Variation in photosynthetic performance and hydraulic architecture
across European beech (Fagus sylvatica L.) populations supports
the case for local adaptation to water stress
Ismael Aranda
1,7, Francisco Javier Cano
2, Antonio Gascó
1,3, Hervé Cochard
4,5, Andrea Nardini
6,
Jose Antonio Mancha
1, Rosana López
2and David Sánchez-Gómez
11Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Centro de Investigación Forestal, Carretera de la Coruña Km 7.5, 28040 Madrid, Spain; 2Unidad de Anatomía, Fisiología y Genética Forestal, Escuela Técnica Superior de Ingenieros de Montes, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain; 3School of Biology, IE University, Cardenal Zúñiga 12, 40003 Segovia, Spain; 4INRA, UMR547 PIAF, F-63100 Clermont-Ferrand, France; 5Clermont Université, Université Blaise-Pascal, UMR547 PIAF, BP 10448, F-63000 Clermont-Ferrand, France; 6Dipartimento di Scienze della Vita, Universita di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy; 7Corresponding author (aranda@inia.es)
Received March 14, 2014; accepted October 26, 2014; published online December 22, 2014; handling Editor Frederick Meinzer
The aim of this study was to provide new insights into how intraspecific variability in the response of key functional traits to drought dictates the interplay between gas-exchange parameters and the hydraulic architecture of European beech (Fagus
sylvatica L.). Considering the relationships between hydraulic and leaf functional traits, we tested whether local adaptation
to water stress occurs in this species. To address these objectives, we conducted a glasshouse experiment in which 2-year-old saplings from six beech populations were subjected to different watering treatments. These populations encompassed central and marginal areas of the range, with variation in macro- and microclimatic water availability. The results highlight subtle but significant differences among populations in their functional response to drought. Interpopulation differences in hydraulic traits suggest that vulnerability to cavitation is higher in populations with higher sensitivity to drought. However, there was no clear relationship between variables related to hydraulic efficiency, such as xylem-specific hydraulic conductivity or stomatal conductance, and those that reflect resistance to xylem cavitation (i.e., Ψ12, the water potential corresponding to
a 12% loss of stem hydraulic conductivity). The results suggest that while a trade-off between photosynthetic capacity at the leaf level and hydraulic function of xylem could be established across populations, it functions independently of the compro-mise between safety and efficiency of the hydraulic system with regard to water use at the interpopulation level.
Keywords: beech, cavitation, chlorophyll fluorescence, drought, gas exchange.
Introduction
Global climate trends indicate a worldwide increase in the risk of acute droughts and heat waves. An important concern in ecology, plant biology and forestry is whether the intraspecific genetic resources of tree species hold enough variability to cope with current climate change through micro-evolutionary processes (Aitken et al. 2008, Alberto et al. 2013). This issue is especially relevant for drought-sensitive temperate forest
tree species, such as European beech (Fagus sylvatica L.; Tognetti et al. 1995, Backes and Leuschner 2000).
Beech is a widespread species that dominates the canopy of many forests throughout its natural distribution range in Europe. However, drought sensitivity limits beech distribution in the Mediterranean basin, where it is confined to mountain ranges with high precipitation (Aranda et al. 2000, Bréda et al. 2006). After unusually dry periods, both acute mortality and a
Research paper
long-term loss of fitness have been recorded for this species. These events can cause changes in the competitiveness of beech and eventually alter forest structure (Cavin et al. 2013). Although these detrimental effects have largely been reported in locations close to the centre of European beech's distribu-tion (Leuzinger et al. 2005), populations at the southernmost range edge, where a large reduction in rainfall and rise in tem-perature are expected, could be particularly vulnerable (Jump et al. 2006, Piovesan et al. 2008).
The preservation of southern range-edge populations could provide a gene bank for traits conferring resistance against harsh environmental conditions through local adaption or assisted migration (Rose et al. 2009, Robson et al. 2012, Sánchez-Gómez et al. 2013). In fact, the wide distribution area of European beech is reflected in the high degree of pheno-typic variation among populations from different localities in traits related to its phenology (Dittmar and Elling 2006, Vitasse et al. 2009, Robson et al. 2013), leaf morphology and growth (Rose et al. 2009, Robson et al. 2012), as well as some func-tional traits (Tognetti et al. 1995, García-Plazaola and Becerril 2000, Peuke et al. 2002, Wortemann et al. 2011, Sánchez-Gómez et al. 2013). The fast expansion of beech distribution following the last glacial period (Demesure et al. 1996, Magri et al. 2006) could be a sign of high adaptive potential in this species and would explain the rapid subsequent establishment of populations that were able to adapt well to particular local conditions.
Local adaptation of populations occurs as a result of phe-notypic plasticity, i.e., the capacity of individual genotypes to produce different phenotypes when exposed to different envi-ronmental conditions (Pigliucci et al. 2006), genotypic variation (i.e., differences in the genome between individuals) or both. Although genetic variation has been claimed to be of lower rel-evance than plasticity for adjustment of some functional traits in beech (Bresson et al. 2011), other studies indicate that intra-specific genetic variability in highly dynamic functional traits, such as those controlling transpiration, can have an important cumulative effect on whole-plant performance (Leonardi et al. 2006). Thus, both phenotypic plasticity and genetic variability in functional traits can be considered relevant mechanisms for adaption to drought (Meier and Leuschner 2008, Rose et al. 2009, Bresson et al. 2011).
Among the multiple traits influencing plant performance dur-ing water deficit, control of water loss by stomata is one of the most important. In the case of beech, stomatal conductance to water vapour is one of the physiological traits most sensitive to water limitation, and it is generally down-regulated in the early stages of drought (Backes and Leuschner 2000, Aranda et al. 2005, Cano et al. 2013). However, the overall water balance of plants results from the difference between supply and loss; thus, the control of water use depends on regulation of the tran-spiration stream throughout the entire plant hydraulic pathway
(Cochard et al. 2002, Brodribb et al. 2003, Aranda et al. 2005). Important advances have recently been made in understand-ing the role of hydraulic traits in tree water economy and their implications from a macro- evolutionary and macro-ecological perspective (Maherali et al. 2004, Choat et al. 2012). However, much less is known at the micro- evolutionary level (but see Martínez-Vilalta et al. 2009, Lamy et al. 2011, López et al. 2013). Additionally, the hydraulic architecture seems not only to govern plant water balance and the maintenance of functional water status within ranges of safety from dehydration of plant tissues, but also to be related to the photosynthetic capacity at the leaf level (Brodribb et al. 2002), although the functional mechanism underlying this last relationship is still unknown.
Although some hydraulic traits are usually conserved within species (Lamy et al. 2011, Wortemann et al. 2011), some authors have reported a degree of genetic and phenotypic variability in plant hydraulic architecture (Mencuccini and Comstock 1997, Kavanagh et al. 1999, Kolb and Sperry 1999, Martínez-Vilalta et al. 2009, Corcuera et al. 2011, López et al. 2013). In beech, both genetic variability and a high degree of phenotypic plasticity to environmental factors, such as light or water availability, have been reported for hydraulic traits (Cochard et al. 1999, Barigah et al. 2006, Herbette et al. 2010, Wortemann et al. 2011).
In this context, there is renewed interest in increasing our knowledge of the role of local adaptation and phenotypic plas-ticity in marginal vs central populations of beech, particularly in populations from the southernmost edge of the species distri-bution area (Leonardi et al. 2012, De Lafontaine et al. 2013). In a previous study with seedlings from the same populations, Sánchez-Gómez et al. (2013) reported that differences in the tolerance of water deficit between provenances were related to the differential expression of leaf functional traits. In the pres-ent study with 2-year-old saplings, we explore the relationship between stem hydraulic plasticity and leaf-level photosynthetic parameters. The following hypotheses were tested: (i) beech populations differ in traits associated with their shoot hydraulic architecture [xylem-specific hydraulic conductivity (Ks), xylem
vulnerability to cavitation and wood density (WD)] and gas-exchange traits; (ii) gas gas-exchange and traits associated with shoot hydraulic architecture are highly plastic with regard to water availability in all populations; and (iii) there is a relation-ship between the control of water loss (inferred from stomatal conductance (gwv)] and xylem vulnerability to cavitation,
esti-mated from water potentials corresponding to 12 or 50% loss of stem conductivity (Ψ12 and Ψ50).
Materials and methods
Plant material and experimental set-up
Two-year-old plants from six European beech populations (see Table 1) were studied, covering central and marginal populations
across a wide range of macroclimatic (Mediterranean, continen-tal and oceanic climates) and microclimatic (soils, altitude) con-ditions (for details, see Table S1 available as Supplementary Data at Tree Physiology Online). The same plant material (ger-minated in spring 2010) was also used in a previous green-house study, in which plants were grown with contrasting water availabilities. In that study, half of the seedlings were kept as control plants, while the other half were submitted to water defi-cit (reported by Sánchez-Gómez et al. 2013). During autumn 2010, plants were kept outside in a nursery for the dormant win-ter period. Between 2 and 4 March 2011, plants were brought back to the greenhouse and transplanted into cylindrical con-tainers filled with 6 l of a 3 : 1 volume mixture of peat Floragard TKS2 (Floragard Vertriebs GmbH, Oldenburg, Germany) and washed river sand. This mixture was supplemented with 2 kg m−3 of Osmocote Plus fertilizer (16-9-12 NPK + 2
micronu-trients; Scotts, Heerlen, The Netherlands). Plants were watered regularly, and bud burst began after 3 weeks. After removal of several dead plants, we randomly selected a total of 204 samples (17 replicates × 2 treatments × 6 populations) from the remaining plants. The experimental layout was based on a factorial design with two factors: population and water availabil-ity. Two levels were established for water availability: ‘control’ and ‘water deficit’, as in the previous study (Sánchez-Gómez et al. 2013). Each plant was kept in the same soil water treat-ment as the previous year. Thus, samples enduring water stress came from plants submitted to drought for two consecutive growing seasons, in order to foster long-term acclimation to drought. The software package CycdesigN 2.0 (CycSoftware Ltd, Ranfurly, New Zealand) was used to arrange the spatial distribution of plants in the greenhouse that was optimized for a row–column design (15 × 14) including the two studied factors (population and watering treatment). The maximal photosyn-thetic photon flux density of sunlight in the greenhouse ranged from 450 to 530 µmol m−2 s−1 on sunny days throughout the
experiment. Relative humidity was kept at 65 ± 4% with a mist-ing system. Temperature varied on a daily and seasonal basis, but it was controlled within ranges close to ambient conditions and not exceeding 28 °C, using the greenhouse climate con-trol systems. The average temperature in the greenhouse was 22.3 ± 4.3 °C (mean ± 1 SD) during the experiment.
The watering protocol was the same as that described by Sánchez-Gómez et al. (2013). Well-watered (ww) seedlings were watered to field capacity regularly throughout the experi-ment. Seedlings from the water-stress treatment were submit-ted to progressive drought by withdrawing irrigation starting on 20 May 2011, when full leaf unfolding had occurred. Drought peaked after 50 days when soil water content reached 8% vol. The volumetric water content of the substrate was individually monitored throughout the experiment by time domain reflec-tometry (TRIME-FM; Imko Micromodultechnik GmbH, Ettlingen, Germany). A similar drop in soil water availability among
individuals was assured by monitoring soil moisture and water-ing on every other day. Waterwater-ing was adjusted accordwater-ing to individual water consumption (for details, see Sánchez-Gómez et al. 2013) in order to maintain comparatively similar draw-down in volumetric water content of the substrate across plants submitted to drought regardless of population or plant size. Measurement of gas exchange and water status
Measurements of gas exchange were carried out twice during the trial, i.e., at the beginning of imposition of water stress and at the peak of the water stress. Gas-exchange and chlorophyll-fluorescence measurements were made on attached leaves of all plants. One fully expanded leaf from the top third of the plant was selected. Measurements were carried out with a Li-Cor 6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA). Leaves were exposed to a CO2 concentration of 400 ppm
using the built-in Li-Cor 6400-01 CO2 mixer (Li-Cor, Inc.) and
a photosynthetic photon flux density of 800 µmol m2 s−1. Gas
exchange was measured at 24 °C and 60–65% relative humid-ity. Leaf chlorophyll fluorescence was recorded immediately after gas-exchange measurement (Li-Cor 6400-40 fluorescence chamber; Li-Cor, Inc.). Measurements were taken from 10:00 to 13:00 h throughout four consecutive days to complete the survey of 204 plants at each measurement time. Special care was taken to ensure an even representation of each popula-tion × treatment combinapopula-tion on each day. Environmental con-ditions did not change significantly among the 4 days when measurements were carried out. Net assimilation rate (An) and
stomatal conductance (gwv) were obtained from gas-exchange
measurements, whereas the effective quantum efficiency of photosystem II (ΦPSII) was obtained from chlorophyll-fluorescence
measurements as follows:
ΦPSII m s
m
= F′F−F
′ (1)
where Fm′ is the light-adapted maximal fluorescence and Fs is
the ‘steady-state’ fluorescence or fluorescence before a sat-urating light pulse. The water status of plants was assessed on each individual by measuring predawn (Ψpd) and midday (Ψmd) water potentials with a pressure chamber at the peak of water deficit (PMS Instrument Co. 7000, Corvallis, OR, USA). One fully expanded leaf nearest to the one chosen for gas-exchange and chlorophyll-fluorescence measurements was detached from shoots at the top of the plant.
Vulnerability to cavitation and hydraulic traits
After the peak of water stress was reached, all plants were watered to field capacity. One week after stress relief, shoots of 40–50 cm length from 10–12 plants per watering treat-ment × population combination were harvested in the early morning. The full harvest was completed in a single day to
avoid timing differences among samples that might inter-fere with subsequent hydraulic analysis. In order to ensure full hydration of the samples, all the plants were watered to field capacity the day before harvest. Shoots were wrapped in wet paper, sealed in black plastic bags to prevent dehy-dration and stored overnight in a cool chamber at 4 °C. The following day, samples were transported to the laboratory at Clermont-Ferrand (France). In the laboratory, 280 mm seg-ments were excised from shoots under water, and 1 cm of bark at both ends of the segments was carefully removed with a razor blade. The diameter was measured in three places, at both ends and halfway along each stem segment. Xylem cavitation was assessed by the Cavitron technique (Cochard et al. 2005). Before spinning, we injected air (<0.1 MPa) at the basal end of the shoot segment while the apical end was immersed in water to estimate whether the maximal vessel length of the segment exceeded 280 mm. We detected bub-bles coming out from the end of the shoot in only a few sam-ples from various populations. Given the high variation within each population and treatment, the vulnerability curves (VCs) of the samples with vessels >280 mm were not significantly different from VCs of the samples with shorter maximal vessel length (data not shown); therefore, all the data were analysed together.
The initial hydraulic conductance of each sample (ki) was
determined at a xylem pressure of −0.1 MPa, measuring the flux of a degassed ionic solution (10 mM KCl and 1 mM CaCl2
in deionized water). Afterwards, hydraulic conductance (kh,
mol s–1 MPa–1) was measured under increasing tensions in the
xylem by increasing the centrifugation speed (for a detailed description of the technique, see López et al. 2013). The per-centage loss of hydraulic conductivity (PLC) was estimated by the step-by-step decrease of kh with regard to ki using the
fol-lowing expression:
PLC i h
i
=100×(k −kk ) (2)
Xylem-specific hydraulic conductivity (Ks; mol s–1 m–1 MPa–1)
was calculated as ki divided by the sapwood area halfway
along the shoot and multiplied by the shoot length. In addi-tion, WD was calculated in 3-cm-long segments cut from the middle part of the shoot used to build VCs. These segments were soaked in degassed water overnight and their saturated volume was determined, according to Archimedes' principle, by immersing each sample in a water-filled test tube placed on a balance. Then, samples were oven dried at 105 °C for 48 h and their dry weight was measured. Wood density was calcu-lated as the ratio of dry weight to saturated volume.
Phenotypic plasticity in response to water availability was estimated for the studied hydraulic traits with a log-response ratio, as described by Hedges et al. (1999):
L=ln(Xww) ln(− Xws) (3)
The term Xww is the population mean of a specific trait in the
well-watered treatment, while Xws is the population mean of a
specific trait in the water-stressed treatment. Statistical analyses
General linear models (GLMs) were fitted to test the effect of population ‘P’, treatment ‘T’ and the interaction P × T on the studied variables. Size effects that might potentially give misleading results were considered by including the initial plant size as a covariate. As a proxy for plant size, root collar diameter was measured at the beginning of the experiment. Significant differences between treatments and/or popula-tions were tested using Fisher's least-significant difference test. Homogeneity of variance (Levene's test) and normality (Shapiro–Wilk test) assumptions were checked beforehand, and log-transformed variables were used when necessary to meet these assumptions. Specific relationships between hydraulic traits and gas-exchange variables across popula-tions were tested by non-parametric Spearman rank correla-tions because most of the studied variables were not normally distributed.
The relationship between PLC and Ψ was fitted to a logistic function (Pammenter and Vander-Willigen 1998), as follows:
PLC (%)= +1 exp( /10025( − ))
50
s Ψ Ψ (4)
where Ψ50 represents the value of Ψ at which 50% of
hydrau-lic conductivity is lost and s is the slope of the VC at Ψ50.
Finally, estimates of xylem water potentials at the beginning of xylem embolism (Ψ12) and full embolism (Ψ88) were
calcu-lated following Domec and Gartner (2001) as follows:
Ψ12 =Ψ50 +50s (5)
Ψ88 = Ψ50−50s (6)
The model, implementing Eq. (4) and assuming a normal dis-tribution (based on previous disdis-tribution fitting analyses), was parameterized separately for each population × water treat-ment combination through a maximum likelihood procedure using a simulating annealing algorithm. This procedure takes into account every single replicate–PLC response as a contri-bution to the overall model. Parameter estimates of the overall model and 95% confidence intervals for each parameter were used to compare models. The most commonly used method of analysing VCs involves computing a single curve for each
replicate, obtaining the estimates for each replicate, and finally, estimating overall average parameters from single replicate estimates. However, in contrast to the maximum likelihood procedure, the main flaw of this traditional approach is that it involves two steps, and consequently, a double penalty on the error terms. The first error term arises from the single repli-cate estimates of the parameters. This error term is, in general, wrongly ignored. The second error term, which is often the only one considered, arises from the overall average estimates of the parameters.
Statistica version 6.0 (StatSoft, Inc., Tulsa, OK, USA) was used for GLM analyses and Spearman rank correlations. The models for PLC (%) as a function of Ψ were built and param-eterized with the R-package ‘likelihood’ using R version 2.9.2 (R Development Core Team 2009).
Results
Water status and gas exchange
At the peak of water stress, the watering treatment had a sig-nificant effect on Ψpd. However, there was no significant
pop-ulation effect on Ψpd, confirming that the watering treatment
was homogeneously applied to all populations (Figure 1 and Table S2 available as Supplementary Data at Tree Physiology Online). The average Ψpd in ws plants was significantly
lower (−0.8 ± 0.1 MPa) than in ww plants (−0.4 ± 0.1 MPa). Homogeneous Ψpd measured among populations within each
watering treatment mirrored the interpopulation homogeneity of volumetric water content of the substrate that was achieved by the watering protocol (25 ± 3 vol% for ww plants and 8.5 ± 0.4 vol% for ws plants). Watering treatment and popula-tion both had a significant effect on Ψmd. However, no significant
interaction (treatment × population) was evident (Table S2 available as Supplementary Data at Tree Physiology Online). In general, the three German populations had significantly lower values of Ψmd than the other populations (−1.65 ± 0.1 vs
−1.35 ± 0.1 MPa on average), although population G1 did not differ significantly from ws plants of population It, nor from ww plants of population Sw (Figure 1).
Before water stress was imposed, there was little dif-ference among populations in terms of gas exchange and chlorophyll fluorescence (data not shown). After 50 days of partial watering withdrawal, there were pronounced dif-ferences owing to both watering treatment and popula-tion (Figure 2), although the main source of variation was provided by the watering treatment (Table S2 available as Supplementary Data at Tree Physiology Online). At the peak of water stress, the population ranking of Anet and gwv for ws
plants was as follows: It ≥ Sp ≥ G2 ≥ Sw > G1 > G3. The rank-ing of Anet and gwv remained similar, with only minor
devia-tions, for ww plants, as follows: Sp ≥ It ≥ Sw ≥ G3 > G2 ≥ G1. Values of ΦPSII decreased significantly with water stress in all
populations, except in G2. Thus, G2 showed no significant plasticity in this trait (the standard error was higher than the estimate of plasticity; Table 2). It should be remarked that ΦPSII was already low for ww plants in this population. The
plasticity of gwv was high (in general >0.4), while plasticity of
ΦPSII was much lower (Table 2).
Hydraulic efficiency and vulnerability to cavitation
A high degree of intrapopulation variability in vulnerability to cavitation was present (Figure 3), but nevertheless several sig-nificant patterns could be identified. Overall, estimates of Ψ50
decreased, and the slope parameter (s) of the VC increased with water stress (Figure 3). Differences in Ψ50 between
water-ing treatments were significant for G2 (Ψ50 = −3.02 ± 0.11
vs −3.41 ± 0.15 MPa for ww and ws seedlings, respec-tively). There were also significant differences between treat-ments for s in the G1, Sp, It and Sw populations. Differences among populations for Ψ50 ranged from −3.02 ± 0.11 (G1) to
−3.50 ± 0.14 MPa (It) for ww plants, and from −3.38 ± 0.12 (Sw) to −3.70 ± 0.13 MPa (It) for ws plants. Absolute differ-ences among populations were low (∼0.3 MPa) but statistically significant. Most of the variation in s was related to the water-ing treatment, i.e., curves for plants under water stress had steeper slopes (Figure 3).
Wood density was generally lower in ww than in ws plants (see Table 1 and negative values in Table 2). The popula-tions with the minimal and maximal variation in WD between Figure 1. Bars represent mean water potential (Ψ; ±1 SEM) measured
predawn (pd) and at midday (md) in well-watered (ww) and water-stressed (ws) plants at the peak of the water-stress cycle in six beech provenances from a wide geographical range (G1, G2, G3, Sp, It and Sw; see Table 1 for description of provenances). A significant effect of treatment but no effect of population was found for predawn water potentials (GLM analysis). A significant effect of both treatment and population was found for midday water potentials (no significant inter-action, GLM analysis). Homogeneous groups (Fisher's least significant difference post hoc test) for midday water potentials are depicted with different letter codes for each watering treatment independently.
watering treatments were G1 and Sw, respectively (i.e., they were the least and the most plastic ones for WD; see Tables 1 and 2). In fact, differences between treatments were significant in the Sw population (Table 1). When populations were com-pared within the same watering regimen, there were significant differences in the population ranking from G1 to Sw for ww plants, and from G2 to Sw for ws plants (Table 1).
There was no consistent trend for the effect of the watering treatment on Ks. Thus, Ks did not show plasticity in response to
water availability (the standard errors for plasticity were larger than the estimate of plasticity itself; Table 2). However, there were differences in Ks among populations for ws plants. In
par-ticular, the Sw population had the lowest Ks, which differed
sig-nificantly from the It population with the highest Ks (Table 1).
There was no significant correlation between WD and either Ψ12 or Ψ50 among populations, but there were two significant
correlations in ws conditions, namely between WD and Ks, and
between Ψ12 and Ψ50 (Figure 4).
In general, the plasticity of the hydraulic architecture in response to water availability was lower than that of gas-exchange variables. No clear trend was apparent for variation in phenotypic plasticity across the studied populations. Relationship between hydraulic traits
and gas-exchange traits
There was a positive correlation between WD and Ψ12
(Spearman's r = 0.89, P < 0.05), and both WD and Ψ12 were
negatively correlated with photosynthetic traits among popula-tions (Figure 4). It should also be remarked that Ψ12 was more
closely correlated than Ψ50 with photosynthetic traits (data
not shown). The general pattern was for an increase in pho-tosynthetic performance with decreasing WD, resulting in its significant correlation with ΦPSII and Anet in ws conditions. The
relationship of ΦPSII and Anet with Ψ12 was similar to WD but,
in this case, it was statistically significant only in ww condi-tions (Figure 4). The general response could be described as an increase in Anet with decreasing vulnerability to cavitation
(lower Ψ12). In ws conditions, the Sw and G2 populations
main-tained relatively high photosynthetic performance despite hav-ing high Ψ12 values, and consequently, these populations were
responsible for the breakdown of the relationship between these two traits.
Discussion
Interpopulation variability in gas-exchange and hydraulic traits
High intrapopulation variability in terms of xylem vulnerability to cavitation was consistent with previous findings for the species (Herbette et al. 2010). This variation arose among individuals experiencing the same environmental conditions, denoting a high degree of intrapopulation genotypic variability in European beech, as already reported for other species (Cochard et al. 2007). This finding tallies with other studies showing high intra-population variability in functional traits of European beech, which in some cases can exceed the variability among popula-tions (Ramírez-Valiente et al. 2009, Aranda et al. 2010, Bresson et al. 2011). For instance, Leonardi et al. (2006) recorded high differentiation in the control of water loss in response to water Figure 2. Mean values (±1 SEM) for net photosynthesis (Anet; in μmol
m–2 s–1), stomatal conductance of water vapour (g
wv; in mol m–2 s–1) and quantum yield (Φ; adimensional) at the peak of water stress. There was no significant effect of either treatment or population for these vari-ables before imposition of water stress. There was a significant effect of both treatment and population (no significant interaction, GLM analysis) for these three variables at the peak of stress. Homogeneous groups (Fisher's least significant difference post hoc test) among populations are depicted separately for each variable and watering treatment with different letter codes. Uppercase letters stand for well-watered (filled bars) and lowercase letters for ws plants (dotted bars).
stress within a full-sibling family of beech. In our study, even despite the contrasting origin of the six populations, interpopu-lation variability for Ψ50 was low (−3.34 to −3.56 MPa for ws
seedlings) and within the range previously reported for beech (Herbette et al. 2010, Wortemann et al. 2011, Barigah et al. 2013). Low vulnerability to cavitation has been reported to be
adaptive in dry sites when a wide range of species was com-pared (Maherali et al. 2004, Choat et al. 2007), though different strategies can be found within the same dry habitat (Jacobsen et al. 2007, Vilagrosa et al. 2014). However, vulnerability to cavitation within a particular species seems to be highly cana-lized (Matzner et al. 2001, Lamy et al. 2011, Wortemann et al. Figure 3. Relationships between the percentage loss of hydraulic conductivity (PLC) and xylem water potential (in MPa) for six beech populations covering a wide geographical range. Filled dots and continuous lines represent ww plants, while crosses and dashed lines depict the responses of ws seedlings. Estimates of Ψ50 and slope (±95% confidence intervals) for PLC models are provided for each population × treatment combination. The letter codes identify homogeneous groups for each estimate among all population × treatment combinations.
2011, but see Maherali et al. 2004). The narrow range of both Ψ50 and Ψ12 among populations under drought (±0.3 MPa) in
our study supports this suggestion of a canalized response. An important point to be mentioned is that we did not flush the samples before constructing the VCs. Our values of midday leaf water potential suggested that tension in the xylem was barely below the minimal values triggering cavitation. However, the relationship between ks and xylem tension suggested slightly
different patterns of native embolism according to population (differences in ks at the beginning of VCs), and we cannot rule
out the possibility that this influenced the apparent vulnerability to cavitation (Figure S1 available as Supplementary Data at Tree Physiology Online).
Populations were similar in terms of vulnerability to cavitation, although their ranking did to some extent reflect differences in drought tolerance, as reported for other traits from a previous study on the same populations (Sánchez-Gómez et al. 2013). Specifically, the Italian and Spanish populations, which had the lowest Ψ50 and Ψ12 values, were the most drought-tolerant
populations in that study. Likewise, the Swedish and G2 German populations, which had the highest Ψ50 and Ψ12 values in the
present study, were also the most drought-sensitive populations in our previous research. Moreover, a recent report of greater leaf damage and reduced growth in response to drought (Thiel et al. 2014) reinforces the idea that the Spanish population is more drought tolerant than those from central and northern Europe.
One important question that arises from these results is whether subtle differences in the absolute values of a
functional trait that, at most, produce marginally significant dif-ferences at the population level, genuinely provide evidence for biologically relevant variation, supporting adaptation at a micro-evolutionary scale (Dudley 1996, Geber and Dawson 1997, Caruso et al. 2005, 2006). Although this question is beyond the scope of the present study, we feel that minor dif-ferences among populations, if maintained over time, could be enough for natural selection to operate at a micro-evolutionary scale. It should be highlighted that often the evaluation of func-tional traits is made from single measurements, but even small differences could have a cumulative effect if maintained over the lifespan of a tree.
The plasticity of hydraulic traits could be an important fac-tor buffering the negative impact of drought on plant function, in particular by maintaining a high capacity for carbon uptake (discussed below). This acclimation capacity could be relevant in a future scenario of increased aridity, because hydraulic fail-ure has been described as a factor causing drought-induced mortality in beech (Barigah et al. 2013) and other species (e.g., Davis et al. 2002, Urli et al. 2013). The results of the pres-ent study indicate a certain degree of hydraulic plasticity in response to water availability; however, significant differences across populations were not apparent. Full acclimation to drier soil conditions is a slow process and might require several grow-ing seasons to be completed (Awad et al. 2010). Consequently, the small changes in terms of resistance to cavitation observed here (a response ranging over merely 0.4 MPa for Ψ50) may be
insufficient to confer resilience against short-term increases in Table 2. Phenotypic plasticity estimates (±SEM) of key functional traits in response to water availability for each population. Positive plasticity denotes that the trait value was higher in well-watered conditions than in water-stressed conditions. The reverse applies for negative plasticity.
Population Phenotypic plasticity
Ks Ψ12 WD gwv ΦPSII G1 −1.6 × 10−1 ± 4.2 × 10−1 9.1 × 10−2 ± 3.4 × 10−2 1.8 × 10−3 ± 4.4 × 10−3 4.5 × 10−1 ± 1.8 × 10−2 7.6 × 10−2 ± 8.7 × 10−3 G2 −1.2 × 10−2 ± 4.3 × 10−1 2.2 × 10−1 ± 4.7 × 10−2 −1.2 × 10−2 ± 5.0 × 10−3 3.7 × 10−1 ± 1.4 × 10−2 1.7 × 10−3 ± 7.0 × 10−3 G3 5.6 × 10−2 ± 3.6 × 10−1 7.0 × 10−3 ± 3.0 × 10−2 −1.7 × 10−2 ± 4.1 × 10−3 5.8 × 10−1 ± 1.5 × 10−2 1.5 × 10−1 ± 6.9 × 10−3 Sp 1.6 × 10−1 ± 3.7 × 10−1 3.6 × 10−2 ± 3.2 × 10−2 −2.0 × 10−2 ± 3.9 × 10−3 4.9 × 10−1 ± 1.3 × 10−2 1.1 × 10−1 ± 6.3 × 10−3 It −2.9 × 10−1 ± 3.0 × 10−1 3.2 × 10−2 ± 2.4 × 10−2 −7.8 × 10−3 ± 4.0 × 10−3 4.4 × 10−1 ± 1.3 × 10−2 1.3 × 10−1 ± 6.7 × 10−3 Sw 1.5 × 10−1 ± 4.7 × 10−1 6.4 × 10−2 ± 3.6 × 10−2 −4.4 × 10−2 ± 4.8 × 10−3 4.5 × 10−1 ± 1.5 × 10−2 1.1 × 10−1 ± 8.0 × 10−3 Table 1. Mean (±SEM) values of hydraulic traits [wood density (WD) and hydraulic conductivity (Ks)] analysed in shoots from the studied beech provenances: Ingolstadt, Germany (G1); Kempten, Germany (G2); Illertissen, Germany (G3); Montejo de la Sierra, Spain (Sp); Belluno, Italy (It); and Falkenberg, Sweden (Sw). The latitude for each population is denoted after each population code. Results are provided for the two watering treat-ments [well watered (ww) and water stressed (ws)]. For each trait, the superscript letter codes denote homogeneous groups (one-way analysis of variance, Fisher's test, P < 0.05) among provenances within a specific watering treatment (lower case) or between watering treatments for a specific provenance (upper case). A total of 8–15 plants per provenance on each watering treatment were measured for WD. The number of repli-cates for hydraulic conductivity was 5–12.
Hydraulic trait Treatment G1 (48°56′N) G2 (47°44′N) G3 (48°11′N) Sp (41°07′N) It (46°02′N) Sw (56°52′N) WD (g cm−3) ww 0.514a(A) ± 0.013 0.471b(A) ± 0.011 0.493ab(A) ± 0.008 0.474b(A) ± 0.010 0.479b(A) ± 0.008 0.466b(B) ± 0.017
ws 0.511ab(A) ± 0.008 0.483b(A) ± 0.010 0.513a(A) ± 0.009 0.497abc(A) ± 0.009 0.488bc(A) ± 0.010 0.516a(A) ± 0.009
Ks (mol m−1 s−1 MPa−1) ww 13.15a(A) ± 3.88 23.35a(A) ± 10.21 18.57a(A) ± 9.36 23.56a(A) ± 5.83 14.22a(B) ± 2.50 17.93a(A) ± 6.09
ws 18.86ab(A) ± 4.00 23.98ab(A) ± 6.08 16.33ab(A) ± 4.08 16.47ab(A) ± 4.93 21.38a(A) ± 4.93 12.74b(A) ± 3.09
stress of the magnitude predicted (Wikberg and Ögren 2007). Given the small and variable responses at both population and drought treatment levels, neither adaptive change nor acclima-tion through phenotypic plasticity seems to make a significant contribution to cavitation resistance in beech. Further cau-tion should be exercised in interpreting our results, because some plastic responses, such as the decrease in Ψ12 or Ψ50
with increased water deficit, might partly be driven not only by structural adjustments and related functional traits, but also by a methodological artefact produced by the use of non-flushed material (Sperry et al. 2012). Nevertheless, the results of this study of beech in controlled conditions agree with the low vari-ability in vulnervari-ability to cavitation observed after many years growing in natural conditions (Herbette et al. 2010).
Figure 4. Relationships between wood density (WD; in g cm–3) or Ψ
12 (in MPa) and different photosynthetic and hydraulic traits among popu-lations. Spearman's R values and fitted lines are shown for the most significant correpopu-lations. Significance is highlighted with one asterisk if 0.10 ≥ P > 0.05 and two asterisks if 0.05 ≥ P > 0.01. Filled symbols denote ww plants and open symbols ws plants. The populations are coded as follows: G1, upright triangles; G2, inverted triangles; G3, stars; Sp, circles; It, squares; and Sw, diamonds.
Regarding gas-exchange parameters, Bresson et al. (2011) identified phenotypic plasticity, rather than local adaptation, as the main source of variation among beech populations along an altitudinal cline. In our study, we found a significant effect of population origin on Amax and gwv, in accordance with previous
reports (Martin et al. 2007, Premoli and Brewer 2007, Robson et al. 2012, Sánchez-Gómez et al. 2013). This may be interpreted as evidence for local adaptation in the phenotypic expression of functional traits, considering the very different genetic back-grounds of the populations tested. Despite the high variability of changes in gas-exchange traits and leaf photochemistry in response to drought (phenotypic plasticity), population origin makes a significant difference to these responses (Peuke et al. 2002, Nielsen and Jørgensen 2003, Rose et al. 2009, Sánchez-Gómez et al. 2013). If local environmental conditions bring about adaptive change consistently across large geographical ranges (Bresson et al. 2011), we would have expected the variation among diverse populations, such as those studied herein, to be evident as adaptive variation in drought resistance (Peuke et al. 2002, Pluess and Weber 2012). This expectation is supported by a recent experiment in common-garden conditions, in which the same Spanish population considered here survived better under water stress than populations from the centre of beeches' range (Thiel et al. 2014).
Interrelationship between gas-exchange and hydraulic traits
At an interspecific level, and across a wide range of species, reducing the risk of cavitation involves higher investment in conduit wall volume per conduit volume, i.e., higher WD, to avoid conduit collapse under negative pressure. This trade-off is responsible for the positive relationship between WD and resistance of the hydraulic system in terms of Ψ50 (Hacke et al.
2001, Jacobsen et al. 2005). A similar relationship has also been reported when comparing WD and Ks, in that species with
higher WD generally have lower specific hydraulic conductivity (Poorter et al. 2010, Bucci et al. 2012). The results of our study indicate that Ks was not coupled with the following: (i) resistance
to cavitation at the interspecific level, as inferred from Ψ12, Ψ88
or, more usually, Ψ50 (Bhaskar et al. 2007); nor with (ii)
con-struction costs, because there was no correlation between Ks
and WD (Hacke et al. 2006). The absence of a clear relation-ship between hydraulic variables has already been highlighted by Tognetti et al. (1995) in a comparison of xeric and mesic beech populations. In our study, Ψ12 and WD tended to have
a negative relationship with carbon assimilation capacity, in that both were negatively correlated with Anet and ΦPSII for ww
plants. A decrease in photosynthesis with increasing WD has been reported to constrain physiological function within rates partly governed by the biophysical structure of wood (Santiago et al. 2004). However, the fact that the relationships of WD with carbon assimilation were significant only when considering Ψ12
as the independent variable (instead of Ψ50, which is more
fre-quently used) could be interpreted as evidence that hydraulic signalling provides an early warning system to elicit drought responses. Those hydraulic systems that are more vulnerable to embolism could benefit from protection by a mechanism such as this to trigger prompt and effective control of water loss in response to water stress. This response would increase resil-ience in environments with frequent drought events. Such a mechanism would be especially relevant for temperate species with a low tolerance for water stress and isohydric performance, such as beech (Aranda et al. 2005). What remains puzzling is that coupling between the hydraulic or mechanical properties of shoots and leaf physiology affects variables directly related to carbon uptake, at the intraspecific level (see Figure 4, relation-ship Ψ12–ΦPSII or WD–ΦPSII). Previous reports considering
inter-specific comparisons have also found a relationship between shoot hydraulic properties and photosynthetic parameters (Brodribb et al. 2002), but without offering a mechanistic expla-nation for the basis of this linkage.
Conclusions
There were small but significant differences in the drought response traits of trees of the provenances tested that were consistent with previous findings. The observed intraspecific differentiation supports the view that the functional traits we measured have potentially adaptive importance for European beech. The WD and vulnerability to cavitation, in terms of Ψ12,
were related to the potential for leaf carbon assimilation inde-pendent of the watering treatment. However, the expected interplay between gas-exchange response to drought and hydraulic function across populations did not seem to be oper-ating in beech, at least with respect to the classical compro-mise between safety and efficiency of the hydraulic system. Considering that forest tree species with wide distribution ranges harbour most of their genetic variability for morphologi-cal, functional, phenological and growth-related traits within populations (Hamrick 2004, Alberto et al. 2013), some of the individual variability we observed within the six populations studied could reflect this important source of variability; this is something that warrants further research.
Supplementary data
Supplementary data for this article are available at Tree Physiology Online.
Acknowledgments
The authors are grateful to G. Huber, R. Övergaard and T.M. Robson, who provided the seeds of some of the studied popu-lations. Dr Robson also kindly reviewed the final version of the
manuscript. The authors thank L. Alté for his invaluable techni-cal assistance in setting up the experiment.
Conflict of interest
None declared.
Funding
This work was supported by the Spanish Economy and Competitivenes Ministry (ECOFISEPI AGL2011-25365). F.J.C. participated in the project thanks to a scholarship from the Autonomous Community of Madrid. A.G. was supported by the Spanish Ministry of Science and Innovation, ‘Juan de la Cierva’ programme associated with the project (AGL2006-03242).
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