Water relations and hydraulic architecture of two Patagonian steppe shrubs: Effect of slope orientation and microclimate

Journal of Arid Environments 75 (2011) 763e772

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Water relations and hydraulic architecture of two Patagonian steppe shrubs: Effect of slope orientation and microclimate Patricia A. Iogna a, b, Sandra J. Bucci a, b, Fabián G. Scholz a, b, *, Guillermo Goldstein a, c, d a

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina Grupo de Estudios Biofisicos y Ecofisiologicos (GEBEF), Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco, Comodoro Rivadavia, Argentina c Laboratorio de Ecología Funcional, Departamento de Ecología, Genética y Evolución, FCEyN, Universidad de Buenos Aires, Buenos Aires, Argentina d Department of Biology, University of Miami, Coral Gables, P.O Box 249118, FL, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 March 2010 Received in revised form 23 March 2011 Accepted 1 April 2011 Available online 4 May 2011

On a local scale, topography influences microclimate, vegetation structure and the morpho-physiological attributes of plants. We studied the effects of microclimatic differences between NE- and SW-facing slopes on the water relations and hydraulic properties of two dominant shrubs of the Patagonian steppe in Argentina (Retanilla patagonica and Colliguaja integerrima). The NE-facing slope had higher irradiance and air saturation deficits and lower soil water availability and wind speed than the SW-facing slope. Predawn and midday JL and osmotic potentials were significantly lower in shrubs on the NEfacing slope. Osmotic adjustment and more elastic cell walls helped the plants to cope with a more xeric environment on NE-facing slope. Higher water deficits on NE-facing slope were partially compensated by a higher leaf and stem water storage. While stem hydraulic efficiency did not vary between slopes, leaf hydraulic conductance was between 40% and 300% higher on the NE-facing slope. Changes observed in leaf size and in SLA were consistent with responses to mechanical forces of wind (smaller and scleromorphic leaves on SW-facing slope). Morpho-physiological adjustments observed at a short spatial scale allow maintenance of midday JL above the turgor loss point and demonstrate that leaves are more responsive to microclimatic selective pressures than stems. Ó 2011 Published by Elsevier Ltd.

Keywords: Colliguaja integerrima Irradiance Leaf water potential Retanilla patagonica Water transport efficiency Wind Wood density

1. Introduction Distribution of plant species is largely controlled by environmental factors, such as water and nutrient availability, light and temperature which may constrain seed germination, seedling survival, growth, and establishment. Vascular plants from arid and semiarid ecosystems are typically exposed to extreme fluctuations in soil water availability driven by seasonality of precipitations and evapo-transpiration (Schwinning et al., 2005). At a short spatial scale, the microclimate experienced by individual plants can vary substantially from the regional climate. In high and medium latitudes, the slope is a determinant factor in the distribution and characteristics of the vegetation, because it modifies the amount of solar radiation impinging on the soil surface (Geiger, 1965). In the Southern Hemisphere, plants growing on north-facing slope are usually subjected to a drier environment, and a more variable * Corresponding author. Grupo de Estudios Biofisicos y Ecofisiologicos (GEBEF), Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco, Comodoro Rivadavia, Argentina. Tel.: þ54 297154327152. E-mail address: [email protected] (F.G. Scholz). 0140-1963/$ e see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.jaridenv.2011.04.001

microclimate than the relatively mesic south-facing slope. These environmental differences due to slope orientation are stronger in arid and semiarid ecosystems and can be intensified by the reduction in annual precipitation and by the increase in mean annual temperature as expected by climate change model predictions (IPCC, 2007). Vascular plants from arid ecosystems, such as the Patagonian steppes in Southern Argentina, are exposed to low soil water availability and low temperatures. Although Patagonian woody species are typically well adapted to these environmental conditions (Bucci et al., 2009), we do not know how the variations in temperature and soil water availability due to topography affect functional traits and plant responses on contrasting slopes. Effects that climate and topography have on plant water relations and hydraulic architecture have been widely studied at regional scales (Barij et al., 2007), but less information exists on how plants respond to local differences in environmental factors (e.g. water availability, wind or incident solar radiation (Barij et al., 2007; Bucci et al., 2006, 2009; Scholz et al., 2008)). To successfully utilize soil water, plants of the same species that grow across a range of environmental conditions must develop adjustments in biomass allocation and hydraulic architecture to maintain the

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hydraulic compatibility with their environment (Scholz et al., 2008). Intra-specific hydraulic changes in stem and leaf xylem function can also occur in environments with different soil moisture (Bucci et al., 2009; Hacke et al., 2000), nutrient availability (Bucci et al., 2006; Scholz et al., 2008), irradiance or temperature (Brodribb and Holbrook, 2004; Sack et al., 2005) which may have an impact on plant gas exchange and growth (Brodribb and Holbrook, 2004). Plants can respond to increasing soil water deficits in xeric environments by increasing sapwood permeability (KS) and by reducing biomass allocation to leaves relative to sapwood (AL:AS) (Maherali et al., 2004). One important consequence of this adjustment is an increase in leaf specific hydraulic conductivity (KL) and a reduction in the soileleaf water potential gradient for a given transpiration rate preventing that xylem water potential may fall below the level that would trigger drought-induced embolism and stomatal closure. In addition to adjustments in hydraulic architecture, plants growing in stressful environments have mechanisms that help to maintain positive turgor in leaf tissue cells. Osmotic adjustment enables plants to acclimate to drought conditions by regulating osmotic potential with a net increase in cell solute concentration and turgor pressure (Kramer and Boyer, 1995). Changes in cell wall elasticity (increases or decreases) are also known responses to variations in water availability. Plants can also exercise a certain level of control over the rate of evaporative water loss by regulating stomatal aperture and maintaining an adequate water economy under low soil water availability conditions and high atmospheric demands (Franks et al., 2007). Three broad categories of stomatal control can be defined: “isohydric” “anisohydric”, and “isohydrodinamic” responses (Franks et al., 2007). Isohydrics plants exert strong stomatal control to regulate transpiration rates resulting in a similar midday leaf water potential independent of environmental conditions (Tardieu and Simonneau, 1998). Stomata of anisohydric plants are less sensitive to air saturation deficits (D) and soil moisture variations and consequently exhibit relatively large fluctuations in midday leaf water potential (JL). Plants with an iso-hydrodynamic behavior exhibits a strong stomatal control resulting in relatively constant internal water potential gradients, but at the same time allows JL to fluctuate on a seasonal basis in synchrony with soil water potential. Patagonia is rain shadowed from the prevailing westerly winds by the Andean mountains resulting in low precipitations not exceeding in most areas 200 mm per year. The Patagonian desert is quite windy as well, a result of the rain shadow effect and descending cool mountain air. This wind makes Patagonia one of the largest, if not the largest, sources of dust over the South Atlantic Ocean. The Patagonian steppe represents an ideal system for studying the responses of plants to changes in microclimatic conditions as determined by differences on slope orientation due to the prevailing winds from the west. Physiological process in Patagonian shrub species at regional level are influenced, among others factors, by low soil water availability during periods when temperatures are favorable for growth (Austin and Sala, 2002; Bucci et al., 2009; Soriano and Sala, 1983). Small variations in environmental factors affecting water availability and temperature such as radiation and wind as a consequence of topography may result in substantial changes in plant functional traits. In this sense, the objective of this study is to understand how microclimatic differences in adjacent slopes with different orientations but similar slope angles influences the water economy and hydraulic architectural traits of two dominant shrub species with contrasting leaf phenology in the Patagonian steppe. Plants in general exhibit some degree of plasticity to acclimate to differences in environmental conditions; however, it is not known if the traits have different threshold for expressing phenotypic plasticity. One question that we

have tried to answer was if there were substantial differences in acclimation capacity between stem and leaf related traits. We hypothesize that leaves in Patagonian shrub species are more responsive to microclimatic selective pressures than stems. We measured leaf water status, osmotic potentials, bulk elastic modulus, leaf capacitance, wood density and leaf and stem hydraulic properties in Retanilla patagonica and Colliguaja integerrima, during the spring of 2008 and the summer of 2009. 2. Materials and methods 2.1. Site and species The research was carried out in a study area (150 m a.s.l; 45 5700 S and 67 3100 W) 2 km from Comodoro Rivadavia city, Chubut, Argentina. Two sites separated by less than 100 m, but on slopes with different exposures (southwest- (SW) and northeast- (NE) facing) and similar elevation (160 m a.s.l) and slope angles (Table 1) were selected for this study. The precipitation falls mainly in the autumn and the mean annual value is 287 mm. The mean annual temperature is 12.9  C and daily mean temperature ranges from 20  C in January (summer) to 7  C in July (winter) (http//dss.ucar.edu/datasets/ds570. 0/data/resent_files/ssm200810.film). Mean annual wind speed is 27 km h1 with mean maximum values during spring and summer mostly coming from the west at about 43 km h1 (Beeskow et al., 1987) The vegetation is a typical Patagonian herbaceous-shrubby steppe from the Golfo San Jorge District with a high abundance of shrubs covering the slopes that descend to the Atlantic. The evergreen C. integerrima Gillies et Hooker ex Hooker (Euphorbiaceae), and the deciduous R. patagonica (Spegazzini) Tortosa (Rhamnaceae), are among the most conspicuous shrubs on the area and were selected for this study. The percentage of bare soil, coverage of plants, height of vegetation and leaf area index were estimated at 5-m intervals along two transects perpendicular to the slope in each site. Leaf area index was estimated using a Ceptometer AccuPAR LP-80 (Decagon Devices). 2.2. Microenvironmental variables and soil water content Relative humidity and air temperature were measured at each site with sensors connected to dataloggers (HOBOs Pro series; Onset Computer Corporation, Pocasset MA, USA) every 60 s for 10 days during November 2008. During February 3, 2009, relative humidity, air and soil temperature, photosynthetic photon flux (PPF) and wind speed and direction were measured in each site with sensors connected to an automatic acquisition data system (CR10X, Campbell Scientific, Inc). Each CR10X was equipped with a Vaísala HMP35C sensor (Vaísala, Helsinki, Finlandia) to measure

Table 1 Slope, bare soil (%), cover of Colliguaja integerrima, Retanilla patagonica and of other species, average height of the vegetation and leaf area index (LAI). Values are means  SE of three points for the slope and ten points throughout transects for the rest of the variables. Significant differences are indicated as *P < 0.1, **P < 0.05 and ***P < 0.01.

Slope (degrees) Bare soil (%) Cover of R. patagonica (%) Cover of C. integerrima (%) Cover of other species (%) Plant height (cm) LAI

SW-facing slope

NE-facing slope

21.05  0.07 23.45  5.85 3.63  0.00 15.2  3.68 57.78  8.07 56.27  6.36 0.67  0.10

25.84  1.12 63.18  5.88** <1.00*** <1.00*** 36.36  5.84* 46.09  5.41 0.29  0.1**

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relative humidity, two thermocouples (type T) to measure soil temperature at 1 and 5 cm depth, a photodiode calibrated against an AccuPAR LP-80 (Decagon Devices) to measure PPF and a cup anemometer (Met One 034A, Grant pass, OR, USA) that was used to measure wind speed and wind direction. Air saturation deficit (D) was calculated from relative humidity and air temperature measurements. The gravimetric soil water potential was determined in three soil profiles per site during November 2008 and February 2009. The soil samples were collected with a Dutch auger every 5 cm through the first 10 cm of the soil profile, then at 10 cm intervals down to 40 cm and finally at 60 cm depth. Gravimetric soil water content was calculated by comparing fresh and dry weights of the soil samples. Dry weights were obtained after placing the soil samples in an oven at 105  C for 72 h. 2.3. Leaf water potential and pressureevolume relations Predawn leaf water potential (JL) and midday JL were measured with a pressure chamber (PMS1000; Corvallis, Oreg.) during November 2008 and February and April 2009. Ten leafy twigs from different individuals, per species and site, were obtained before dawn and at midday. Predawn JL of five covered (nontranspiring) leafy twigs from five different plants per species and slope were measured simultaneously with predawn JL of exposed leaves during April 2009. Leaves for non-transpiring JL were enclosed in plastic bags and aluminum foil during the late afternoon prior to the measurement day. Pressureevolume curves were determined for four leafy twigs per species and site using the bench drying technique during November 2008 (Tyree and Hammel, 1972). The twigs were cut in the field, recut immediately under water and covered with black plastic bags to avoid dehydration. Samples were non-hydrated to avoid alteration in water relation characteristics observed in species of arid ecosystems (e.g. Meinzer et al., 1986) and in previous studies with the two studied species (Bucci unpublished results). After each determination of balancing pressure with the pressure chamber, twigs were immediately weighed to the nearest 0.001 g and until the next measurement left to transpire freely on the laboratory bench. After all balancing pressureeweight measurements were completed; the twigs were oven-dried at 70  C to a constant mass and weighed. Saturated weights of non-hydrated samples were estimated by determining hydrated/dry weight ratios for parallel samples obtained from the same individual on the same date. Pressure readings, fresh mass at each reading, saturated mass and dry mass for each twig were entered into a pressureevolume relationship analysis program developed by Schulte and Hinckley (1985). The tissue water relation parameters calculated from moisture release curves were osmotic potential at full turgor (p100) and turgor loss point (TLP), relative water content at turgor loss point (RWCTLP), symplastic fraction (SF), maximum bulk elastic modulus (e), solute content and leaf capacitance (Cleaf). The symplastic solute content per unit dry mass was determined as follows: tissue dry mass was subtracted from tissue fresh mass to get tissue water content which was then multiplied by SF to obtain the symplastic water volume. Saturated osmotic potential was converted to osmolality by multiplying saturated osmotic potential by 410 milliosmol MPa1. Osmolality was then multiplied by symplastic water volume and divided by the dry mass of the sample (Tyree et al., 1978). The leaf capacitance was obtained from the slope of the relationship between RWC and JL. Calculation of KLeaf (see below) requires that Cleaf be expressed in absolute terms and normalized by leaf area (Brodribb and Holbrook, 2003). To do this, capacitance calculated from the PV curve was multiplied by the saturated mass

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of water in the sample and then divided by leaf area. In practice, the ratios of leaf dry weight:leaf area and saturated mass of water:leaf dry weight were determined for each species and used to calculate the leaf area normalized absolute capacitance:

Cleaf ¼ dRWC=dJl  ðDW=AL Þ  ðWW=DWÞ=M where DW is leaf dry weight (g), AL is leaf area (m2), WW is mass of leaf water at 100% RWC (g), and M is molar mass of water (g mol1). 2.4. Wood density The density and saturated water content of the terminal branch sapwood were measured using four branch cores per species and slope. Wood density (r) was determined by dividing the dry mass by the fresh volume of the sample as described in Scholz et al. (2007). 2.5. Specific leaf area For specific leaf area (SLA; fresh area/dry mass) determinations, 50e100 fully expanded sun leaves, depending of the total number of leaves per plant, were collected from five individuals per species and slope. After the fresh surface areas (AL) were determined with a scanner, the leaves were oven-dried at 70  C for 72 h and weighed to obtain their dry mass. 2.6. Leaf hydraulic conductance Leaf hydraulic conductance (KLeaf) was determined using the timed rehydration method described in Brodribb and Holbrook (2003), which involved the use of the following equation based on an analogy between rehydrating a leaf and recharging a capacitor:

.
t KLeaf ¼ Cleaf ln JLo =JLf where Cleaf ¼ leaf capacitance (estimated from pressure volume curves), JLo ¼ leaf water potential before partial rehydration, JLf ¼ leaf water potential after partial rehydration and t ¼ duration of rehydration. Branches approximately 50 cm long were collected from plant at dawn before significant transpirational water loss and were transported back to the lab, recut under water and allowed to rehydrate for at least 2 h. Shoots were dried on the bench laboratory for varying lengths of time, placed in a plastic bag and sealed and then kept in the dark for at least 1 h to equilibrate. Measurements of leaf water potential were conducted on excised leafy twigs for initial values (JLo) and for final values after a period of rehydration of t seconds (JLf). The KLeaf value used in this study was the mean value when JLo was over the positive cell turgor range. 2.7. Stem hydraulic conductivity and AL:AS The hydraulic conductivity was measured in four long branches per species and slope collected at midday during November, 2008, re-cut under water to avoid embolisms and transported to the laboratory with both extremes under distilled water. The proximal extreme of each segment was connected to a hydraulic conductivity system (Tyree and Sperry, 1989) using a hydraulic head of 38e49 cm in height and a perfussing fluid of deionized and degassed water. A graduated micropipette (0.001 ml) was connected to the open extreme and when the flow of water was constant, the time required for the meniscus to cross 5 consecutive marks of the pipette graduation (0.005 ml) was recorded. Hydraulic conductivity (kg m s1 MPa1) was calculated as Kh ¼ J/(DP/DX), where J is the flow

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2.8. Statistic analyses The SPSS 11.5 statistical package (SPSS Inc.) was used for statistical analysis. Physiological traits did not show normal distribution and ManneWhitney non-parametric tests were used for assessing for differences within species among slopes. Differences between means for microclimatic variables among slopes were analyzed using t-test. One-way ANOVA was applied to test differences in water content between soil depths within one site. Once it was determined that differences existed among the means, Bonferroni’s pairwise multiple comparison test was applied. Linear regressions between saturated water content vs. wood density and leaf capacitance vs. wood density were calculated using Sigma Plot 11 (Systat Software, Inc.).

Temperature ( o C)

28

Microclimate conditions differed between NE- and SW-facing slopes during spring of 2008 (November) and summer of 2009 (February) (Figs. 1 and 2). The air temperature during November varied from 4 to 28  C and was generally higher on the NE-facing slope during the morning and the early afternoon compared to the SW-facing slope (P < 0.0001; Fig. 1A). The differences in air temperature between slopes were up to 5  C. The relative humidity (RH) followed the inverse pattern of temperature variations during the spring with lower RH during the morning and higher RH during the late afternoon on the NE-facing slope compared to the RH of the SW-facing slope (P < 0.0001; Fig. 1B). However, during the summer, RH was always higher on the SW-facing slope (data not show). Maximum differences in RH between slopes were about 15% in both periods. The air saturation deficit varied from 0 to 3 kPa and was higher on the NE-facing slope (P < 0.01; Fig. 1C). During February 3, wind velocity was 45% higher on SW-facing slope than on NE-facing slope (P < 0.0001; Fig. 2A) and wind direction was predominantly W or SW (78% of the time, data not

60

A

SW NE

A

SW NE

24

3. Results

50

-1

rate through the branch segment (kg s1) and DP/DX is the pressure gradient across the segment (MPa m1). Specific hydraulic conductivity (KS, kg m1 s1 MPa1) was obtained as the ratio of Kh and the cross sectional area of the active xylem, and leaf specific hydraulic conductivity (KL, kg m1 s1 MPa1) was obtained as the ratio of Kh and leaf surface area (AL) distal to the stem section. The active xylem area (AS) for water transport measured in the middle of stem segments was obtained by introducing a dye to stem segments from one cut end.

Wind velocity (km h )

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20 16 12

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90 80

10

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s -1 )

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3

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PPF (µ mol m

1500

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500 1

11/4/08

11/5/08

11/6/08

11/7/08

11/8/08

Time (days) Fig. 1. (A) Temperature ( C), (B) relative humidity (%), and (C) air saturation deficit (kPa) for NE (dashed line) and SW (solid line)-facing slopes during 5 days of November of 2008 (spring).

10 00 11 00 12 00 13 00 14 00 15 00 16 00 17 00

0

0

Ladera SO Ladera NE

90 0

Air saturation deficit (kPa)

Relative humidity (%)

4

Time (h) Fig. 2. (A) Wind velocity (km h1) and (B) photosynthetic photon flux (PPF) (mmol m2 s1) for NE (dashed line) and SW (solid line)-facing slopes during one day in February, 2009.

P.A. Iogna et al. / Journal of Arid Environments 75 (2011) 763e772

shown). The photosynthetic photon flux (PPF) was higher on the NE-facing slope than on SW-facing slope throughout the day (P < 0.0001). Maximum PPF values were recorded near midday on the NE-facing slope and were about 2800 mmol m2 s1, while on the SW-facing slope they were about 1800 mmol m2 s1 (Fig. 2B). Soil water availability and temperature across all depths were also different between slopes (P < 0.05; Fig. 3). The soil water content varied with depth being lower at 5 cm depth than at higher depths (P < 0.001) and between slopes and periods of measurements (November and February) (Fig. 3A). The largest differences between slopes were observed at 10 and 60 cm depth in November 2008 and were up to 5% different. Water content at 60 cm was always higher on the SW-facing slope (approximately 19% in November and 17% in February) than on the NE-facing slope

Soil gravimetric water content (%)

0

5

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Depth (cm)

**

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40 SW(Nov) NE(Nov) SW(Feb) NE(Feb)

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SW(1 cm depth) NE(1 cm depth) SW(5 cm depth) NE(5 cm depth)

Soil temperature (°C)

35 30 25 20 15 10

B 5

8

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10 11

12

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14 15

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Time (h) Fig. 3. (A) Soil gravimetric water content (%) at 5, 10, 20, 30, 40 and 60 cm depth for NE- and SW-facing slopes during November, 2008 (white circle: NE and black circle: SW) and February, 2009 (white triangle: NE and black triangle: SW) and (B) soil temperature ( C) at 1 and 5 cm depth for NE- and SW-facing slopes (white circle: NE at 1 cm depth, white triangle: SW at 1 cm depth, black circle: NE at 5 cm depth, black triangle: SW at 5 cm depth) during February 3, 2009. Each point in (A) represents the mean value  SE of three depth profiles. Statistically significant differences are indicated as **P < 0.05.

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(approximately 15% in November and 10% in February). Soil temperatures at 1 and 5 cm depth were in both cases significantly higher in the NE-facing slope (P < 0.001) (Fig. 3B). At 5 cm depth the temperature differed up to 13  C between slopes, while at 1 cm depth the differences were up to 20  C. Community structure also differed between sites (Table 1). The NE-facing slope exhibited lower plant cover including lower coverage of R. patagonica and C. integerrima. Bare soil represented 63% of the total cover on NE-facing slope, while on SW-facing slope it was only 23%. Mean height of vegetation and leaf area index were higher on the SW-facing slope (56.3  6.4 cm and 0.68  0.10 m2 m2, respectively) than on the NE-facing slope (46.1  5.4 cm and 0.29  0.10 m2 m2, respectively) (Table 1). Predawn leaf water potential (JL) of covered leaves (nontranspiring leaves were 3.1 and 2.65 MPa for R. patagonica and C. integerrima, respectively), did not differ significantly from predawn JL of transpiring leaves (3.0 and 2.5 MPa for R. patagonica and C. integerrima, respectively), suggesting that equilibration along the soil to leaf continuum was achieved at night for the two species on both slopes. R. patagonica had predawn and midday JL significantly more negative (between 3 and 5 MPa, respectively) that C. integerrima (between 2.5 and 3.5 MPa, respectively) in both slopes (P < 0.01; Fig. 4A and B). Predawn and midday JL were significantly more negative in the NE-facing slope than in the SW-facing slope for both species (about 1.0 MPa; P < 0.001). There was no statistical difference in the soil to leaf water potential gradient (DJsoileleaf) between sites (1.2 and 1.1 MPa for R. patagonica and 1.2 and 1.0 MPa for C. integerrima). Turgor loss point (TLP) for both species was significantly more negative in the NE-facing slope (P < 0.001; Fig. 4C). For example, for C. integerrima, TLP was 7.5 MPa on the NE-facing slope while it was 4.5 MPa on the SW-facing slope. Consistent with TLP differences, osmotic potential at full turgor (p100) was significantly more negative in the NE-facing slope for both species (Dp100 ¼ 0.07 and 0.95 MPa for R. patagonica and C. integerrima, respectively) (Fig. 5A). The symplastic solute content on a dry matter basis was higher on the NEfacing slope for both species, but the differences were only significant for R. patagonica (Fig. 5B). The mean TLP for both species was observed at water contents higher than 55% and it was always higher on the SW-facing slope (P < 0.001; Fig. 5C). The bulk leaf modulus of elasticity across species was marginally higher (more rigid walls) in the SW-facing slope (P < 0.1; Fig. 5D). Leaves of C. integerrima were larger than leaves of R. patagonica (P < 0.001) and both species have larger leaf size on the NE-facing slope that on the SW-facing slope (Fig. 6A). The specific leaf area (SLA) varied between 45 and 85 cm2 g1 and R. patagonica had the highest value on both slopes (Fig. 6B). While SLA for R. patagonica was significantly higher in the NE-facing slope, SLA for C. integerrima was higher on the SW-facing slope. Leaf capacitance (Cleaf) varied between 0.1 and 0.6 mol m2 MPa1 and it was higher for R. patagonica (Fig. 7A). On the NEfacing slope Cleaf values were about two times the values of the SW-facing slope (0.52 and 0.3 mol m2 MPa1 for R. patagonica and 0.28 and 0.1 mol m2 MPa1 for C. integerrima, on NE-facing and SW-facing slopes, respectively). Stem wood density was a good predictor of the water storage capacity measured as sapwood saturated water content (y ¼ 201  202x; R2 ¼ 0.69; P < 0.0001). R. patagonica had lower wood density and higher saturated water content than C. integerrima (Fig. 7B and 80  3% and 60  4% of saturated water content, respectively (data not shown)). For both species, wood density was higher on the SW-facing slope, but the differences were only significant for C. integerrima (P < 0.1). There was a negative and marginally significant correlation (y ¼ 3.28  4.56x; R2 ¼ 0.87; P < 0.07) between Cleaf and wood density across individuals and slopes.

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0

-3

*** ***

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Fig. 4. (A) Predawn leaf water potential (MPa), (B) midday leaf water potential (MPa) and (C) osmotic potential at turgor loss point (MPa) (C) for R. patagonica and C. integerrima for NE (white bars) and SW (black bars)-facing slopes during November, 2008. The bars are means values  SE of ten individuals per species and slope. Significant differences are indicated as **P < 0.05 and ***P < 0.01.

Leaf hydraulic conductance (KLeaf) was significantly higher on the NE-facing slope for both species (up to three fold in C. integerrima; Fig. 8A). Nevertheless, despite leaf specific hydraulic conductivity (KL), a branch level measurement of water transport efficiency, tended to be higher on the SE-facing slope, there were no statistical differences between adjacent slopes (Fig. 8B). Similarly, specific hydraulic conductivity and AL:AS (a morphological index of the potential water demand for transpiration in relation to the water transport system; Bucci et al., 2005) did not show significant differences between slopes (data not shown). 4. Discussion This study showed that small variations in environmental factors affecting water availability and temperature such as

SW SO NE NE

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0.15 0.10 0.05 0.00 58

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Relative water content at turgor loss point (%)

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12 10 8 6 4 2 0 R. patagonica

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Fig. 5. (A) Osmotic potential at full turgor, (B) simplastic solute content on a dry matter basis, (C) relative water content at the turgor loss point and (D) bulk leaf modulus of elasticity of R. patagonica and C. integerrima in SW(black bars) and NE (white bars)facing slopes during November, 2008. Each bar represents the mean value  SE of three to four individuals per species and slope. Significant differences are indicated as **P < 0.05.

radiation and wind as a consequence of topography may result in substantial changes in plant functional traits. Microclimatic differences between contrasting slope orientation in the Patagonian steppe had no effects on wood density, neither on stem hydraulic capacity. However, water relations of leaf tissues and leaf hydraulic

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0.6

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Leaf area (cm2)

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0.68 0.66 0.64 0.62

20 0.60 R. patagónica

0 R. patagonica

C. integerrima

Fig. 6. (A) Leaf area and (B) specific leaf area of R. patagonica and C. integerrima in SW (black bars) and NE (white bars)-facing slopes during November, 2008. The bars are mean values  SE of five individuals per species and slope. Significant differences are indicated as *P < 0.1, **P < 0.05 and ***P < 0.01.

conductance were substantially affected by radiation, soil and air temperature, air saturation deficit and soil water content. 4.1. Microenvironment and slope orientation Significant differences in environmental variables in two sites separated by less than 100 m with similar physical soil properties and slope angles but with opposite slope orientation (SW vs. NE) were observed in the Patagonian steppe. Solar radiation dominates the surface energy balance and influences near-surface temperatures, evaporative demand and soil moisture content. In this study, the higher incoming radiation on the NE-facing slope resulted in higher air and soil temperatures and lower soil water availability compared to the SW-facing slope. The SW-facing slope, even though it was exposed to higher wind velocity, had higher vegetation cover, higher soil water content and lower air saturation deficits. Higher plant cover, as observed on the SW-facing slope, should absorb and reflect more solar radiation resulting in less solar energy reaching the ground. On the NE-slope this energy is partitioned into sensible heat, latent heat and soil heat fluxes which are used to heat the air close to the ground, evaporate soil water and heat the soil, respectively. The higher soil water content at the site with high plant cover and lower radiation (SW-facing slope) suggests that transpiration is not the major determinant of the water balance in this arid ecosystem. Transpiration rates are very low and represent 34% of the incoming annual precipitation in Patagonian steppes (Paruelo and Sala, 1995). Consistent with the slope-specific differences in incoming solar radiation, the soil water content in the SW-facing slope was higher

C. integerrima

Fig. 7. (A) Leaf capacitance and (B) wood density of R. patagonica and C. integerrima in NE (white bars) and SW (black bars)-facing slopes during November 2008. The bars are mean values  SE of three to four individuals per species and slope for capacitance and of ten individuals per species and slope for wood density. Significant differences are indicated as *P < 0.05 and ***P < 0.01.

compared to the NE-facing slope. The upper portion of the soil is of crucial biological importance because it is richer in nutrients, receives short-term rainfall pulses, and is the place where decomposition, mineralization and soil carbon dynamics occur. In addition, emergence and survival of seedlings, both key processes for the maintenance of plant abundance (Lauenroth et al., 1994), are mainly controlled by water availability in the top soil layers and the micro-environment close to the ground (Lauenroth et al., 1994). Consequently, small spatial variations in resource availability and physical factors in the upper soil layers and in the soil boundary layer may result in important changes at community and plant levels, as observed in this study between the NE- and SW-facing slopes. Although recent studies have indicated an absence of response to drought in herbaceous plants with roots constrained to the top soil in Patagonia (Cipriotti et al., 2008), this behavior could be different for woody species that have deeper roots and potentially more adaptations for maintaining survival and growth under water deficit conditions. 4.2. Water relations and leaf morphology A previous study showed that Patagonian shrubs exhibit different degrees of stomatal control according with rooting depth (Bucci et al., 2009). Species with shallow roots have an any-sohidric behavior of its minimum JL, while species with deeper roots shown a similar leaf water status (isohydric response) in dry summers and wet summers (Bucci et al., 2009). In the present study, we found that the two species studied have the third mode of response according to Franks et al. (2007) (“isohydrodinamic”), where soil to leaf water potential gradients (DJsoileleaf) was maintained relatively constant

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20

***

Leaf hydraulic conductance -2 -1 -1 (mmol m s MPa )

SW NE 15

*

10

5

0 Specific leaf hydraulic conductivity -1 -1 -1 (Kg MPa m s )

A

B

0.0020

0.0015

0.0010

0.0005

R. patagónica

C. integerrima

Fig. 8. (A) Mean daily leaf hydraulic conductance and (B) specific leaf hydraulic conductivity of R. patagonica and C. integerrima in SW (black bars) and NE (white bars)facing slopes during November, 2008. The bars in (A) are mean values  SE of leaf hydraulic conductance in the range of positive turgor obtained from leaf vulnerability curves. Bars in (B) are mean values  SE of three to four terminal branches per species and slope. Significant differences are indicated as *P < 0.1 and ***P < 0.01.

between sites with different soil and atmospheric moisture, despite minimum leaf water potential (JL) being more negative on the NEfacing slope. We consider predawn JL of both species as a proxy for water potential in the soil explored by roots as long as there is no nocturnal transpiration, which is the case with both species as observed by comparing covered and uncovered predawn JL (Bucci et al., 2004). This similar DJsoileleaf suggests that the driving force for water uptake was similar in plants growing in sites with different soil moisture; therefore, other physiological mechanisms such as utilization of stored water could contribute to plant water relations in the drier site maintaining the homeostasis in DJsoileleaf. Across sites it was observed that both species have the capacity to adjust leaf tissue physiological characteristics. This was revealed by a decrease in osmotic potential at saturation and at turgor loss point (TLP) and by an increase in osmotically active solutes and the elasticity of tissues in the NE-facing slope plants. Even though osmotic adjustment can be energetically costly due to organic or inorganic solute accumulation particularly in an ecosystem poor in nutrients such as Patagonia (Austin et al., 2004), relatively low JL in plants on NE-facing slope is required for extracting water from drier soil (the NE-facing slope had relatively low soil water content). Osmotic adjustment can be advantageous in ecosystems characterized by summer rain pulses such as in Patagonia (Fravolini et al., 2005), where a rapid response to water availability is needed. On the other hand, a higher elasticity of tissues (lower elasticity modulus) allowed the plants to maintain turgor at reduced water content. The ability to osmotically adjust or change cell elasticity with changes in environmental conditions has been observed in plants from several arid or semiarid ecosystems (e.g. Mitchell et al., 2008). However, in some ecosystems where there is a strong seasonality in precipitation, such as tropical savannas, plants do not

exhibit osmotic adjustments (Bucci et al., 2008) because most woody species are isohydric (maintain similar minimum JL throughout of year) due to a strong stomatal control of transpiration water losses, reduction in total leaf area and by roots that have access to more stable water source in the soil (Bucci et al., 2008). Mechanisms of drought tolerance are also related to leaf morphology and anatomy. For example, species with higher seasonal changes in the TLP (Mitchell et al., 2008) or with more negative TLP (Bucci et al., 2004) have more scleromorphic leaves (low specific leaf area, SLA). We found that the species with the lowest SLA (C. integerrima) also had the largest shifts in TLP between slopes with different soil water availability (3 MPa more negative on NE-facing slope). This relationship between TLP and SLA across species suggests that turgor maintenance may be crucial to protect leaves with large structural costs under water limitations. On the other hand, a consistent relationship between SLA and TLP was not observed across sites, probably due to the effects of other physical factor such as wind that may play a role in determining leaf physiology and morphology of species exposed to the strong winds common in Patagonia. Consistent with our finding, Mishio et al. (2007) observed that leaves of Boninia grisea exposed to strong wind and low radiation has lower SLA than leaves protected from wind and with higher radiation levels. Other studies found also that wind affects size leaf being leaves smaller in sites with high wind speed (Niklas, 1996; Smith and Ennos, 2003). 4.3. Leaf and stem hydraulics traits Resource allocation related to adjustments in water transport efficiency may be affected by irradiance. Simple adjustments in plant architecture, such as a reduction in the leaf: sapwood area ratio (AL:AS), can enhance water transport efficiency per unit leaf area (Bucci et al., 2005). In this study, the changes in hydraulic architecture did not significantly affect the stem specific hydraulic conductivity (KS) or the pattern of carbon allocation to photosynthesis vs. sapwood tissues; nevertheless there was an increase in the leaf hydraulic conductance (higher KLeaf) in the site with higher radiation. While coordinated changes between stem and leaf specific hydraulic conductivity (both hydraulic architectural features related to long distance water transport to the leaves) and leaf hydraulic conductance (an hydraulic architectural feature related to water transport within the leaves) have been observed in other ecosystems such as Neotropical savannas (Bucci et al., 2008), in this study with two shrub species such coordination between KS or KL and KLeaf was not observed. The hydraulic architecture of R. patagonica and C. integerrima were adjusted to the higher radiation and higher D on the NE-facing slope through changes in KLeaf. We assumed that genetic differences among plants of the same species growing in both slopes were relatively small and that the responses to microenvironmental variables were substantially larger than intra-specific genotypic differences. There is a great body of evidence that the overall KLeaf is substantially affected by light, but as yet the mechanisms of changes in KLeaf as a function of light levels are poorly understood (Nardini et al., 2005). For example, Lo Gullo et al. (2005) showed that KLeaf was positively correlated with PPF in evergreen and deciduous trees. In laboratory experiments, Sack et al. (2002) demonstrated that KLeaf of Quercus leaves was larger under high irradiance (>1200 mmol m2 s1) than that measured under ambient light condition. Here we found that KLeaf increased up to three times in the site with higher irradiance. The positive response of KLeaf to radiation and air temperature could be the result of a more effective leaf vascular system characterized by a higher vein density, vessel size or conduit density, high permeability of the symplastic pathway, changes in water viscosity (Sack and Frole, 2006) and/or

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by abundance of aquaporins (Nardini et al., 2005). The temperature and irradiance dependent adjustments in KLeaf could contribute to the coordination between liquid and gaseous phase conductance because well exposed plants, having higher leaf temperature (as a consequence of high radiation) will require a more efficient water supply system to compensate for a higher rate of transpiration than plants growing on sites with lower irradiance (Sperry et al., 2003). In this sense the increase in efficiency of water transport within leaves could help to maintain a stable soil to leaf water potential gradient, even though the transpiration rate could increase as a consequence of higher D on the NE-facing slope plants. Increasing water supply to maintain favorable tissue water status has the advantage of allowing continued photosynthesis but it may lead to rapid depletion of soil water reserves. In addition to drought tolerance mechanisms, the two species have adaptations to escape low soil water availability at the end of summer. C. integerrima has relatively deeper roots systems (Bucci et al., 2009) and R. patagonica is a deciduous species that drops its leaves during the summer. These adaptations should allow them to maintain high KLeaf in response to higher radiation and soil water deficits. We are hypothesizing that changes in KLeaf is one of the main responses of plants to changes in the physical environment at short spatial scales. 4.4. Leaf capacitance and wood density Water storages in leaves and stems of many woody species from different ecosystems are correlated with a suite of hydraulic traits (Bucci et al., 2004; Meinzer et al., 2008; Scholz et al., 2007). For example leaf capacitance (Cleaf) is positively correlated with KLeaf (Sack and Tyree, 2005) and stem capacitance is associated with apparent leaf area specific hydraulic conductance in the soileleaf pathway (Scholz et al., 2007) and with sapwood area specific hydraulic conductance in the soil to terminal branches (Meinzer et al., 2003). Although leaf water storages contribute only a small percentage of the total transpired water on a daily basis, it can help to buffer the imbalance between water demand by the transpiring leaves and the soil water sources (Sack and Tyree, 2005). In this study a high Cleaf helped plants to prevent JL to fall below the TLP and allowed the maintenance of a relatively high KLeaf at the slope with higher D and lower soil water availability. Even though wood density is considered to be species-specific it can vary under different growing conditions, as observed in this study. Generally, wood density is considered to be a proxy of water storage in branches and trunks (Bucci et al., 2004; Stratton et al., 2000) and is negatively correlated with stem sapwood capacitance (Scholz et al., 2007). The high metabolic cost of having dense wood and consequently a low stem capacitance is generally associated with lower efficiency in the water transport system (Bucci et al., 2004; Stratton et al., 2000) and higher resistance to cavitation (Hacke et al., 2001; Pratt et al., 2007). However, in this study there was no trade off between wood density and stem water transport capacity, suggesting that anatomical changes that affected wood density have no effects on stem related hydraulic traits. Despite the fact that Patagonia is a region characterized by very strong winds, there are no studies evaluating the effects of this mechanical perturbation on plants morpho-physiological traits in this ecosystem. Plants growing on windy sites tend to have, per example, a highly tapered shape, increased radial growth or more resources invested into production of stronger wood for efficiently resisting mechanical forces and maintaining the mechanical stability (Niklas, 1993; Telewski, 1990). Here we found that at the expense of lower water storage capacity plants on wind exposed slope had a higher mechanical strength (denser wood). This finding suggests that wind could be an important factor in determining mechanical properties of Patagonian steppe woody plants.

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5. Conclusions The microclimate and vegetation structure differed significantly despite the short distance between the NE- and SW-facing slopes study sites. The higher radiation and D and the lower soil water availability on the NE-facing slope resulted in relatively higher water deficits and drought. These differences observed in the microclimatic variables at a short spatial scale influenced the characteristics of the community, the water relations, and the hydraulic architecture of the two woody species studied. Plants on the NE-facing slope exhibited higher drought resistance (osmotic adjustment and changes in the elastic properties of the tissues), leaf hydraulic efficiency, leaf capacitance and stem water storage which could help to buffer the higher water deficits. On the other hand, there were no differences in the stem hydraulic properties between plants growing on the two different slope orientations. These findings suggest that changes in leaf traits associated with improved drought resistance are not necessarily coupled to coordinated changes in stem hydraulic properties. Morphological leaf traits (smaller leaves and more scleromorphic on the SW-facing slope plants) were more consistent with responses to high wind (mechanical stimuli) than responses to high radiation or low water availability on the NE-slope. Overall our results indicate that leaves of Patagonian shrubs are more responsive to microclimatic selective pressures than stems and consequently the leaf characteristics may provide a better understanding of plant eco-physiological responses than stems. References Austin, A., Sala, O., 2002. Carbonenitrogen dynamics across a natural precipitation gradient in Patagonia, Argentina. Journal of Vegetation Science 13, 351e360. Austin, A.T., Yahdjian, L., Stark, J.M., Belnap, J., Porporato, A., Norton, U., Ravetta, D.A., Schaeffer, S.M., 2004. Water pulses and biogeochemical cycles in arid and semiarid ecosystems. Oecologia 141, 221e235. Barij, N., Stokes, A., Bogaard, T., Van Beek, R., 2007. Does growing on a slope affect tree xylem structure and water relations? Tree Physiology 27, 757e764. Beeskow, A.M., Del Valle, H.F., Rostagno, C.M., 1987. Los sistemas fisiográficos de la región árida y semiárida de la provincia del Chubut. S. C de Bariloche SECYT Delegacion Regional Patagonica, p. 173. Brodribb, T.J., Holbrook, N.M., 2003. Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiology 132, 2166e2173. Brodribb, T.J., Holbrook, N.M., 2004. Stomatal protection against hydraulic failure: a comparison of coexisting ferns and angiosperms. New Phytologist 162, 663e670. Bucci, S.J., Goldstein, G., Meinzer, F.C., Scholz, F.G., Franco, A.C., Bustamante, M., 2004. Functional convergence in hydraulic architecture and water relations of tropical savanna trees: from leaf to whole plant. Tree Physiology 24, 891e899. Bucci, S.J., Goldstein, G., Meinzer, F.C., Franco, A.C., Campanello, P., Scholz, F.G., 2005. Mechanisms contributing to seasonal homeostasis of minimum leaf water potential and predawn disequilibrium between soil and plant water in Neotropical savanna trees. Trees 19, 296e304. Bucci, S.J., Scholz, F.G., Goldstein, G., Meinzer, F.C., Franco, A.C., Campanello, P.I., Villalobos-Vega, R., Bustamante, M., Miralles-Wilhelm, F., 2006. Nutrient availability constrains the hydraulic architecture and water relations of savanna trees. Plant Cell and Environment 29, 2153e2167. Bucci, S.J., Scholz, F.G., Goldstein, G., Hoffmann, W.A., Meinzer, F.C., Franco, A.C., Giambelluca, T., Miralles-Wilhelm, F., 2008. Controls and stand transpiration and soil water utilization along a tree density gradient in a Neotropical savanna. Agricultural and Forest Meteorology 148, 839e849. Elsevier. Bucci, S.J., Scholz, F.G., Goldstein, G., Meinzer, F.C., Arce, M.E., 2009. Soil water availability and rooting depth as determinants of hydraulic architecture of Patagonian woody species. Oecologia 160, 631e641. Cipriotti, P.A., Flombaum, P., Sala, O.E., Aguiar, M.R., 2008. Does drought control emergence and survival of grass seedlings in semi-arid rangelands? An example with Patagonian species. Journal of Arid Environment 72, 162e174. Franks, P.J., Drake, P.L., Froend, R.H., 2007. Anisohydric but isohydrodynamic: seasonally constant plant water potential gradient explained by a stomatal control mechanism incorporating variable plant hydraulic conductance. Plant Cell and Environment 30, 19e30. Fravolini, A., Hultine, K.R., Brugnoli, E., Gazal, R., English, N.B., Williams, D.G., 2005. Precipitation pulse use by an invasive woody legume: the role of soil texture and pulse size. Oecología 144, 618e627. Geiger, R., 1965. The Climate Near the Ground. Harvard University Press, Cambridge, Mass. Hacke, U.G., Sperry, J.S., Ewers, B.E., Ellsworth, D.S., Schafer, K.V.R., Oren, R., 2000. Influence of soil porosity on water use in Pinus taeda. Oecologia 124, 495e505.

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