within eucalyptus species - brodribb lab · 2016-03-11 · lites (psms) are compounds that can have...

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Tree Physiology 00, 1–15doi:10.1093/treephys/tpv106

Responses to mild water deficit and rewatering differ among secondary metabolites but are similar among provenances within Eucalyptus species

Adam B. McKiernan1,2,4, Brad M. Potts1,2, Timothy J. Brodribb1, Mark J. Hovenden1, Noel W. Davies3, Scott A.M. McAdam1, John J. Ross1, Thomas Rodemann3 and Julianne M. O’Reilly-Wapstra1,2

1School of Biological Sciences, University of Tasmania, Private Bag 55, Hobart, TAS 7001, Australia; 2National Centre for Future Forest Industries, University of Tasmania, Private Bag 55, Hobart, TAS 7001, Australia; 3Central Science Laboratory, University of Tasmania, Private Bag 74, Hobart, TAS 7001, Australia; 4Corresponding author ([email protected])

Received March 16, 2015; accepted September 8, 2015; handling Editor Ülo Niinemets

Water deficit associated with drought can severely affect plants and influence ecological interactions involving plant secondary metabolites. We tested the effect of mild water deficit and rewatering on physiological, morphological and chemical traits of juvenile Eucalyptus globulus Labill. and Eucalyptus viminalis Labill. We also tested if responses of juvenile eucalypts to water deficit and rewatering varied within species using provenances across a rainfall gradient. Both species and all provenances were similarly affected by mild water deficit and rewatering, as only foliar abscisic acid levels differed among provenances during water deficit. Across species and provenances, water deficit decreased leaf water potential, above-ground biomass and formylated phloroglucinol compound concentrations, and increased condensed tannin concentrations. Rewatering reduced leaf carbon : nitrogen, and total phenolic and chlorogenic acid concentrations. Water deficit and rewatering had no effect on total oil or individual terpene concentrations. Levels of trait plasticity due to water deficit and rewatering were less than levels of constitutive trait variation among provenances. The overall uniformity of responses to the treatments regardless of native provenance indicates limited diversification of plastic responses when compared with the larger quantitative variation of constitutive traits within these species. These responses to mild water deficit may differ from responses to more extreme water deficit or to responses of juvenile/mature eucalypts growing at each locality.

Keywords: abscisic acid, drought, intraspecific, phenol, tannin, terpene.

Introduction

The changing global climate is predicted to alter rainfall patterns, and decreasing rainfall in many regions will lead to more frequent, prolonged and/or severe drought events ( Collins et al. 2013). Drought causes soil water deficit, and plants may avoid dehydra-tion by conserving within-plant water ( Maximov 1929). Conser-vation of plant water can be achieved by regulating guard cell turgor that reduces the aperture of stomatal pores ( Schroeder et al. 2001). Stomatal closure is signalled by the phytohormone abscisic acid (ABA), the foliar levels of which increase in response to soil water deficit ( Tardieu and Simonneau 1998). While complete or partial stomatal closure inhibits water loss through stomata, this also limits the uptake of carbon dioxide

( Schroeder et al. 2001). Prolonged soil water deficit leading to long-term stomatal closure can result in a negative carbon (C) balance as plant C stores are depleted ( McDowell et al. 2008), thereby limiting resources available for use by the plant.

Susceptibility to water deficit varies among plant species ( Pizarro and Bisigato 2010, Brodribb et al. 2014). Some species have highly developed physiological mechanisms to avoid water stress and/or morphological traits that enable tolerance of water stress (e.g., Bréda et al. 2006, McDowell et al. 2008, Blackman et al. 2010, Nardini and Luglio 2014). Drought susceptibility also varies among genetically distinct populations within a species (e.g., Tuomela 1997, Dutkowski and Potts 2012, Taeger et al. 2013, Cochrane et al. 2014, de la Mata et al. 2014), likely due to

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quantitative variation of traits common to the species. Plant spe-cies occurring over a wide environmental gradient can adapt to local environments, so that individuals from drier or more drought-prone regions are less affected by drought than individuals from wetter regions ( Taeger et al. 2013, Anderegg 2015). For example, genotypes of Fagus sylvatica (European beech) from dry regions are more drought resistant than F. sylvatica from wetter regions ( Thiel et al. 2014). Likewise, within-species variation in drought susceptibility has been described in numerous species including Eucalyptus microtheca ( Tuomela 1997, Li et al. 2000) and Euca-lyptus globulus Labill. ( Dutkowski and Potts 2012), as well as in Pinus ( Tognetti et al. 1997, Chambel et al. 2007) and European grasses ( Beierkuhnlein et al. 2011). However, responses to drought can vary among species of the same genus (e.g., Pinus spp., Chambel et al. 2007), and populations of a species from dry regions are not always the most drought tolerant (e.g., Beierkuhnlein et al. 2011, Lamy et al. 2011, Cochrane et al. 2014).

In addition to chemical compounds such as ABA that have a role within the plant, water deficit can influence compounds that have diverse effects outside of the plant. Plant secondary metabo-lites (PSMs) are compounds that can have important ecological influences such as contributing to the decomposition, flammability and defence of plants or plant parts ( Wiggins et al. 2006, Ormeño et al. 2009, Youngentob et al. 2011, Chomel et al. 2014). The influence of PSMs can be concentration dependent ( Jensen et al. 2014), and PSM concentrations vary naturally among prove-nances within some plant species (e.g., Moore and Foley 2005, O’Reilly-Wapstra et al. 2010, McKiernan et al. 2012). The con-centrations of many PSMs are plastic and vary due to environmen-tal variables such as water deficit ( Miles et al. 1982, Gleadow and Woodrow 2002, Zhang et al. 2012). However, the effect of water deficit on PSM concentrations can be species-, experiment-, PSM class- or even compound-specific ( Pizarro and Bisigato 2010, Zhang et al. 2012, McKiernan et al. 2014). To unravel the com-plexity of species × population × PSM responses to water deficit, research that incorporates within-species provenances that vary in drought tolerance and constitutive PSM concentrations will aid elucidation of overall species responses, provenance level responses and flow-on ecological effects ( Tognetti et al. 1997, Staudt et al. 2008, Said et al. 2011).

Eucalyptus is a dominant tree genus consisting of over 700 species ( Brooker 2002), and PSMs vary qualitatively and quan-titatively among Eucalyptus species ( Li et al. 1996, Nicolle et al. 1998, Steinbauer 2010). Plant secondary metabolites also vary quantitatively among genetically distinct provenances within Eucalyptus species ( O’Reilly-Wapstra et al. 2010, Andrew et al. 2013) and among individual trees ( Moore and Foley 2005). Eucalyptus species often grow naturally over a vast area encom-passing considerable environmental variation ( Austin et al. 1990, Warren et al. 2005, Andrew et al. 2007). Previously, E. globulus ( Dutkowski and Potts 2012) and Eucalyptus viminalis Labill. ( Ladiges 1974) genetically based drought tolerance was

shown to vary among localities across a wide area. As such, vari-able drought tolerance, intraspecific genetically based quantita-tive PSM variation and environmental gradients make Eucalyptus an ideal genus on which to study the effect of water deficit.

Here, E. globulus and E. viminalis provenances from the same four locations across the species’ natural range were used in a glass-house-based experiment, incorporating a water deficit treatment and also a rewatering period. Previous work showed that many C-based PSMs (terpenes and phenolics) remained stable in juvenile E. globulus and E. viminalis during water deficit ( McKiernan et al. 2014). However, this previous work used seed from one location per species, and limited inferences could be made about species-level juvenile responses or about variation in responses within spe-cies ( McKiernan et al. 2014). Here, we used juveniles from multiple provenances per species to test whether the findings presented by McKiernan et al. (2014) were representative of juveniles of each species or whether genetic variation among provenances and envi-ronmental variation among locations would lead to a range of responses to water deficit. We hypothesized that eucalypts of both species from the drier locations would tolerate water deficit and avoid water stress, and also that morphology and concentrations of selected PSMs would remain relatively stable due to adaptation to dry conditions. In contrast, we hypothesized that eucalypts from wetter locations would become water stressed and that this would be reflected in quantitative changes to morphological and PSM traits due to limited adaptation to naturally dry soils. We also included a rewatering period after water deficit to assess the stabil-ity of changes to plant traits resulting from water deficit.

The specific aims of this study were to determine whether (i) constitutive plant traits of these two species differed among localities, (ii) responses to water deficit differed depending on locality, (iii) traits of different types (morphological or chemical; particular PSM classes) were differentially affected by water deficit and (iv) relief from soil water deficit (rewatering for 2 weeks) would affect morphological and chemical plant traits.

Materials and methods

Plant material

Open-pollinated E. globulus and E. viminalis seed were collected from seven trees per species at each location (Southern Tasmania [ST], Queens Domain [QD], St Helens [SH] and King Island [KI]), totalling 28 trees per species (Table 1). Seed of a single ‘mother’ tree was pooled and termed a ‘family’. Mother trees of each spe-cies were at least 100 m apart to avoid sampling highly related individuals. Mother trees on KI were spread across the island.

Eucalyptus globulus and E. viminalis seed from all 56 families were germinated and grown for 1 month in a naturally lit glass-house (mean light: 1120 µmol m−2 s−1; mean day/night tempera-ture: 23/16 °C). Uniform size seedlings (cotyledons and one leaf pair) were selected (eight individuals per family = 448 plants total) and transplanted into individual plastic pots with potting mix

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containing eight parts composted fine pine bark: three parts coarse river sand and N : P : K (19 : 2.6 : 10) at 1 g−1 l−1 potting mix. Seedlings were grown for 10 weeks, then repotted into larger pots (base: 115 mm diameter, top: 138 mm diameter, height: 250 mm) containing 900 g of moist potting mix (430 g−1 dry weight [DW]; pH 6.0) with extra fertilizer (5 g Osmocote® 3–4 months [N14 : P6.1 : K11.6] per pot) and wetting agent (Everris Hydraflo® at 1.35 l m−3 potting mix). All pots (n = 448) contained equal bulk density of potting mix and a single plant, and they were arranged in a randomized split-plot design of 12 replicates, each containing a control and a water deficit plot to simplify watering. Each plot contained equal numbers of E. globulus and E. viminalis from each locality (randomly selected with regards to family). Eight replicates contained plots of 16 plants (two plants per local-ity [n = 4] from each species [n = 2]), and four replicates con-tained plots of 24 plants (three plants per locality [n = 4] from each species [n = 2]). Plants were randomized within plots and plot position randomized within replicates. All plants were grown for a further 7 weeks and watered daily to field capacity before experimental treatments were applied.

Experimental treatments

Treatments began in January 2013 (summer) when eucalypts were 26 weeks old and at least 50 cm tall. Daily evapotranspi-ration was monitored gravimetrically and species means calcu-lated using six random control plants per species. A percentage (50%) of the 3-day rolling mean evapotranspiration was cal-culated every 2 days, then provided to water deficit plants of

each species ( Mitchell et al. 2013). Control plants used to determine evapotranspiration were randomly reselected weekly, and all control plants were watered daily to field capac-ity. Following this method, all water deficit plants of a species were provided the same amount of water regardless of indi-vidual plant water usage. Due to the hydrophobic nature of dry potting mix, plastic bags were placed over the base of all water deficit plants to eliminate drainage, while the pot remained open to facilitate evaporation. No waterlogging occurred as a result of plastic bags.

Treatments were maintained for 3 months, then six random replicates were destructively harvested (Harvest 1; Figure 1) over 6 days. Plastic bags were removed from all remaining water deficit plants and these were rewatered daily to field capacity for 2 weeks along with remaining controls in each replicate (Figure 1). The remaining six replicates (each containing one control plot + one rewatered plot) were then destructively har-vested over 6 days (Harvest 2). The eight individual plants from each family were evenly but randomly divided among the two treatments (control and water limited) and the two sampling periods (Harvests 1 and 2; Figure 1).

Sample collection

Samples for foliar ABA analysis and leaf water potential (Ψleaf; 143 plants) were primarily collected at Harvest 1 (control and water limited plants), as foliar ABA levels and vascular ten-sion of all plants were expected to be similar at Harvest 2 ( Correia et al. 2014). Plants were placed outside in full sunlight

Responses to mild water deficit and rewatering 3

Table 1. Details of four localities from which E. globulus and E. viminalis open-pollinated seed were collected.1 KI, King Island; ST, Southern Tasmania; QD, Queens Domain; SH, St Helens.

Locality

KI ST QD SH

Coordinates Latitude −39.865881° −43.028629° −42.864423° −41.271634° Longitude 143.980070° 146.890798° 147.322510° 148.312977°Mean altitude (m) E. globulus 35 326 102 117 E. viminalis 27 263 120 28Mean annual rainfall (mm)2 927.4 960.0 601.6 817.72Mean number of days of rain1 162.6 176.2 58.7 152.1Rainfall driest quarter (mm)2 126.7 179.2 126.6 180.5Rainfall wettest quarter (mm)2 350.0 309.5 174.8 234.2Rainfall seasonality (C of V)2 37.8 20.9 15.0 14.7Drought susceptibility3 Highly susceptible Susceptible Intermediate TolerantMean maximum temperature (°C)1 17.0 16.6 16.9 18.0Mean minimum temperature (°C)1 10.0 5.7 7.8 8.9Mother trees per species 7 7 7 7Individuals per mother 8 8 8 8Total number of plants grown 112 112 112 112

1Data obtained from closest Bureau of Meteorology (Australia) weather station.2Data from ANUCLIM (Xu and Hutchinson 2010) using geographic coordinates.3From Dutkowski and Potts (2012).C of V, coefficient of variation.

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(1500–2000 µmol m−2 s−1) for >30 min (between 11.30 am and 2.00 pm). Mid-day Ψleaf was measured on a single fully expanded leaf using a Scholander pressure chamber (PMS, Albany, OR, USA). For foliar ABA quantification, fully expanded juvenile leaves were taken from multiple positions on each plant; leaves of three plants were pooled (same species, locality and treatment), immediately immersed in liquid nitrogen (N) and then stored at −70 °C. Due to the large number of plants, foliar ABA levels were used as a proxy measure of stomatal adjust-ment during water deficit ( McAdam and Brodribb 2015a).

Remaining parts of each plant were then destructively sam-pled in turn. A single fully expanded leaf from the fourth node below the apex was frozen (−12 °C) for measurement of leaf mass per area (LMA). Next, the stem was cut and total fresh above-ground biomass was determined. From this cut stem, we were only interested in leaves that developed during the treat-ment period, and so harvested only the top (youngest) eight fully expanded E. globulus leaf pairs or the top 50% of fully expanded E. viminalis leaves. Leaves of a plant were pooled and mixed, disregarding leaves that were damaged. A random sub-sample was frozen for oil analysis, then the remaining pooled leaves were weighed, frozen, freeze-dried, reweighed, scanned using near-infrared spectroscopy (NIRS; see below), ground in a Cyclotec™ 1093 cyclone mill (Foss, Hillerød, Denmark) and passed through a 1-mm sieve for analysis of C and N levels (Thermo Finnigan EA 1112 Series Flash Elemental Analyser, Thermo Fisher Scientific, Milan, Italy), total phenols, condensed tannins (CTs), chlorogenic acid and formylated phloroglucinol compounds (FPCs). Sample mass prior to and post lyophiliza-tion was used for the quantification of fresh leaf water content expressed as percentage fresh weight (% FW). The remaining above-ground plant section (fresh stem and un-harvested leaves) was weighed and oven-dried at 40 °C until constant mass. Total dry above-ground biomass was determined using the dry stem and leaf mass and the calculated dry mass of harvested leaves of each sample. The below-ground portion of each plant remained in the pot, including lignotubers, which are storage organs developed from cotyledonary and early seedling leaf nodes from which regenerative shoots develop after fire or drought ( Whittock et al. 2003). The lignotuber was collected and frozen for morphological assessment. Roots were discarded.

Foliar ABA

Eighty ABA samples from Harvest 1 were assayed (five samples of pooled leaves × two species × four localities × two treatments [con-trol and water limited]). Extraction of foliar ABA followed the method described by McAdam and Brodribb (2014) using an internal stan-dard, except that ∼0.2 g of pooled leaves was used per sample. Endogenous ABA was physico-chemically quantified using a Waters ultra-performance liquid chromatograph coupled to a Xevo triple quadrupole mass spectrometer (UPLC–MS), with results expressed as ng g−1 DW using the calculated water content of each sample.

Lignotuber and leaf dimensions

Lignotuber size was calculated for all plants using the maximum lignotuber width minus the stem diameter and expressed as the ratio of lignotuber to stem diameter ( Ladiges and Ashton 1974). Leaf mass per area was calculated on all plants using dry mass and lamina area of a single leaf. Leaf area was determined using a scanned leaf image and ImageJ (version 1.48). Leaf mass per area was expressed as g m−2.

Secondary chemistry

We quantified chemical groups and individual compounds assayed previously by McKiernan et al. (2014), which have known ecological roles ( Wiggins et al. 2006, Ormeño et al. 2009, Youngentob et al. 2011, Chomel et al. 2014) to investi-gate the impact of mild water deficit and rewatering. Due to the large number of samples (∼800) from this and two other con-current experiments (same mother trees, plant age, glasshouse— A.B. McKiernan, J.M. O’Reilly-Wapstra, B.M. Potts, M.J. Hovenden, T.J. Brodribb, S.A.M. McAdam, N.W. Davies, T. Rodemann, unpub-lished data), a subset of samples (156–204 samples depend-ing on assay) from the three experiments were used for chemical assays, then the chemical data were correlated to NIR spectra to create independently validated calibration models for each trait (see below; Table 2). The subset of samples used in chemical assays included equal quantity of random samples from each species, locality and treatment.

Extraction, analysis, identification and quantification of the total oil (component list in Table S1 available as Supplementary Data at Tree Physiology Online) and five of the consistently most abundant

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Figure 1. Experimental design. A total of 448 seedlings were used in the experiment, with 224 plants sampled at Harvest 1 (control and water limited treatments), then the remaining 224 plants sampled at Harvest 2 (control and rewatered treatments).

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monoterpenes (1,8-cineole, α-pinene and limonene) and sesqui-terpenes (globulol and aromadendrene) from fresh leaf material followed the methods outlined by McKiernan et al. (2012). The total oil was calculated from the sum area of all peaks detected by gas chromatography–mass spectrometry relative to the n- heptadecane internal standard, and the peak area of each major component was calculated as a relative percentage of the total oil area. Concentrations of 1,8-cineole and α-pinene were expressed as mg g−1 DW using standards, while total oil and other oil compo-nents were expressed as mg g−1 DW cineole equivalents using the DW of each individual sample. Phenols were extracted, then total phenolics (TPs) and CTs were quantified in duplicate as described by McKiernan et al. (2014). Total phenolics were expressed as mg g−1 DW gallic acid equivalents, and CT results were expressed as mg g−1 DW sorghum tannin equivalents using purified sorghum tannin ( Hagerman and Butler 1980). We assayed two specific FPCs (macrocarpals A and G) by high performance liquid chro-matography following the methods of Wallis and Foley (2005). A group of relatively late-eluting FPCs (eluting just before and after macrocarpal G) were also quantified. Macrocarpal A was expressed as mg g−1 DW, while macrocarpal G and the group of late-eluting FPCs were expressed as mg g−1 DW macrocarpal A equivalents, using standards described by Eyles et al. (2003).

Chlorogenic acid

Chlorogenic acid was assayed as it has a known role in alleviating oxidative water stress damage ( Živkovic et al. 2010). Phenols were extracted from ground samples using aqueous acetone ( McKiernan et al. 2014). Supernatant of each sample was passed through a new 0.22-µm nylon syringe filter directly into a

2-ml autosampler vial. Chlorogenic acid was quantified using a Waters Acquity H-series UPLC, Waters Acquity Photodiode Array detector, an Acquity BEH C18 1.7 µM column (2.1 × 100 mm), a chromatogram at 325 nm (retention time: 1.18 min) and a pharmaceutical grade chlorogenic acid reference standard (Sigma-Aldrich, St. Louis, USA). A flow rate of 0.35 ml min−1 and a mobile phase gradient of 90% A/10% B to 50% A/50% B (mobile phase A [1% acetic acid] and mobile phase B [acetoni-trile]) at 3 min followed by 3-min re-equilibration were used. Results were expressed as mg g−1 DW.

Near-infrared spectroscopy

All freeze-dried whole leaf samples (∼800 samples from this and two other concurrent unpublished experiments) were measured using the standard optical fibre of a Bruker MPA Fourier Trans-form NIR spectrometer (Bruker, Billerica, USA) and a TE-InGaAs NIR detector. Two different positions (at either end of leaf on opposite sides of midrib) were measured on two different leaves per sample and averaged to one single spectrum. Each spectrum was recorded using a spectral range between 12,500 and 4000 cm−1 with a spectral resolution of 8 cm−1 and four scans. Background spectra were measured using 64 scans on the stan-dard Bruker reference for the fibre optic cable that was used for a subsequent maximum of 2 h. Quantitative chemical data com-bined with the corresponding NIR spectra for each sample were used to create a partial least squares (PLS) calibration model for each trait using The Unscambler X (version 10.1, CAMO Soft-ware, Oslo, Norway). The models for total oil, 1,8-cineole, α-pinene, globulol, aromadendrene and limonene used 156 samples and were randomly cross-validated using 20 segments.


Table 2. Details of predictive models calibrated using NIRS and complimentary laboratory assayed chemical data for each chemical trait in Eucalyptus leaves.1 C-Val, cross-validated (random with 20 segments); MSC, multiplicative scatter correction; 1stD, first derivative using Savitzky–Golay filter; Smooth, smoothing using Savitzky–Golay filter; SNV, standard normal variate; RMSEV, root mean standard error of validation; Bias indicates systematic errors of measured and predicted values.

Model Number of calibration samples

Number of validation samples

Factors Spectral pre-processing

Smoothing points

RMSEV r2 Bias

1,8-Cineole 156 C-Val 8 MSC + 1stD 25 4.47 0.87 −0.01α-Pinene 156 C-Val 18 MSC + 1stD 25 1.74 0.85 0.03Aromadendrene 156 C-Val 7 MSC + 1stD 17 0.92 0.78 −0.02Globulol 156 C-Val 7 MSC + 1stD 9 0.50 0.78 <0.01Limonene 156 C-Val 6 MSC + 1stD 9 0.53 0.79 <0.01Total oil 156 C-Val 8 MSC + 1stD 25 8.14 0.83 −0.05Chlorogenic acid 154 50 9 MSC + 1stD 9 1.43 0.62 −0.33CTs 154 50 18 Smooth 17 3.52 0.64 0.14TPs 154 50 4 MSC 0 21.33 0.80 −0.90Macrocarpal A 154 50 9 1stD 17 0.39 0.63 0.04Macrocarpal G 154 50 10 1stD 17 1.23 0.64 0.23Late-eluting FPCs 154 50 10 SNV 0 5.33 0.50 −0.24C 120 60 7 MSC + 1stD 9 0.40 0.94 −0.01N 120 60 4 1stD 17 0.20 0.87 0.09

1Samples for model calibration were randomly selected but were chosen to include equal numbers of samples from each species, locality and treatment. The resulting global predictive model for each chemical trait was then used to predict values on samples regardless of species, provenance or treatment.

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Modelling all other chemical traits used 204 samples total, with 154 samples randomly selected for model calibration and 50 samples used for validation. Spectra and chemical data for each trait were checked for clustering at the experiment, species, local-ity, treatment or harvest level using principal component analysis and no clustering was identified. Therefore, a single global model for each trait was used to predict values of samples regardless of experiment, species, locality or treatment. Spectral pretreatment was used to optimize the individual PLS calibration models with most using a combination of multiplicative scatter correction or normalization with Savitzky–Golay smoothing or first derivatiza-tion (Table 2). Models for all chemical traits performed well dur-ing validation (Table 2); for each trait model, the root mean squared error of prediction was only marginally higher than mean lab errors calculated from triplicate samples. Near-infrared spec-tra of remaining samples were used to predict chemical values for each compound in turn. The complete data set (assayed chemical data and NIRS predicted values of this and two unpub-lished experiments) was then separated by experiment.

Statistical analysis

All analyses were completed using SAS statistical software pack-age (version 9.2, SAS Institute Inc., Cary, NC, USA). A component of the data was tested using only a fixed-effects model (species, locality, treatment and sampling time [harvest]; PROC GLM of SAS) including Ψleaf and ABA. A large number of Ψleaf data were collected at Harvest 1, and so species (E. globulus and E. viminalis), locality (KI, ST, QD and SH), treatment (control and water limited) and their interactions were fitted in the model for Harvest 1. Leaf water potential data from Harvest 2 were analysed separately due to the small number of samples (n = 19), with only treatment fitted in the model. Abscisic acid data were collected only at Har-vest 1, and samples were pooled rather than from a single plant, so only species, locality, treatment and their interactions were fitted in the ABA model. For all other traits, species, locality, treat-ment, Harvest (1 and 2) fixed effects and their interactions were tested in a mixed model (PROC GLIMMIX of SAS with a normal residual distribution), where mother within locality for each spe-cies (mother [locality × species]) and interactions between mother (locality × species) and harvest, or treatment, or both (harvest × treatment × mother [locality × species]) were fitted as random effects. Residuals for all variables were checked for assumptions of normality and heterogeneity of variances and no transformations were required. Due to the number of individual mixed model analyses performed (n = 17), the false discovery rate was controlled following Benjamini and Hochberg (2000).

Results

Mid-day Ψleaf

The mean mid-day Ψleaf of water limited plants (−0.88 MPa) was lower than control plants (−0.24 MPa; F1,65 = 56.1; P < 0.001),

indicating minimal plant stress at Harvest 1. No difference in mean Ψleaf was detected between species (F1,65 = 0.02; P = 0.90) or among localities (F3,65 = 1.75; P = 0.15), and no significant interactions were detected. At Harvest 2, no difference was detected between the mean mid-day Ψleaf of control and rewatered plants (F1,10 = 0.21; P = 0.66; mean −0.35 MPa).

Foliar ABA

Water deficit increased foliar ABA levels compared with control plants (1954.2 and 1483.4 ng g−1 DW, respectively; F1,74 = 13.52; P < 0.001), and the foliar ABA levels in both spe-cies were altered similarly by water deficit (no species × treat-ment interaction; F1,74 = 0.33; P = 0.57). The four localities responded differently to mild water deficit (F3,74 = 3.67; P = 0.02; Figure 2) as large increases to foliar ABA levels were observed in leaves of juvenile eucalypts from ST (wet locality; mean 2566.9 ng g−1 DW) compared with controls (mean: 1459.8 ng g−1 DW), yet no significant change in foliar ABA lev-els were detected in the other three localities. No significant species × locality × treatment interaction influenced foliar ABA levels (F3,74 = 0.67; P = 0.58); however, this interaction is pre-sented as Figure 2 to enable species responses to be viewed within the significant locality × treatment interaction.

Results of the main mixed model analysis

The remainder of plant traits (n = 17) was analysed in turn and no significant three- or four-way interactions of the main fixed effects (species, locality, treatment and harvest) were detected for any trait. As such, results of three- and four-way interactions are not presented in Table 3, but see Tables S2 and S3 available as Sup-plementary Data at Tree Physiology Online for least-squares means (±SE). The main effect of harvest and the species × harvest inter-action will not be discussed (although results are included in Table 3) as harvest confounds the effect of time and a changed watering regime. Rather, it is the treatment × harvest interaction that indicates a differential response to water deficit compared with controls and signals a response to rewatering (Table 3).

Genetic-based trait variation

Quantitative genetic differences were detected for all traits at either species or locality level, or both (Table 3). Between spe-cies, LMA was greater in E. viminalis (64.5 g m−2) than E. globulus (59.2 g m−2) and above-ground biomass was greater in E. globulus (33.9 g−1 DW) than E. viminalis (24.5 g−1 DW; Table 3). Eucalyptus globulus leaves contained higher 1,8- cineole (23.43 mg g−1 DW) and chlorogenic acid (3.5 mg g−1 DW) con-centrations than E. viminalis leaves (22.24 and 2.3 mg g−1 DW, respectively). In contrast, E. viminalis leaves contained higher total oil (56.0 mg g−1 DW), limonene (2.11 mg g−1 DW) and globulol (2.13 mg g−1 DW) concentrations compared with E. globulus (total oil: 45.6 mg g−1 DW, limonene: 1.82 mg g−1 DW and globulol: 1.63 mg g−1 DW).

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Among the four localities, concentrations of 1,8-cineole ( Figure 3a), α-pinene (Figure 3b), limonene (Figure 3c), total oil (Figure 3d) and the late-eluting FPCs (Figure 3e) were highest in eucalypts from the wetter locations (KI and ST) compared with the drier locations (QD and SH) regardless of species. However, chlorogenic acid content was lowest in leaves from the wet KI location compared with ST (also wet), and also QD and SH (drier locations; Figure 3f). In contrast to these consistent patterns across species, the concentrations of CTs, TPs and macrocarpals, leaf C : N and lignotuber size differed among localities, and the pattern was different for the two species (spe-cies × locality interaction; Table 3). Specifically, while no varia-tion was detected among E. globulus from the locations, E. viminalis CT concentrations were highest in KI and QD juve-niles (Figure 4a), while TP concentrations were greatest in SH, intermediate in ST and lowest in KI and QD E. viminalis ( Figure 4b). There was minimal variation in macrocarpal concen-trations among localities (Figure 4c and d). Interestingly, macro-carpal A and G concentrations varied between species from ST (higher concentrations in E. globulus leaves) but not from KI, QD or SH (Figure 4c and d). Likewise, C : N of E. globulus from KI, ST and QD was higher than E. viminalis from each respective location, yet no significant difference in C : N was detected between species from SH (Figure 4e). We also detected varia-tion in lignotuber size among localities and species (Table 3), where eucalypts of both species from drier locations (particularly

SH) generally had larger lignotubers than eucalypts from wetter locations (particularly KI), yet with some species variation in the intermediate ST and QD localities (Figure 4f).

Effect of water limitation, rewatering and development on plant traits

The rarity of significant interactions of species or locality terms with the harvest or treatment terms (Table 3) indicates that for most traits, the response to treatment and rewatering was similar regardless of species or locality. Using results of the treat-ment × harvest analysis (Table 3), we first identified decreased LMA (Figure 5a), CT (Figure 5b) and TP (Figure 5c) concentra-tions in the controls at Harvest 2 compared with controls at Harvest 1. These changes are attributed to normal plant growth/development over the 2 weeks ( Goodger et al. 2006, Loney et al. 2006). Second, water limitation increased mean LMA (70 g m−2) and CT (6.6 mg g−1 DW) concentrations above that of controls (64 g m−2 and 5.1 mg g−1 DW, respectively), then rewatering returned LMA and CTs to control levels (Harvest 2; Figure 5a and b). In contrast to LMA and CT responses, leaf water content decreased with water availability (69.1% FW in controls; 64.7% FW in water limited plants) and rewatering increased leaf water content (70.7% FW) slightly above controls (69.6% FW; Figure 5d). A different pattern was observed in the TP, C : N and chlorogenic acid responses to treatments, where water deficit (Harvest 1) did not quantitatively affect these traits,


Figure 2. Least-squares mean foliar ABA levels in leaves of juvenile E. globulus (dark bars) and E. viminalis (light bars) from four localities grown under control or water limited (provided 50% of the control plant evapotranspirational water loss) treatments. Error bars are standard errors (+SE). Letters indicate significant differences (P ≤ 0.05) among all treatment and locality combinations (disregarding species within each treatment) after Tukey’s post hoc tests. No significant species × locality × treatment effect was detected (F3,74 = 0.67; P = 0.58), however the interaction is shown to illustrate species foliar ABA levels in relation to the significant locality × treatment interaction (F3,74 = 3.67; P = 0.02).

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8 McKiernan et al.

Tabl

e 3

. Re

sults

of

mix

ed m

odel

ana

lyse

s fo

r va

riatio

n of

che

mic

al a

nd m

orph

olog

ical

trai

ts in

juve

nile

E. g

lobu

lus

and

E. v

imin

alis

leav

es b

etw

een

spec

ies,

loca

litie

s (K

I, ST

, QD

, SH

), w

ater

trea

t-m

ents

(co

ntro

l, w

ater

defi

cit)

, sam

plin

g pe

riod

(Har

vest

s 1

and

2)

and

two-

way

inte

ract

ions

.1 L

MA

, lea

f m

ass

per

unit

area

; Lig

notu

ber :

ste

m, l

igno

tube

r di

amet

er in

rel

atio

n to

ste

m d

iam

eter

ex

pres

sed

as a

rat

io; C

: N

, the

rat

io o

f C to

N; F

PCs,

form

ylat

ed p

hlor

oglu

cino

l com

poun

ds. N

o si

gnifi

cant

(P ≤

0.0

5)

thre

e- o

r fo

ur-w

ay in

tera

ctio

ns o

f th

e m

ain

effe

cts

wer

e de

tect

ed in

the

full

mix

ed m

odel

ana

lysi

s, a

nd s

o on

ly in

divi

dual

mai

n ef

fect

s an

d tw

o-w

ay in

tera

ctio

ns o

f th

e m

ain

effe

cts

are

pres

ente

d he

re fo

r co

ncis

enes

s.

Trai

tSp

ecie

sLo

calit

ySp

ecie

s ×

loca

lity

Trea

tmen

tTr

eatm

ent ×

ha

rves

tSp

ecie

s ×

treat

men

tLo

calit

y ×

harv

est

Loca

lity ×

treat

men

tH

arve

st2

Spec

ies ×

harv

est2

F 1,4

7P

F 3,4

7P

F 3,4

7P

F 1,4

7P

F 1,4

7P

F 1,4

7P

F 3,4

7P

F 3,4

7P

F 1,4

7P

F 1,4

7P

Plan

t mor

phol

ogy

Abo

ve-g

roun

d bi

omas

s143.9

***

1.6

2.1

32

.9**

*5

.03

3.7

1.1

1.4

22

0.1

***

17

.3**

*

Lign

otub

er :

stem

10.1

**15.2

***

3.7

*0

.80

.53

.20

.20

.68

.3**

0.1

LM

A18.3

***

1.4

0.7

0.9

17

.7**

*0

.11

.61

.21

01

.6**

*1

2.5

***

Leaf

wat

er a

nd e

lem

ents

Le

af w

ater

con

tent

138.9

***

2.1

0.9

42

.0**

*1

09

.2**

*0

.40

.70

.11

49

.2**

*7

.0*

C

: N

57.3

***

3.2

38

.5**

*2

8.7

***

10

.4**

1.8

0.8

2.1

38

.6**

*0

.2Se

cond

ary

chem

istr

yTe

rpen

oids

To

tal o

il89.3

***

7.8

***

1.0

0.1

0.1

1.0

2.2

0.1

7.0

*0

.8

M

onot

erpe

noid

s

1,8

-Cin

eole

8.7

**5.0

**1

.52

.51

.00

.80

.40

.35

4.4

***

0.6

α-

Pine

ne1.7

5.5

**2

.61

.60

.10

.30

.20

.27

.5*

0.0

Li

mon

ene

15.8

***

9.2

***

1.4

1.0

3.2

1.5

0.4

0.1

65

.4**

*6

.03

Sesq

uite

rpen

oids

Aro

mad

endr

ene

82.3

***

1.1

2.3

3.1

0.0

2.6

0.7

0.4

0.6

6.5

*

Glo

bulo

l100.1

***

1.5

3.1

32

.60

.32

.00

.40

.81

.53

.93

Phen

ols

Chl

orog

enic

aci

d73.2

***

15.9

***

2.6

1.3

15

.7**

*2

.91

.31

.31

1.0

**2

.4

Con

dens

ed ta

nnin

s146.5

***

3.2

38

.3**

*6

.0*

9.5

**1

.22

.20

.14

7.7

***

1.8

TP

s115.1

***

6.8

***

9.7

***

2.1

16

.1**

*0

.12

.31

.46

7.5

***

4.7

3

FPCs

La

te-e

lutin

g FP

Cs

0.4

5.4

**2

.93

8.9

**2

.00

.20

.70

.43

.92

.7

Mac

roca

rpal

A0.7

2.5

5.0

**1

3.3

***

1.0

0.1

1.1

0.7

3.4

0.0

M

acro

carp

al G

1.9

2.6

4.4

**1

3.1

***

3.9

30

.21

.20

.80

.12

.1

1Bl

ank

P-va

lues

indi

cate

no

sign

ifica

nce

(P >

0.0

5).

2H

arve

st a

nd th

e in

tera

ctio

n be

twee

n sp

ecie

s an

d ha

rves

t are

not

dis

cuss

ed a

s w

ithou

t the

inte

ract

ion

with

trea

tmen

t, ha

rves

t in

isol

atio

n co

nfou

nds

the

effe

ct o

f tim

e (p

lant

dev

elop

men

t) a

nd c

hang

es

to w

ater

ing

regi

me

(rew

ater

ing

afte

r wat

er d

efici

t).

3Si

gnifi

cant

(P ≤

0.0

5)

afte

r po

st h

oc te

sts

(Tuk

ey’s

) bu

t no

long

er s

igni

fican

t fol

low

ing

cont

rol f

or fa

lse

disc

over

y ra

te (

Benj

amin

i and

Hoc

hber

g 2

00

0).

* P ≤

0.0

5.

**P <

0.0

1.

***P

< 0

.001.

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yet rewatering did (Figure 5c, e and f). Specifically, rewatering (Harvest 2) decreased leaf C : N from 23 : 1 to 19 : 1 (Figure 5f), and concentrations of chlorogenic acid (2.4 mg g−1 DW) and TPs (182.7 mg g−1 DW) in rewatered plants were lower than in controls (3.0 and 201.2 mg g−1 DW, respectively). Concentra-tions of terpenes (mono- or sesquiterpenes) were not altered by the mild water deficit or by rewatering (Table 3).

The effect of 12 weeks water limitation then 2 weeks rewatering on plant traits

The main treatment effect compared all control eucalypts (pooled Harvests 1 and 2 control data) against eucalypts that had altered water availability (pooled water limited [Harvest 1] and water limited/rewatered [Harvest 2] data). The altered water treatment decreased mean plant biomass by 14% compared with controls (Figure 6a) and decreased late-eluting FPC concentrations by 7% (Figure 6b). Likewise, mean macrocarpal A (Figure 6c) and

macrocarpal G (Figure 6d) concentrations were 10–11% lower in altered watering plants compared with controls.

Discussion

We expected the effect of water deficit on both species from the wet localities to be larger than on dry localities based on eucalypt adaptation to local rainfall patterns, and also known variation in adult E. globulus drought tolerance ( Dutkowski and Potts 2012). However, responses (quantitative increases, decreases and stability) to water deficit and rewatering were largely common across species and localities for all measured morphological, physiological and PSM traits. The only exception was the effect of water deficit on foliar ABA levels, where ABA levels in leaves of eucalypts from ST increased, but levels in all other localities remained stable. Foliar levels of the phytohor-mone ABA signal stomatal closure to avoid plant water stress


Figure 3. Least-squares mean concentrations of (a) 1,8-cineole, (b) α-pinene, (c) limonene, (d) total oil, (e) late-eluting FPCs and (f) chlorogenic acid in juvenile E. globulus (dark bars) and E. viminalis (light bars) from four localities (KI, King Island; ST, Southern Tasmania; QD, Queens Domain; SH, St Helens). Error bars are standard errors (+SE), and letters indicate significant difference (P ≤ 0.05) after Tukey’s post hoc test among localities. Con-centrations of 1,8-cineole, α-pinene and chlorogenic acid expressed as mg g−1 DW. Concentrations of total oil and limonene expressed as mg g−1 DW cineole equivalents. Late-eluting FPCs expressed as mg g−1 DW macrocarpal A equivalents. Note different scales on y-axis among individual graphs.

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( Brodribb et al. 2014); thus, it appears that juvenile eucalypts from the cooler and moister ST (Table 1) are either more sensi-tive to mild water deficit than other locations or they exploited the uniform amount of water more quickly than other localities. High foliar ABA levels were not detected in juvenile eucalypts from the second wet site, KI, suggesting that the response of ST Eucalyptus is not solely related to rainfall patterns as initially hypothesized. Adaptation to low temperatures at the ST site may have influenced foliar ABA signalling of ST eucalypts in response to the high vapour pressure deficit within the glass-house ( McAdam and Brodribb 2015b). We did not quantify stomatal conductance given the number of plants involved, and neither Ψleaf nor leaf water content differed among localities

during water deficit. As such, we were unable to define the strategy that eucalypts from ST employed when experiencing mild water deficit.

Genetically determined variation in traits between species and localities

Morphological and chemical traits could be divided into three groups based on patterns of genetically based constitutive vari-ation. These patterns are (1) traits that differed among localities and patterns were consistent across species (Figure 3), (2) traits that differed between species with little variation among localities and (3) traits that varied among localities, but the pat-tern of variation was species specific (Figure 4).

10 McKiernan et al.

Figure 4. Least-squares mean concentrations of (a) CTs, (b) TPs, (c) macrocarpal A, (d) macrocarpal G, (e) C to N ratio (C : N) and (f) the lignotuber to stem ratio in juvenile E. globulus (dark bars) and E. viminalis (light bars) from four localities. Error bars are standard errors (+SE), and letters indicate significant difference (P ≤ 0.05) after Tukey’s post hoc test. Note different scales on y-axis among individual graphs. Condensed tannin results expressed as mg g−1 DW sorghum tannin equivalents. Total phenolic results expressed as mg g−1 DW gallic acid equivalents. Macrocarpals A and G expressed as mg g−1 DW macrocarpal A equivalents.

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Traits that differ consistently among localities (Pattern 1) may be evidence of biophysical limitations relating to climate ( McLean et al. 2014) or geology ( Austin et al. 1990) that differs among these localities and have led to analogous population divergence ( O’Reilly-Wapstra et al. 2010). Primarily, the terpenes followed Pattern 1, and wet localities had higher foliar terpene concentrations than dry localities (Figure 3). Given the wet/dry dichotomy of terpene concentrations among localities, a biotic agent such as high fungal/pathogen levels in wet environments ( Ribera and Zuñiga 2012) may also have played a role in determining these genetic-based patterns. In contrast, traits (biomass, LMA, aromadendrene and globulol concentrations) that differed between Eucalyptus species with

little or no variation among localities (Pattern 2) suggest trait conservation across each species range. Consistent above-ground biomass and LMA among localities may represent lim-ited adaptive benefit of variation in these traits for very young eucalypts, or that variation only becomes evident with plant growth. For example, genetically based variation in height (of 4-year-old plants) and leaf morphology (of 1.5-year-old plants) has been reported among older E. globulus ( Dutkowski and Potts 1999). The concentrations of two sesquiterpenes (aro-madendrene and globulol) did not differ among localities ( Pattern 2), and diversification of genetically based concentra-tions of these compounds may be restricted by biochemical factors ( Keszei et al. 2008) that do not restrict diversification


Figure 5. Least-squares mean (a) LMA, (b) CT concentrations, (c) TP concentrations, (d) leaf water content, (e) chlorogenic acid concentrations and (f) C to N ratio (C : N) of juvenile Eucalyptus (E. globulus and E. viminalis) leaves from plants grown under control (full water—light bars) and water limited (dark bars) treatments. Sampling period (x-axis) refers to plants harvested after 12 weeks (Harvest 1; control or water limited treatments) or after 14 weeks (Harvest 2; control or water limited and then rewatered). Different individual plants were sampled at Harvests 1 and 2. Bars are standard errors (+SE), and letters indicate significant difference (P ≤ 0.05) after Tukey’s post hoc test. Condensed tannin results expressed as mg g−1 DW sor-ghum tannin equivalents. Total phenolic results expressed as mg g−1 DW gallic acid equivalents. Leaf water content expressed as percentage fresh leaf mass (% FW). Note different scales on y-axes.

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of monoterpene and other PSM concentrations in juveniles of these species. While our explanation of Patterns 1 and 2 quan-titative variation is speculative, identifying mechanisms driving trait variation showing Pattern 3 are possibly even more difficult to explain. Genetically based variation in macrocarpal, TP and CT concentrations among juveniles of these species, and pat-terns that vary among localities, indicate a potential interaction of multiple genetic, biochemical and biophysical factors on con-centrations of these PSMs.

Responses to water deficit differed depending on the trait

Water deficit altered traits associated with water stress (Ψleaf and foliar ABA level), decreased above-ground biomass and leaf water content as expected ( Li et al. 2000, McKiernan et al. 2014) and increased LMA as shown in some other eucalypts ( Ayub et al. 2011). More uncertain was the influence that water deficit would have on leaf PSM concentrations, and we found that responses were compound specific and support the hypoth-esis proposed by McKiernan et al. (2014) and tested here. We know that groups of like compounds such as α-pinene, 1,8- cineole and limonene ( Keszei et al. 2010) or macrocarpals ( Pass et al. 1998) share common biosynthetic pathways. As such, common responses to water deficit of compounds with biosynthetic links are expected as opposed to independent com-pound responses. While related compounds (e.g., monoter-penes) may respond similarly to mild water deficit due to biosynthetic association, the level of trait plasticity and the type of response (increased or decreased concentrations) vary among PSM groups. This response variation may result from genetic controls or biochemical limitations that differ between compounds/compound groups.

Plastic changes to plant chemistry during natural water deficit may have ecological impacts. If the changes to FPC (decreased) and CT (increased) concentrations evident in potted juveniles of these species also occur in juveniles/adults during drought peri-ods at each locality, the altered PSM concentrations may flow on to affect the local community. For example, FPCs influence her-bivore tree use and browsing patterns ( Moore and Foley 2005, O’Reilly-Wapstra et al. 2010, Matsuki et al. 2011) and can have antifungal ( Lau et al. 2010, Tian et al. 2014) or antibacterial ( Nagata et al. 2006) properties. Tannins can affect soil microbes ( Ushio et al. 2013), invertebrate mid-gut bacterial communities ( Mason et al. 2015) and mammal food intake ( DeGabriel et al. 2009). However, given the relatively modest changes to phenol (CT and FPC) concentrations observed here during water deficit, the effect of these same changes in naturally growing eucalypts on plant-mediated interactions such as herbivory may be equally modest. Perhaps changes to leaf C : N, decreased leaf water content and retarded plant growth rates during water deficit would have more of an impact on herbivory than the modest changes to CT and FPC concentrations.

12 McKiernan et al.

Figure 6. Least-squares mean (a) above-ground biomass and concen-trations of (b) late-eluting FPCs, (c) macrocarpal A and (d) macrocarpal G in juvenile eucalypts grown under control conditions (water daily to field capacity) throughout two sampling periods (Harvests 1 and 2) or altered water availability (plants grown under 50% water and sampled [Harvest 1] plus plants grown under 50% water then rewatered for 2 weeks at field capacity [Harvest 2]). Bars are standard errors (+SE), and letters indicate significant difference (P ≤ 0.05) after Tukey’s post hoc test. Note different scales on y-axis among individual graphs. Late-eluting FPCs and macrocarpal G expressed as mg g−1 DW macrocarpal A equivalents. Above-ground biomass expressed as g−1 DW.

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Quantitative trait changes due to rewatering after water deficit

Rewatering had contrasting effects on juvenile Eucalyptus leaf chemistry. First, traits that changed quantitatively due to water deficit (increased CT concentration) returned to control levels after rewatering, demonstrating relatively rapid (2 weeks) responses to water availability. Second, rewatering influenced traits not altered by water deficit. Specifically, concentrations of TPs and chlorogenic acid and the C : N decreased during rewa-tering, even though C assimilation rates of eucalypts are over-compensated when plants are rewatered (increase above control plant assimilation rates; Correia et al. 2014). Decreased leaf C : N during rewatering resulted from increasing leaf N content. High leaf N may provide a valuable source of nutrition to herbi-vores after natural drought periods ( McArthur et al. 2003), depending on the protein-binding action of concurrent increases to tannin concentrations in those same leaves. The observed decreases to chlorogenic acid and TP concentrations are linked, as chlorogenic acid is included as a small proportion (∼1.5%) of the TPs. Phenols are natural antioxidants and are utilized during cell rehydration to limit cell damage ( Živkovic et al. 2010). As water deficit increases, levels of reactive oxygen species (ROS) in plant cells also increase, damaging cell components and caus-ing oxidative stress ( Beckett et al. 2012, Voss et al. 2013). This oxidative stress increases further at the beginning of plant recovery as plant metabolism resumes, maximizing ROS abun-dance ( Živkovic et al. 2010). Given the antioxidant properties of phenols, the decrease in TP concentrations (including chloro-genic acid) during rewatering may represent the activity of phe-nolic compounds while scavenging ROS.

Conclusion

Periods of drought are predicted to become more frequent and prolonged in many regions ( Collins et al. 2013). We hypothesized that juvenile Eucalyptus of genetically differentiated provenances from higher rainfall localities would be more susceptible to soil water deficit and would exhibit greater water stress, and that plant traits would be altered to a greater extent than traits of eucalypts from drier localities. We found that while quantitative variation in many constitutive traits among provenances suggests localized adaptation, plastic responses of juveniles are still largely con-served among provenances of these two species. The only evi-dence of intraspecific variation in responses to mild water deficit was the elevation of foliar ABA levels to signal stomatal adjust-ment, yet this intraspecific response pattern may differ with more severe levels of water deficit. The overall uniformity of provenance level responses to mild soil water deficit within juveniles of these two Eucalyptus species would simplify predicting drought impacts on these species if the same response uniformity was to be found in juveniles and mature trees growing at each locality.

Supplementary data

Supplementary data for this article are available at Tree Physiology Online.

Acknowledgments

We thank Stephen Bresnehan at Hobart City Council for access to Queens Domain for seed collection, Michelle Lang and Tracy Winterbottom for glasshouse assistance and Hugh Fitzgerald for invaluable laboratory support. We thank anonymous reviewers for their comments on improving the manuscript.

Conflict of interest

None declared.

Funding

This work was supported by funding from the National Centre for Future Forest Industries (NCFFI) awarded to A.B.M. and an ARC Discovery Grant (DP120102889) awarded to J.M.O.-W.

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