no significant differences were found for this trait across populations, the mean population cavitation resistance being remarkably constant in each trial. We found a significant site effect and a significant site × population interaction, suggesting that cavitation resistance has a high phenotypic plasticity and that this plasticity is under genetic control. The implications of our findings for beech forest management in a context of climate change are discussed. Keywords: common garden experiment, Fagus sylvatica L., genetic variability, P50, phenotypic plasticity, vulnerability to cavitation.

Introduction Droughts of exceptional magnitude like those that struck Europe in 1976 and 2003 have caused severe forest diebacks and substantial economic losses (Bréda et al. 2006). The risk of such damage may increase in the future, as global climate models predict that extreme drought periods will be more frequent in the next decades (IPCC 2007). The sustainability of the current forest ecosystems is thus clearly challenged. This issue is of paramount importance for the many foresters who need to take key decisions now for the management of forest ecosystems likely to experience several extreme drought events before they are harvested, typically in a hundred years’ time. How successfully current species can acclimate to drier conditions is not well documented. Replanting forest stands with genotypes or ecotypes of the same species that are better adapted to water stress is appealing, but very little information is available on the

genetic diversity of drought tolerance in tree species, mainly because sound criteria and operational techniques to evaluate tree drought tolerance were lacking until recently. There is now abundant evidence that xylem cavitation resistance is a key physiological trait closely correlated with local soil water conditions (Maherali et al. 2004, Meinzer et al. 2009) and associated with the capacity of tree species to survive extreme drought events (Tyree and Sperry 1989, Bréda et al. 2006, Cochard et al. 2008, Brodribb and Cochard 2009). Techniques have also been developed to measure cavitation resistance on a larger scale (Cochard et al. 2005). However, comprehensive information on the genetic diversity and phenotypic plasticity of this trait remains very partial (Neufeld et al. 1992). As far as we know, only two Pinus species have been documented (Martinez-Vilalta et al. 2009, Corcuera et al. 2011, Lamy et al. 2011). We do not know whether the results

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1176  Wortemann et al. of these studies are specific to Pinus species or generic to other woody species. However, the ecology and the history of European forest species being very diverse, it is unsafe to hypothesize uniform patterns across them. The main objective of this study was to evaluate, for the first time, the genetic diversity of cavitation resistance in a broadleaf tree across its entire natural range, and the phenotypic plasticity of this trait. We selected European beech (Fagus sylvatica L.) as a model species for several reasons. First, this major forest species has a very widespread distribution in Europe and thus occurs in contrasting climatic areas (Figure 1). It is also known to be relatively sensitive to drought (Magnani and Borghetti 1995, Bréda et al. 2006) and therefore vulnerable to cavitation (Cochard et al. 1999). This may have favored the selection of ecotypes adapted to the local pedo-climatic conditions. Beech is also known to exhibit high phenotypic plasticity of its physiological traits, including cavitation resistance (Cochard et al. 1999, Herbette et al. 2010). Finally, we recently found that beech showed a broad phenotypic variability in nature for vulnerability to xylem cavitation (Herbette et al. 2010), but the relative contributions of genotypic variability and plasticity in this variability are still unknown. To evaluate the genetic variability of cavitation resistance within and across populations, and its phenotypic plasticity

across sites, we measured the cavitation resistance of beech trees from different populations across a large part of their natural distribution. The trees were grown in three different common gardens under contrasting climatic conditions. To determine whether the contrast between populations was purely neutral or the result of adaptive variations, we analyzed the differentiation of neutral chloroplastic markers between populations. The implications of our findings in terms of forest management in the context of a changing climate are discussed.

Materials and methods Plant material All the experiments were conducted on 15-year-old beech trees (F. sylvatica L.) from three ex situ trial populations located in France, Spain and Slovakia (Mátyás et al. 2009). Seeds were collected in 1993–1995 in 17 natural populations representing the distribution range of the species (Figure 1). The seeds were grown in a nursery and samples were randomly distributed during 1995 in each trial to form, for each population, three randomized blocks of 50 trees each. The location and climatic data of each trial and population are presented in Table 1. The French trial was used to assess the overall variability of xylem cavitation within beech species by using 17

Figure 1. ​Current distribution map of F. sylvatica in Europe. The different populations evaluated in this study are identified by black circles and trials by black stars.

Tree Physiology Volume 31, 2011

Genotypic variability and phenotypic plasticity of cavitation  1177 Table 1. ​Characteristics of three trials (*) and of the 17 beech populations evaluated in this study. Populations tested in all three experimental sites are in bold. Country

Locality

Code

Global positioning

Altitude (m)

Total annual precipitation (mm)

Spain* Slovakia* France* Germany Italy Czech Republic Czech Republic Poland France Slovakia Romania France Denmark Germany Germany Spain Germany Germany Germany France

Burgette Vrchdobroc Lyons-la-Forêt Kaufbeuren Veneto Kladskà Chvalsiny Stary Sacz Beffenares Trenc In Prisaca Léoncel Glorup Osterholz Gransee Anguiano Pferdestall Appenthal Ettenheim Fougères

Sp Sk F 101G 108I 110CR 111CR 115PO 12F 130SK 146RO 19F 26DK 36G 46G 5SP 84G 92G 94G 9F

43°00′N 1°20′W 48°36′N 19°38′E 49°29′N 1°36′E 47°55′N 10°35′E 46°8′N 12°13′E 50°2′N 12°37′E 48°51′N 14°15′E 49°31′N 21°41′E 46°11′N 2°57′E 48°53′N 18°E 46°41′N 22°16′E 44°55′N 5°11′E 55°11′N 10°41′E 53°14′N 8°48′E 53°N 13°10′E 42°15′N 2°45′E 50°57′N 13°34′E 49°22′N 7°57′E 48°12′N 7°55′E 48°23′N 1°10′E

910 840 190 700 1150 690 750 660 575 600 265 1350 70 25 70 950 365 405 445 180

835 625 692 986 1362 755 714 708 854 681 663 1106 568 721 590 731 563 721 826 761

populations. A subset of six contrasting populations for cavitation resistance was then measured in each site to evaluate the effect of local environments on this trait. This same subset of six contrasting populations for cavitation resistance was used for the molecular analysis in the French trial.

Chloroplastic DNA diversity Neutral genetic differentiation across and within populations was assessed on the same subset of six populations used for the analysis of the vulnerability to cavitation in the three trials (see Table 1). Fifteen individuals per population were sampled. The material (leaf or stem bark) was collected and stored at −80 °C. DNA was extracted from fresh material using the protocol developed by Novaes et al. (2009). DNA amount was tested on 1% agarose gels after staining with SYBER Safe. Chloroplast microsatellites were amplified using six primers: ccmp2, ccmp3, ccmp4, ccmp6, ccmp7 and ccmp10 (Weising and Gardner 1999). The amplification was performed on the genotyping platform GENTYANE of INRA Clermont-Ferrand. Fragment sizes were determined with GeneMapper™ 3.5 from Applied Biosystems. Haplotypes were identified based on variations in the six polymorphic chloroplast microsatellites. Analysis of molecular variance (AMOVA) was performed with GenAlEx 6.3 software.

Vulnerability curves Vulnerability curves were established on randomly selected trees for each population in each trial. As no significant

­ ifferences were found between blocks in the French trial, d the data for all blocks were pooled for all sites and populations. Stems were collected from sunlit second-order branches in the upper part of the crown using a pole pruner. The branches were 1–3 years old, and no significant age effect was detected (data not shown). To maximize the number of replicates between trees of the same population, we sampled only one branch per tree. However, to estimate the variation of cavitation resistance within a tree, we sampled four or five sunlit branches per tree on a subset of eight trees from two populations in the French trial (5SP and 101G). After sampling, the stems were wrapped in wet paper and sealed in black plastic bags to prevent dehydration. To minimize any temporal effects on hydraulic traits, all the samples were collected in one day on each site. On arrival in the laboratory, the stems were stored at 5 °C until processing. All the stems underwent measurements within 3 weeks after collection, a time lapse known to have no effect on cavitation resistance in beech (Herbette et al. 2010). Xylem cavitation was assessed with the Cavitron technique (Cochard 2002, Cochard et al. 2005) following the procedure described by Cochard et al. (2010). The technique uses centrifugal force to increase the water tension in a xylem segment and at the same time measures the decrease in hydraulic conductance. Just before analysis, the stems were cut in the air into segments 28 cm long, which were placed in the rotor of the centrifuge. During centrifuging, samples were injected with an ionic solution of 10 mM KCl and 1 mM CaCl2. Xylem pressure (P) Tree Physiology Online at http://www.treephys.oxfordjournals.org

1178  Wortemann et al. was first set to reference pressure (−1 MPa) and the sample maximal conductance (Kmax) was determined. The xylem pressure was then set to a more negative pressure by increasing the rotational velocity, and the conductance K was determined once more. The sample percent loss of conductance was then computed as PLC = 100 × (1 − K/Kmax). The procedure was repeated for more negative pressures, with −0.5 MPa step increments, until PLC reached at least 90%. Rotor velocity was monitored with an electronic tachymeter (10 rpm resolution) and xylem pressure was adjusted at approximately ±0.02 MPa. The PLC versus xylem water tension represents the sample’s vulnerability curve. Following Pammenter and Vander Willigen (1998), a sigmoid function was fitted to each curve:

PLC = 100/(1 + exp( s/25(P − P50 ))),

where P50 is the pressure causing a PLC of 50% and s is a slope parameter. The pressures at 12% (P12) and 88% (P88) loss of conductivity were obtained from the following equations:

P12 = P50 + 50/s,



P88 = P50 − 50/s.

P12 is an estimate of the xylem pressure at which embolism begins (Sparks and Black 1999) and P88 is an estimate of the xylem pressure at critical embolism level (Domec and Gartner 2001). The degree of native level of embolism (PLCnative) is known to impact vulnerability curves (Awad et al. 2010). We measured PLCnative for all the samples from the Slovak and Spanish sites, and on half the samples in the French site with a Xyl’em apparatus. On average, PLCnative equaled 2.45% with no significant differences between populations, which eliminated the risk of bias due to PLCnative in our study.

Results Neutral genetic variability The neutral genetic variability was addressed by the analysis of 90 genotypes from six populations. The AMOVA analysis shows that 89% of genetic variability is explained by intra-population variations with relatively small genetic differentiation between populations. Chloroplast markers revealed no structure between populations and no difference between populations. In other words, if we detected differences in xylem cavitation between populations, then they could be attributed to adaptation by natural selection and not to neutral drift.

Genetic variability for xylem cavitation within and between populations The genetic variability of cavitation in beech was assessed by establishing the vulnerability curves of 10 individuals from 17 populations in the French trial. The average P50 for all the populations was relatively constant (in the range −2.8 to −3.2 MPa, with a CV of 4.12%) and not significantly different (F = 1.21, P = 0.27) (Figure 2). As a corollary, no correlation was found between these P50 values and the annual precipitation at the area of origin of the populations (r 2 = 0.0159, P = 0.62). The same conclusion was drawn by comparing the P12 and P88 values (data not shown). Similarly, no significant difference was detected between populations in the other two trials (Spanish trial F = 1.49, P = 0.22 and Slovak trial F = 1.08, P = 0.38; Figure 3). However, in all three trials, a broad variability of P50 values was found within each population, with a CV of 12.6%, with differences as large as 1 MPa between extreme individuals of a population.

Statistical analysis Before statistical analyses, all data were tested for normal distribution (Shapiro–Wilk test) and homogeneity of variance (Bartlett test). The significance of population effects and site effects was determined by variance analysis (ANOVA). Data were analyzed using ANOVA and a Tukey HSD (honestly significant difference) test (α = 0.05) to determine the significance of population offsets and site effects. The mean P50 values among populations were compared using one-way ANOVA, and the mean P50 values among populations and trials were compared using two-way ANOVAs. We also used coefficients of variation (CVs) to compare the distribution of P50 values at different levels of observation.

Tree Physiology Volume 31, 2011

Figure 2. ​ Genotypic variability of cavitation resistance (P50, MPa) across 17 beech populations grown in the same common garden in France. The plot indicates the mean (open circle) and the 0.95 confidence intervals for each population. No statistically significant differences were found between populations.

Genotypic variability and phenotypic plasticity of cavitation  1179

Figure 3. ​Phenotypic plasticity of cavitation resistance for six beech populations grown under contrasting conditions in three trials (different symbols). The left panel indicates the mean and the 0.95 confidence intervals for each population in each site, and the right panel the average values per site.

Phenotypic plasticity of xylem cavitation The P50 values measured on different branches of the same tree were close, indicating a relatively small phenotypic plasticity (average CV 9.6%). This CV being less than the CV between trees inside these populations (12.9%), significant differences existed between trees in a given population. The phenotypic plasticity of cavitation resistance was assessed by comparing the vulnerability curves of the 15–20 individuals of the same six populations planted in three different sites with contrasting climatic conditions. Significant differences (F = 20.62, P = 0.00) were found between trials for the vulnerability to cavitation of all the populations (Figure 3b). However, the site effect varied across populations, indicating a site–population interaction (F = 2.45, P = 0.01) (Figure 3a). Overall, our results indicate that 82.4% of the total P50 variance was found within each population, 10.5% was attributable to a site effect, 6.2% to a population–site interaction and only 0.9% to a population effect (Table 2).

Discussion Exploring the genotypic and phenotypic variability of key ­physiological traits is an important task that may offer a better understanding of forest tree responses to global climate Table 2. ​Two-way ANOVA for cavitation resistance for six different populations grown in three trials.

Trial Population Trial × population Intra-population

df

MS

F

P

% var

2 5 10 324

2.063 0.068 0.245 0.100

20.61 0.68 2.45

0.000000 0.639834 0.007869

10.5 0.9 6.2 82.4

df, degrees of freedom; MS, mean square; F, statistical value; P, probability; % var, part of total variance.

change. However, this variability is still very poorly documented, mostly because the techniques for phenotyping these traits are usually laborious and time-consuming. With the development of the Cavitron technique we were able to explore, for the first time on a large scale, the variability of xylem cavitation resistance within and among populations of a major temperate angiosperm tree. Our results indicate that this variability is broad but also clearly structured within and between populations concerning vulnerability to cavitation.

Genotypic variability of vulnerability to cavitation A substantial variability of cavitation resistance was found across individuals native to different populations but grown in the same common garden. This variability most probably reflected the genotypic variability of this trait: in such a ­common garden, the environmental variability and thereby the phenotypic variability of the trait is minimized. We know from a previous study (Herbette et al. 2010) that vulnerability to ­cavitation varies little between branches taken in the same part of a tree crown. The same observation was made in the present study. To demonstrate the genotypic variability between genotypes more precisely, it would have been necessary to grow several clonal copies of each genotype, which is technically difficult with beech. We know from our previous studies on other species for which clonal material is available (Sangsing et al. 2004, Dalla-Salda et al. 2011, Fichot et al. 2011) that P50 values are very strongly conserved between copies of the same clone. We therefore consider that the variability measured in these field trials reflects genotypic differences between individuals. Clearly, most of the variability lies at this intra-population level (at least 80% of the total variability). This finding is consistent with the results of two recent surveys of cavitation resistance conducted on a Pinus species (Corcuera et al. 2011, Lamy et al. 2011). This finding is also

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1180  Wortemann et al. consistent with the large genotypic variability found in apple progeny derived from the crossing of two varieties (Lauri et al. 2011). By contrast, the genotypic variability between beech populations was remarkably small. The broad variability of P50 values within each population largely explains why the differences were not statistically different across them, but the mean values for each population were strikingly close in all three sites. Lamy et al. (2011) have recently proposed that the similar lack of genetic differentiation for cavitation resistance across Pinus populations is an indication of a ‘uniform selection’, canalizing this trait to buffer its genetic ­ ­variation. The same mechanism may also explain our findings in beech. Our results on cavitation resistance are in agreement with those of Hamrick (2004), who observed that most of the genetic variation for most traits lay within populations, with only limited differences between populations. This finding is also substantiated by our genetic diversity analysis of chloroplastic DNA, showing that most of this neutral genetic variability is located within each population and with little genetic differentiation between populations.

Phenotypic plasticity of vulnerability to cavitation We found in this study that beech trees grown in different sites exhibited contrasting cavitation resistance. These variations were not caused by a genetic bias, as the same pools of seeds were randomly distributed in each site. They more probably reflect the phenotypic plasticity of cavitation resistance in beech; cavitation resistance in trees is known to exhibit a large phenotypic plasticity in response to variations in environmental conditions. In a beech crown for instance, the light regime has a strong impact on branch cavitation resistance (Cochard et al. 1999, Herbette et al. 2010). Similarly, cavitation resistance can be influenced by soil dryness (Beikircher and Mayr 2009, Awad et al. 2010). Here, the ‘site effect’ accounted for about 10% of the total P50 variability. Trees grown in Spain were the most vulnerable to cavitation, and those grown in Slovakia the least vulnerable. We could not clearly attribute this plasticity to specific climatic variables available to us. Thus the Spanish trial received much more precipitation than the other sites, but the difference was small between the two other sites. This indicates that phenotypic plasticity in beech is under complex environmental control, and more work is needed to understand how local pedo-climatic conditions shape the xylem structure and function. Our results also suggest that the phenotypic variability of cavitation resistance in beech may be under genetic control, as a small but significant site × population interaction was found, representing more than 6% of the total variance. This result agrees with the findings of Fichot et al. (2010), who also showed that phenotypic plasticity in poplar was strongly genotype-dependent. Here again, we could not clearly attribute

Tree Physiology Volume 31, 2011

the degree of plasticity across populations to local climatic variables.

Conclusions This study represents the first extensive survey of the variability of xylem cavitation in an angiosperm species. The results indicate that in beech species this trait varies substantially within each population, but very little genetic differentiation is seen across populations. Phenotypic plasticity of cavitation resistance is significant between sites, the degree of this plasticity possibly varying between populations. These results suggest that the large phenotypic variability measured in nature by Herbette et al. (2010) was more probably caused by phenotypic plasticity than by genetic variability between populations. Beech trees thus have the capacity to acclimate their hydraulic traits to local pedo-climatic conditions, but the drivers of this acclimation remain to be elucidated. These conclusions are consistent with those recently arrived at for Pinus (MartinezVilalta et al. 2009, Corcuera et al. 2011, Lamy et al. 2011). This consistency is striking, considering the marked phylogenic and physiologic contrasts between these species: gymnosperm versus angiosperm, early versus late successional, heliophilous versus shade tolerant, drought tolerant versus drought avoidant, etc. The relevance of our findings may be more general and apply to many European tree species. Our study has several important implications for beech forest management in a context of global climate change. First, it is probably unrealistic to seek to identify more drought-­performing populations or ecotypes for this species on the basis of the hydraulic traits we have used in this study. However, this does not preclude the existence of such ecotypes, as drought performance is a very complex process conferred by the combination of many other functional traits not measured in our study. Our data suggest that the genetic differentiation is actually very low between the populations we have evaluated in this study. However, the possibility remains that in more marginal beech populations, more extreme climatic conditions may have favored the selection of more strongly contrasting ecotypes. Nevertheless, our data suggest that there is a large reserve of genetic diversity of cavitation resistance within each beech population. The functional and ecological significance of this diversity remains to be elucidated. For instance, it has not yet been demonstrated experimentally that more cavitation-­resistant genotypes are necessarily more drought-resistant. Under this hypothesis, we may speculate that during extreme drought conditions the most cavitation-resistant genotypes will perform better and may then be used as genitors to regenerate beech forests. Finally, our work demonstrates that beech trees have the capacity to acclimate their xylem hydraulics in response to local pedo-climatic conditions. If the effect of global change is progressive, then we can speculate that beech trees will also

Genotypic variability and phenotypic plasticity of cavitation  1181 progressively acclimate to new conditions. This acclimation may also be favored by appropriate silvicultural practices, for instance by manipulating the local environmental conditions. However, more work is needed to understand how environmental conditions shape tree hydraulics. This information is also crucial for the development of more realistic models predicting the effect of climate change on species distribution.

Acknowledgments We thank Sylvaine Labernia, Brigitte Girard, Christophe Serre, Pierre Conchon and Christian Bodet for their technical support and assistance in the field, Nathalie Bernard, Lydia Jaffrelo and Charles Poncet from the genotyping platform GENTYANE of INRA Clermont-Ferrand for their help in the genetic analysis, and the Cost Action E52 network for facilitating access to trials.

Funding This research was funded in part by the PitBulles project (ANR no. 2010 Blan 171001). Rémi Wortemann was supported by a doctoral fellowship from the French Ministry of Research.

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