Hydraulically integrated or modular? Comparing whole-plant-level hydraulic systems between two...

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142 New Phytologist (2009) 183: 142–152 © The Authors (2009)142 www.newphytologist.org Journal compilation © New Phytologist (2009)

Blackwell Publishing LtdOxford, UKNPHNew Phytologist0028-646X1469-8137© The Authors (2009). Journal compilation © New Phytologist (2009)282810.1111/j.1469-8137.2009.02828.xMarch 200900142???152???Original ArticleXX XX

Hydraulically integrated or modular? Comparing whole-plant-level hydraulic systems between two desert shrub species with different growth forms

Susana Espino and H. Jochen SchenkDepartment of Biological Science, California State University Fullerton, PO Box 6850, Fullerton, CA 92834-6850, USA

Summary

• Hydraulic systems of shrubs vary between hydraulically integrated and modulararchitectures; the latter divide the shrub into independent hydraulic units. Hydraulicsystems of two common North American desert shrub species, the multi-branchedAmbrosia dumosa and the single-stemmed Encelia farinosa (both Asteraceae), werecompared to test for division into independent hydraulic units and the implicationsof such a division for water loss through leaves and roots.• Hydraulic systems of mature shrubs in the field were characterized using dyetracers and by documenting the degree of stem segmentation. Young pot-grownshrubs were subjected to heterogeneous and homogeneous watering. Spatial within-canopy variation of leaf water potentials and stomatal conductances, as well as soilwater contents, were measured in response to manipulated soil water heterogeneity.• Results show that young Ambrosia shrubs are divided into independent hydraulicunits long before they physically split into separate ramets as mature shrubs, and thatyoung and mature Encelia shrubs possess integrated hydraulic systems. No hydraulicredistribution was detected for eitherspecies.• Our study shows that functional segmentation into independent hydraulic unitsprecedes physical axis splitting, rather than being the consequence of split axes, andsuggests that mature shrubs with round basal stems are likely to be hydraulicallyintegrated.

Abbreviations: CV, coefficient of variation; F, hydraulic integration index defined inEqn 2; gs, stomatal conductance (mmol m−2 s−1); HM, homogeneous; HT, hetero-geneous; IHUs, independent hydraulic units; S, stem segmentation index definedin Eqn 1; T, temperature (°C); Tsoil, soil temperature (°C); WT, watering trial; θv,volumetric soil water content (vol.%); θv,pre, mean θv (vol.%) at 04.00 h in the maincompartment averaged over the 3 d before watering; θv,post, mean θv (vol.%) at04.00 h in the main compartment averaged over the 3 d after watering; θv,diff,θv,post − θv,pre; Ψleaf, predawn leaf water potential (MPa); Ψsoil, soil water potential(MPa).

Author for correspondence:Susana EspinoTel: +1 657 278 5288Email: [email protected]

Received: 21 November 2008Accepted: 15 February 2009

New Phytologist (2009) 183: 142–152 doi: 10.1111/j.1469-8137.2009.02828.x

Key words: Desert shrubs, hydraulic architecture, hydraulic integration, hydraulic redistribution, independent hydraulic units (IHUs), leaf water potential, plant water relations, stomatal conductance.

Introduction

For the growth and survival of plants, it is essential to maintainan uninterrupted water transport system throughout thexylem (Pockman & Sperry, 2000). Desert plants, in particular,have developed adaptations that prevent hydraulic failurecaused by the introduction of air bubbles (emboli) into thexylem by resisting the formation of embolisms, repairingembolisms and maintaining hydraulic redundancy (Holbrook

& Zwieniecki, 1999; Cruiziat et al., 2002; Sack et al., 2008).Some shrubs are structurally segmented and divided intoindependent hydraulic units (IHUs) (Schenk, 1999), leadingto a modular type of hydraulic redundancy. Segmentation orphysical splitting of stems and roots, also known as axissplitting (Fahn, 1964; Jones & Lord, 1982), is known to bethe dominant growth form in arid ecosystems (Schenk et al.,2008). Although this phenomenon appears to be a commonoccurrence in desert ecosystems, there has been remarkably

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little study of the effects of the development of functionaland structural axis splitting on canopy-level physiology. Thepurpose of this study was to test for division into IHUs in twodesert shrub species, Ambrosia dumosa A. Gray Payne andEncelia farinosa A. Gray ex Torr, document the effects of sucha division on canopy water relations in young plants, and testthe hypotheses that division into IHUs reduces water lossthrough leaves and roots.

Woody plants from dry environments that divide intoIHUs can be exceedingly long lived, ranging from manydecades to hundreds, even thousands, of years (Vasek, 1980;Schenk, 1999; Larson, 2001). Division into IHUs couldpotentially increase the chance of genet survival during adrought by concentrating water from small pockets of soilmoisture in a single IHU, and thereby allowing IHUs tosurvive, rather than spreading the water out over a wholewater-stressed canopy (Jones, 1984). Other potential benefitsof IHUs include a reduced loss of water to the soil as a resultof lower hydraulic redistribution, because of the lack ofhydraulic connections between roots with access to water androots in dry soil, reduced loss of water through stomata byallowing independent stomatal regulation, and restrictedspread of embolisms (Tyree & Sperry, 1998) and pathogens(Chatelet et al., 2006). In this article, we focus on the rela-tionship between division into IHUs and water loss throughleaves and roots.

Ambrosia dumosa and E. farinosa are very common andoften coexisting plant species in the Sonoran and MojaveDeserts of North America, and are similarly sized, drought-deciduous semi-shrubs in the Asteraceae. Differences betweenthem include a shorter lifespan, larger leaves and single basalstems in E. farinosa, compared with greater longevity, smallerleaves and branching from the ground in A. dumosa (Muller,1953). Ambrosia dumosa tends to split along its axes intoseparate ramets as it matures (Jones & Lord, 1982), withplants of more than 0.5 m in diameter being normally split(Schenk, 1999), and mature shrubs have been found torespond to heterogeneous watering by developing a highlyheterogeneous water status in the canopy (Jones, 1984). Itremains an open question as to whether physical splittingin this species is necessary to achieve a functional divisioninto IHUs, or whether it is merely a consequence of possessinga wood anatomy that functionally divides a young planteven before the axes split (Schenk, 1999). Encelia farinosashrubs are very different from A. dumosa in that they possesssingle, round, basal stems (Schenk, 1999; Schenk et al.,2008), a trait that is more typical for shrubs from wetenvironments, in which it is normally associated with ahigh degree of hydraulic integration (Schenk et al., 2008).However, the round stems of E. farinosa could be anatomicallydivided into separate IHUs. The two species stood out in aprevious study (Schenk et al., 2008) among all desert shrubsas possessing the most extreme opposites in their degree ofstem segmentation.

This study was designed to compare the hydraulic systemsof A. dumosa and E. farinosa plants, including young plants ofboth species with undivided stems, predicting that youngA. dumosa shrubs would be divided into IHUs and thatE. farinosa would have an integrated hydraulic system. Wetested the hypotheses that, in the presence of heterogeneouswatering, a division into IHUs would cause water not to beshared among all canopy branches, creating greater waterstatus variability within the canopy, and causing lower overallconductance, as several branches would be at lower water statuswhen compared with a homogeneous watering treatment.We also tested the hypothesis that division into IHUs woulddecrease the degree of hydraulic connectedness among rootsand thereby reduce water loss from the roots as a result ofhydraulic redistribution.

Materials and Methods

Field studies of mature Ambrosia and Encelia shrubs

The degree of physical segmentation of the stems and thehydraulic integration of five shrubs of each species, A. dumosaand E. farinosa, with canopy diameters of greater than 0.5 m,were investigated in January 2005 at a field site on thesouthern slope of the Cottonwood Spring Mountains in thetransition area between the Mojave Desert and the SonoranDesert in California, using the methods described in Schenket al. (2008). Briefly, for each shrub, a single lateral woodyroot with a diameter of c. 4 mm (± 2 mm) was excavated at asoil depth between 0.1 and 0.3 m, and inserted into a vialcontaining acid fuchsin dye (0.5% in water) for 24 h. Theshrub was then cut at the most compact section of the basalstem, usually right at the soil surface, and digital photographsof transverse sections of the basal stem (e.g. Fig. 1) at thispoint were analyzed using the software SigmaScan Pro(version 5.0, SPSS Inc., Chicago, IL, USA). Shrub growthforms were characterized by measurements of canopy heightand widths, and canopy volume (calculated as an ellipsoid). Thedegree of physical segmentation of shrubs was characterizedby the relationship between the cross-sectional area andperimeter of the basal stem’s living sapwood and associatedundecayed heartwood at the basal stem’s most compact point,using the formula:

Eqn 1

(p, stem perimeter; A, basal stem cross-sectional area, suchthat S = 1 for a perfectly circular area). The degree ofhydraulic integration within the basal stem was characterizedby measuring the fraction of basal stem cross-sectional area(without bark) colored by dye and calculating a hydraulicintegration index as:

F = d/A Eqn 2

S p= ( )π π/ Α2


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(d, area of stem cross-section colored by dye; A, total area ofthe basal cross-section).

Controlled pot experiments of young Ambrosia and Encelia shrubs

To investigate the hydraulic architecture of young Ambrosiaand Encelia plants, an experiment was designed to grow youngplants under conditions of manipulated soil water heterogeneityat a soil depth of 0.2–0.5 m (Barbour et al., 1977; Nobel,1997). Dormant young Encelia and young Ambrosia shrubs(canopy diameters, < 0.24 m) with round basal stems thatshowed no external signs of axis segmentation were collectedin September 2005 from the same location as used for thefield studies described above. They were transported to the

outdoor area of the glasshouse complex at California StateUniversity Fullerton and planted into pots made of polyvinylchloride pipes (1.2 m tall, 0.24 m diameter; see Fig. 2). Fiftypots were placed vertically within a 17-m2 area in 10 groupsof five pots each (Fig. 2b), which were held together in a circle(Fig. 2c), with groups spaced c. 0.6 cm apart. Pots were filledto the top of the pot with a mixture of soil and washed sand,resulting in a texture of loamy sand similar in texture to thatfrom the field site, with 10.3% clay, 3.7% silt and 86.0%sand, and a field capacity of c. 14% volumetric soil moisturecontent (θv, %). The field capacity, defined as the soil moisturecontent at a soil water potential of 0.033 MPa, was calculatedfrom the soil texture data according to Saxton et al. (1986).Each group included one control pot without a plant [inthe heterogeneous (HT) or homogeneous (HM) watering

Fig. 1 Transverse sections of basal stems of mature shrubs of Ambrosia dumosa (top) and Encelia farinosa (bottom) cut at their most compact point. To better show the shape of xylem, bark and phloem were edited out of the images using Photoshop software (Adobe Systems, version 7.0). Acid fuchsin dye-tracer distribution is shown in purple.

Fig. 2 (a) Design of pots for controlled heterogeneous and homogeneous watering treatments. (b) Set-up of the pots in 10 groups of five pots. (c) Close-up view of the top of a group of five pots with the no-plant control covered to prevent the infiltration of water.



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treatment], two pots with one Encelia each (one each for theHT and HM treatments), and two pots with one Ambrosiaeach (one each for the HT and HM treatments). Half of the10 controls and half of the Encelia and Ambrosia shrubs(20 Ambrosia and 20 Encelia) were randomly assigned tothe HT treatment; the other half were assigned to the HMtreatment.

Each pot included an internal cylindrical compartment(28 cm tall, 12.7 cm diameter) attached to the interior side ofthe pot, with its upper opening 19 cm from the top of the potand top of the soil (Fig. 2a), creating a heterogeneous soilwater environment in the HT watering treatment. For HTwatering treatments, internal compartments were closed atthe bottom, whereas, in HM treatments, they were sealedwith Nitex nylon cloth (TETKO, Inc., Briarcliff Manor, NewYork, USA) with 25-µm openings to restrict root growth(as in the HT treatment), but to allow water flow out of theinternal compartment. Three irrigation tubes (0.6 cm indiameter) were placed vertically from the top of the soil surfacedown to 25 cm, and their bottom halves were connectedto porous irrigation tubes closed at the lower ends. One tubewas placed so that it delivered water directly to the internalcompartment, and the other two delivered water to the maincompartment. A layer of small river rocks was placedbetween 15 and 19 cm from the top, just above the internalcompartment, to decrease upwards and lateral capillary move-ment of water.

Measurements of soil water contents

Each pot was equipped with at least two soil moisture probes(model ECH2O-10, Decagon Devices Inc., Pullman, WA,USA), inserted parallel to the ground through slits cut into thepot, and θv was recorded every 30 min. Both probes weresituated 30 cm from the top of the pot, one centered withinthe internal compartment and the second directly opposite thefirst, in the main compartment (Fig. 2a). In addition, 10Encelia and 10 Ambrosia pots were randomly selected for theinsertion of three additional sensors. One was inserted verticallyinto the soil surface and therefore integrated measurements

over the top 10 cm; the other two were placed parallel to theground at depths of 60 and 90 cm (Fig. 2a).

Soil moisture probes were calibrated for effects of soil typeand soil temperature (T) by placing them into containers withthe soil mixture with a range of gravimetrically determined θv,and by placing them into a growth chamber and graduallyincreasing T. The calibration equation [θv as a function ofprobe output (in mV) and T] was created by nonlinearregression using the software TableCurve 3D (version 3, SPSSInc., Chicago, IL, USA). Soil temperature data for the potexperiment were only available after 2006, and therefore themissing Tsoil data for 2006 were estimated from an empiricalrelationship developed using TableCurve 3D between datafor minimum daily T of the current day, maximum daily Tof the previous day at Fullerton Airport, and existing Tsoilmeasurements at 04.00 h Pacific Standard Time.

Watering treatments

For 6 months after planting, all plants were watered frequentlyand exposed to natural rainfall to facilitate plant establishmentand promote vigorous root growth throughout the pots andinternal compartments. On 1 April 2006, watering wasdiscontinued and shrubs were covered during subsequent rainevents to allow the soil to dry. Root activity in the compartmentswas evaluated by monitoring changes in θv in the internaland main compartments. The presence of active roots ina compartment was detected as a decrease in θv during thedrying-out period, whereas a lack of decrease in θv wasinterpreted to indicate the absence of active roots in thecompartment. Consequently, 12 shrubs per species (half inthe HM and half in the HT treatments) were chosen fromthe initial 20 because they appeared to have roots in bothcompartments. Watering trials (WTs) and measurementsof plant water status began on 9 July 2006 (Table 1), after θvhad declined to less than 2% in all replicates, becauseprevious measurements had shown a θv value of 2% to be theminimum water content in these soils (Fig. 3). For eachmeasurement trial, 24 shrubs were measured within a 3-dperiod; per day, eight randomly selected shrubs were measured.

Table 1 Dates for measurements of stomatal conductance, leaf water potentials and volumetric soil water contents for hydraulic redistribution experiments (watering treatments were applied during the evenings before the dates listed)

Watering trial

Dates measured

Shrubs 1–8 Shrubs 9–16 Shrubs 17–24

Initial (no watering) 10 July 2006 11 July 2006 12 July 20061 13 July 2006 14 July 2006 15 July 20062 10 August 2006 11 August 2006 12 August 20063 24 August 2006 25 August 2006 26 August 20064 11 September 2006 12 September 2006 13 September 20065 25 April 2007 (no shrubs measured, soil water contents only)6 24 July 2008 (no shrubs measured, soil water contents only)



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Six watering treatments (WT 1–6, see Table 1) were appliedvia porous watering tubes (Fig. 2a) using a 60-ml syringe theevening before the day on which the measurements of plantwater status were taken. Watering amounts for both treatmentswere determined as the volume of water that would bring θvin the internal compartment up to field capacity. In HTtreatments, this volume was added only to the internalcompartment, bringing it up to near field capacity. In HMtreatments, this volume was evenly distributed over the internaland main compartments via the three watering tubes, therebycreating the same below-field capacity θv in the internal andmain compartments (Fig. 2).

Measurements of predawn leaf water potentials (Ψleaf) and leaf stomatal conductance (gs)

The psychrometric dew-point technique was used to measureΨleaf (in MPa) at 04.00 h Pacific Standard Time using a water

potential system (model HR-33T, Wescor Inc., Logan, UT,USA) and psychrometric sample chambers. Five leaves ofAmbrosia or portions of five leaves for Encelia (c. 1 cm2) werecollected from four different compass directions on the plant(N, E, S, W) and from the top of the canopy for psychrometrymeasurements. Psychrometric chambers were then placedinside a styrofoam box, left to equilibrate for 12 h beforemeasuring. Leaf gs (in mmol m−2 s−1) was measured at 09.00 hwith a steady-state leaf porometer (Decagon Devices Inc.,Pullman, WA, USA). For measurements of the small Ambrosialeaves, the porometer was equipped with a custom-builtattachment that reduced the opening for gas exchangemeasurements to either 1 or 2 mm diameter, and which wascalibrated by measuring moistened filter paper with openingsranging from 1 to 6.35 mm. For each shrub, eight leaves fromdifferent compass directions (N, NE, E, SE, S, SW, W, NW)were randomly chosen for porometry measurements. Rates ofnocturnal gs were also measured occasionally.

Fig. 3 Mean volumetric soil water contents (θv) measured daily at 04.00 h for pots with Ambrosia dumosa and Encelia farinosa shrubs in heterogeneous and homogeneous watering treatments from 2 July 2006 to 13 September 2006. For each watering trial, the six pots within each treatment were watered over a 3-d period, two pots per day. To synchronize the dates according to the watering schedule, day 1 on the x-axes corresponds to either July 2, 3 or 4, depending on the first date of initial measurements. Grey arrows indicate the day of initial plant water relations measurements and black arrows indicate the days of the watering trials. (a) θv for Ambrosia shrubs at 30 cm depth. (b) θv for Encelia shrubs at 30 cm depth. (c) θv for Ambrosia and Encelia shrubs at 60 cm. (d) θv for Ambrosia and Encelia at 90 cm.



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Measurements of hydraulic redistribution

Hydraulic redistribution was tested during the WTs in 2006described above, and again in April 2007 and June 2008. Thepots used for measurements of hydraulic redistribution werethose in the HT treatment (six per species), which wereequipped with a soil moisture probe in the upper 10 cm ofthe soil, in addition to the two sensors at 30 cm depthplaced into the internal and main compartments (Fig. 2a). Asignificant increase in θv in the top 10 cm of the soil surface,or at 30 cm depth in the main compartment, during the 3 dafter watering into the internal compartment was interpretedto indicate hydraulic redistribution from the internalcompartment. To determine whether water could move fromthe internal into the main compartment without the aid ofplant roots, WTs 5 and 6 included a control treatment thatconsisted of watering into the internal compartment of potswithout shrubs.

Statistical analyses

Indices of stem segmentation (S) and hydraulic integration (F)were compared between mature shrubs of the two species usingt-tests. To test for effects of treatments on measures of leafwater status, two separate multivariate repeated measuresanalyses of variance (MANOVA) were performed usingSYSTAT (version 12.02, SYSTAT Software Inc., San Jose, CA,USA), one with mean Ψleaf and gs (per individual), and thesecond with coefficients of variation (CVs) of Ψleaf and gs (perindividual) of the test plants as dependent variables. Bothanalyses had species and treatments as fixed effects, withmeasurements repeated five times (days from initial mea-surements: 0, 3, 31, 45, 63), followed by planned comparisonsto test for treatment effects at each measurement date withinspecies and for the treatment × species interaction.

To test for hydraulic redistribution from the internalcompartment to the main compartment after watering, themean θv value in the main compartment measured at 04.00 h,averaged over the 3 d before watering (θv,pre), was comparedwith the mean θv at 04.00 h of the main compartment,averaged over the 3 d after watering (θv,post). The differencesbetween these two measures (θv,diff = θv,post − θv,pre) was usedas a measure of hydraulic redistribution if positive, or absenceof hydraulic redistribution if equal to or less than zero. Paired,one-tailed t-tests for each species and each water trial were usedto test for the presence of hydraulic redistribution within trialdates, and significance at the level of P < 0.05 was determinedby adjusting for false discovery rate in multiple comparisons(Benjamini & Hochberg, 1995; Verhoeven et al., 2005). Totest for differences in hydraulic redistribution between species,the θv,diff data measured between 0 and 10 cm depth andat 30 cm depth were analyzed in MANOVA using SYSTATwith species as the independent variable and measurementsrepeated five times (no measurements of θv were available for

WT 2). θv,diff data for WT 5 were also analyzed in a separateanalysis of variance using SYSTAT. This analysis included ano-plant control treatment in addition to the two species.

Results

Field measurements of adult shrubs

Adult shrubs of the two species that were sampled for stemsegmentation measurements and dye tracer experiments wereof similar sizes (means ± standard errors: Ambrosia: height,43 ± 5 cm; width, 95 ± 12 cm; canopy volume, 0.23 ± 0.07 m3;Encelia: height, 64 ± 4 cm; width, 84 ± 12 cm; canopyvolume, 0.26 ± 0.07 m3). Ambrosia shrubs had highlysegmented stems and were physically split into several subunits(segmentation index S = 5.2 ± 0.4; Fig. 1), whereas basalstems of Encelia were nearly round (S = 1.3 ± 0.1; Fig. 1).Mean indices S differed between species (P < 0.001). Acidfuchsin dye tracer provided to a single root was detected inbasal stems of three of five Ambrosia shrubs, with between 0.6and 3.4% (mean, 1.7 ± 0.9%) of the wood cross-sectional areacolored by dye. Dye tracer was detected in all five Enceliashrubs sampled, and between 0.3 and 12.7% of the wood areawas colored by dye (mean, 4.1 ± 2.3%). Mean F indices didnot differ significantly between the two species (P = 0.482),but the maximum radial and tangential spread of dye wasmuch greater in Encelia than in Ambrosia, in which the dye wasphysically restricted to small lobes of light-colored sapwood(Fig. 1).

Soil water contents

Volumetric soil water contents (θv, %) in the controlledoutdoor experiments declined to c. 2% by the beginning ofthe watering treatments in July 2006 (Fig. 3a–d) and, exceptfor short-term increases by watering, remained at theselevels throughout the experiment. Watering into the internalcompartment of the HT watering treatment resulted in θv of14–17% for Encelia and 10–15% for Ambrosia during thenight following watering; by contrast, the main compartmentfor both species remained constant at c. 2% θv throughout themeasurement period. HM watering treatments increased θv inthe main and internal compartments to c. 6–9% θv (Fig. 3a,b).

Predawn leaf water potentials (Ψleaf)

The Ψleaf value in summer 2006 decreased over the time ofthe experiment for both shrub species (Fig. 4a). The wateringtreatment had no effect on the absolute values of Ψleaf of eitherspecies across all measurement times (P = 0.421; Table S1, seeSupporting Information), but absolute values of Ψleaf weresignificantly lower for Ambrosia than for Encelia (P = 0.001;Table S1). Canopy variability in Ψleaf of Ambrosia and Enceliashrubs, as characterized by CV, was not different for Encelia



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shrubs in the HT treatment vs HM treatment at any time(Fig. 5a; Table S2B, see Supporting Information). No differencesbetween treatments were observed for Ambrosia shrubs in theinitial measurements or in WTs 1 and 2; however, significanttreatment effects developed after WTs 3 and 4 (Fig. 5a;Table S2B), when Ambrosia shrubs in the HT treatments hadhigher canopy variability in Ψleaf than shrubs in the HMtreatment.

Leaf stomatal conductance (gs)

Absolute values of gs for Ambrosia peaked around mid-Augustand showed a tendency to decline over time, whereas gs valuesfor Encelia stayed more or less constant (Fig. 4b). The wateringtreatments had no effect on the absolute gs values of eitherspecies (Table S1). However, Ambrosia leaves had higher gsvalues than did Encelia leaves (P = 0.001; Table S1). Canopyvariability in gs of Encelia shrubs, as characterized by CV, didnot differ in response to HT vs HM treatments (Fig. 5b;Table S2B). Canopy variability in gs of Ambrosia shrubsshowed a significant treatment effect in WTs 2, 3 and 4, whenAmbrosia shrubs in the HT treatments had higher canopy

variability in gs than did shrubs in the HM treatment (Fig. 5;Table S2B). Overall, there were significant differencesbetween the canopy variability in gs and Ψleaf of the twospecies in their reaction to the watering treatments (significantspecies × treatment interaction in Table S2A, see SupportingInformation).

Hydraulic redistribution measurements

After watering into the internal compartments of pots in theHT treatment, significant decreases in θv in the maincompartments of pots with Encelia and Ambrosia shrubs andwithout shrubs were observed (Fig. 6a,b), indicating normaldrying of the soil. No considerable increases in θv were foundfor either species at any depth of the main compartment,indicating that these shrubs did not perform hydraulicredistribution from the side chamber to the main chamber(Figs 3a–d, 6a,b) under the experimental conditions (Table S3,see Supporting Information). WTs 5 and 6, which included a

Fig. 4 (a) Mean [± standard error (SE)] predawn leaf water potentials and (b) mean (± SE) stomatal conductance for Encelia farinosa and Ambrosia dumosa shrubs before watering treatments were applied and in response to watering treatments in four watering trials (WT 1–4) from 16 July 2006 to 13 September 2006. There was no significant treatment for either predawn leaf water potential or stomatal conductance, but there were significant differences between species (Table S1, see Supporting Information).

Fig. 5 Mean (± standard error) coefficients of variation in predawn leaf water potentials (a) and stomatal conductance (b) for Ambrosia dumosa and Encelia farinosa shrubs before watering treatments were applied and in response to watering treatments in four watering trials (WT 1–4) from 16 July 2006 to 13 September 2006. Statistically significant differences within a species between the homogeneous vs heterogeneous treatments are indicated by an asterisk (P < 0.01).



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no-plant control treatment in addition to the two species, alsoshowed no evidence of hydraulic redistribution (Table S4, seeSupporting Information), except for one Encelia plant in WT6, which showed a nonsignificant increase in θv,diff at 10 cm(Fig. 6a).

Discussion

The physical segmentation and splitting of mature Ambrosiashrubs divided them into complete IHUs (Fig. 1), as observedpreviously (Jones & Lord, 1982; Jones, 1984; Schenk, 1999),whereas the round stems of mature Encelia shrubs providedno morphological or anatomical indication for division intoIHUs. Dye in at least some Encelia plants spread widely aroundthe stem within several growth rings (Fig. 1), indicatingabundant radial and tangential connections between vessels.Despite these structural differences in mature shrubs, thefraction of wood cross-sections colored by dye did not differbetween the two species. This raises the question as to whetherphysical splitting is required for dividing plants into IHUs, orwhether the physical splitting follows from an anatomicaldivision into IHUs that is already present in young plants.

In answer to that question, this study showed that divisioninto IHUs can be present long before physical splitting ofstems is externally apparent. Young Ambrosia shrubs showedsignificantly higher canopy variability in leaf water potentials(Ψleaf) and stomatal conductance (gs) in the HT than HMwatering treatments, especially in the latter (Fig. 5), indicating

that young Ambrosia shrubs with undivided stems arefunctionally divided into IHUs. Axis splitting in this speciesapparently does not break any functioning connectionswithin the vascular system, and it occurs because the decayingheartwood readily succumbs to mechanical stress (Jones &Lord, 1982). For Encelia, no significant differences in canopyvariability of Ψleaf and gs values occurred between the HTand HM treatments, indicating that young Encelia shrubspossess integrated hydraulic systems. This type of hydraulicarchitecture allows unrestricted transfer of water throughoutthe shrub, creating a relatively homogeneous water statuswithin the canopy, regardless of soil water heterogeneity.

Division into IHUs could potentially reduce transpirationof the whole plant if IHUs with access to soil water openedtheir stomata, whereas other IHUs kept their stomata closed.However, mean gs values between the HT and HM wateringtreatments in Ambrosia did not differ, because this species doesnot appear to close its stomata very much, even when underdrought stress, as observed previously (Jacobsen et al., 2008).It remains to be seen whether division into IHUs in otherspecies reduces water loss, but it may be that a modularhydraulic system is primarily a risk-spreading strategy thatincreases the probability of survival for the whole genet byspreading the risk of mortality over independent modules,which cannot negatively affect each other (Watkinson &White, 1985; Eriksson & Jerling, 1990; Schenk, 1999). Inour experiment, plants were probably not stressed to the pointthat some IHUs would desiccate and die.

Few other studies have investigated within-plant heteroge-neity in water status and its relationships to hydraulic traits.In temperate tree and shrub species, different rates of watersupply to leaves and branches have been interpreted to becaused by restricted vascular pathways (Orians et al., 2004,2005; Zanne et al., 2006). It has been hypothesized that theserestrictions may be caused by a lack of intervessel connections(Ellmore et al., 2006; Zanne et al., 2006), by hydraulicisolation of vessels in a matrix of nonconducting cells, such asair-filled fibers, that surround the vessels (Sano et al., 2005;Schenk et al., 2008), or by the presence of heartwood betweenfunctional strands of sapwood (Keeley, 1975; Burgess & Bleby,2006).

The significant effects of heterogeneous watering on spatialcanopy water relations of Ambrosia occurred when themean Ψleaf value decreased to less than − 2 MPa (Figs 4a, 5).A decreasing Ψsoil over time would be expected to cause adecrease in Ψleaf (Turner, 1974). However, throughout themeasurement period, θv remained constant at c. 2% (Fig. 3a–d),except during days after watering, suggesting that some otherfactor, perhaps increasing vapor pressure deficit, must havecaused the declining Ψleaf. Depletion of water stored in planttissues over the course of the experiment, i.e. capacitance(Meinzer et al., 2001), could be an alternative explanation forthe decreasing Ψleaf. Because young Ambrosia shrubs appear tobe divided into IHUs, in the HT treatment, only the roots in

Fig. 6 Differences between mean volumetric soil water content (θv,diff) in the main compartment averaged over two 3-d periods before and after watering (n = 6) for all six watering trials (see Table 1). (a) Top 0–10 cm soil depth; (b) 30 cm depth. Statistically significant differences within a species between before and after watering are indicted by an asterisk (P < 0.05). Black bars, Ambrosia dumosa; grey bars, Encelia farinosa; white bars, control.



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the internal compartments had access to additional water,which may have kept tissues of IHUs that were supplied bythose roots hydrated. The other IHUs with roots in the maincompartment (Fig. 3a), and subjected to low θv (2%), mayhave used most of the available internally stored water tostay functional. In the HM watering treatments, the overalldeclining Ψleaf in Ambrosia suggests that transpirationdemands were not met by watering, causing water status todecrease, and presumably depleting internally stored water.

Mean Ψleaf in Encelia also decreased over the course of theexperiment, albeit to a lesser degree than in Ambrosia shrubs(Fig. 4a). Encelia shrubs may have also depleted internallystored water, but their stems possess abundant phloemparenchyma, which, in well-watered plants, forms a succulenttissue around the xylem. This probably causes the stemcapacitance of Encelia to be higher than that of Ambrosia, andthe integrated hydraulic system may have allowed Enceliashrubs to refill water storage cells throughout the plant inresponse to both watering treatments.

It may be expected that, in the HT treatment, branches andleaves located directly over the internal compartment wouldshow higher gs and Ψleaf. To test this concept, we used circularstatistics to test for a directional distribution of gs and Ψleafwith reference to the directional location of the internalcompartment. No directional effects were found (resultsnot shown), which came as no surprise, because Ambrosiastems often intertwine spirally and irregularly (Jones & Lord,1982), and because our results show that Encelia appears to behydraulically integrated.

The degree of spatial canopy variability may be expected todiffer between Ψleaf at predawn and gs during the day. Predawnmeasurements are made when water status within a canopy hascome as close to equilibrium as the hydraulic architecture ofthe plant and environmental conditions allow. By contrast,daytime gs may vary depending on the exposure of leaves to

solar radiation and wind. Stomatal conductance may alsovary in response to heterogeneous soil water when hightranspirational demand causes axial xylem water flow to vastlyexceed any radial or tangential water flow, thereby preventingthe equilibration of water status among vessels. Axial hydraulicconductance tends to be orders of magnitude higher thanradial or tangential conductance, even in hydraulicallyintegrated plants (Zwieniecki et al., 2001). In contrast withthese expectations, the observed CVs for Ψleaf and gs in thisexperiment were similar within and between species(Table S2), possibly because both species had substantialrates of gs at night (Encelia, 35–49 mmol m−2 s−1; Ambrosia,39–64 mmol m−2 s−1), which may have reduced canopyequilibration at night.

The anatomical traits that may confer different degrees ofhydraulic isolation of vessels in Encelia and Ambrosia shrubsare revealed in tangential sections of their basal stems (Fig. 7).Xylem in basal stems of Ambrosia has very straight, parallelvessels, with bands of thick-walled libriform fibers separatingthe vessels from each other and from ray cells. Fibers areconsidered to be nonconducting cells (Tyree & Zimmermann,2002; Sano et al., 2005), acting as a barrier for water transfer,especially when they are air-filled (Utsumi et al., 1998;Umebayashi et al., 2007). Because vessel groups are arrangedin radial bands in Ambrosia, the fiber bands probably restricttangential flow in the xylem of this species, thereby creatingradial vessel groups that function as IHUs. By contrast, thebasal stem xylem of Encelia shrubs has wavy vessels with greaterpotential for vessel-to-vessel contact, which should allow forradial and tangential flow among vessels (Fig. 7). Moreover,the vessels are in direct contact with ray parenchyma cells,which are thought to aid in intervessel water transport (Tyree& Zimmermann, 2002). The wood anatomical traits found inEncelia are clearly indicative of a shrub with an integratedhydraulic system.

Fig. 7 Tangential wood sections from basal stems of mature Encelia farinosa (right) and Ambrosia dumosa (left) shrubs. Encelia has wavy vessels in direct contact with libriform fibers and ray parenchyma cells. Ambrosia has straight vessels isolated from one another and from rays by thick-walled libriform fiber cells (bar, 350 µm). Images reproduced by permission of Hugo I. Martínez Cabrera, University of Connecticut.



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Reduced water loss caused by hydraulic redistribution maybe expected to be one of the many potential benefits of divisioninto IHUs for plants in a water-limited environment, as therewould be less opportunity for water exchange within the rootsystem. Our experiments yielded no evidence for hydraulicredistribution within root systems of either of the two species(Fig. 6a,b). Compared with average changes in θv of c. 7%,observed as a result of hydraulic redistribution in Artemisiatridentata (Ryel et al., 2003), the differences observed here(θv < 0.7%) are negligible. Hydraulic redistribution has beenreported previously for Ambrosia shrubs in the Mojave Desert(Yoder & Nowak, 1999). However, the evidence from thatstudy was not strong, because it lacked measurements of Ψsoilfluctuations in bare soil without plants. Two other studiesfound circumstantial evidence for hydraulic redistributionin Ambrosia based on the fact that annuals and other shrubspecies benefited from their close spatial association withAmbrosia (Holzapfel & Mahall, 1999; Schenk & Mahall,2002). We know of no previous research on hydraulic redistri-bution in Encelia.

Why did hydraulic redistribution not occur in the shrubsof this study? Rectifier properties in roots that allow waterflow into roots, whilst minimizing reverse flow (Caldwell et al.,1998), may be a reason. Night-time transpiration is anotherplant characteristic that potentially could restrain the mag-nitude or pattern of hydraulic redistribution (Ryel et al., 2002;Hultine et al., 2003; Bauerle et al., 2008). Both species inthis study had substantial rates of nocturnal gs. Shrubs thattranspire significant amounts of water at night are likely toprevent loss of water from roots to the soil because their xylemΨ will not rise above Ψsoil (Hultine et al., 2003). This seemsto be the most probable explanation for a lack of hydraulicredistribution in our study.

The degree of connectedness within a plant’s hydraulicsystem, i.e. hydraulic integration, is becoming a major focus ofplant hydraulics research (Pratt et al., 2008). Connectednessallows water sharing between plant modules that differ inwater status (Marshall, 1990) but, at the same time, it allowsembolisms to spread within a hydraulic system (Wheeler et al.,2005; Hacke et al., 2006; Loepfe et al., 2007), and does notallow for the hydraulic isolation of vessels that is required forembolism repair whilst the xylem is under tension (Holbrook& Zwieniecki, 1999). From this, one would predict that a lowdegree of hydraulic integration should be a common trait indesert shrubs, and this seems to be the case (Waisel et al., 1972;Schenk, 1999; Lambert, 2007; Schenk et al., 2008). Shrubsdivided into IHUs tend to have high longevity (Schenk, 1999),suggesting that this trait is adaptive for perennial plants ina desert environment. Results from this study show thatfunctional segmentation into IHUs precedes physical axissplitting, at least in Ambrosia, rather than being the con-sequence of split axes. Our study also supports the finding ofSchenk et al. (2008) that shrubs with round basal stems arelikely to be hydraulically integrated. Division into IHUs allows

independent stomatal regulation of IHUs in Ambrosia, but thisdid not lead to reduced leaf water loss under our experimentalconditions (Fig. 4b), and we found no effect of the degree ofhydraulic integration on water loss from roots as a result ofhydraulic redistribution. Determining whether division inIHUs translates into increased fitness or survivorship in thefield will require long-term studies of branch-level and genet-leveldemography.

Acknowledgements

We thank David Ackerly and three anonymous reviewers fortheir helpful comments and suggestions. We owe thanks toEd Read, Daisha Ortega, Christine Goedhart, Greg Pongetti,Jessica Hoang, Nichole Cervin, Maya Mezon, Calvin Threat,Victor Herrera, Ana Espino and Rosa Hernandez for theirgenerous help in conducting the research. We also thank HugoI. Martínez-Cabrera for providing the anatomical picturesused in this paper, Cynthia S. Jones for helpful comments onthe manuscript, and Darren R. Sandquist and Sean E. Walkerfor their comments on earlier versions of the manuscript.Funding for this research was provided by grants from theAndrew W. Mellon Foundation and the National ScienceFoundation (IOS-0641765) to H.J.S.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Table S1 Multivariate, repeated measures analysis of variancewith predawn leaf water potentials and stomatal conductancesas the dependent measured variables

Table S2 Multivariate, repeated measures analysis of variance,with coefficients of variation, with predawn leaf water potentialsand stomatal conductances as the dependent measured variables

Table S3 Multivariate, repeated measures analysis of varianceto test for hydraulic redistribution

Table S4 Analysis of variance to test for hydraulic redistributionduring watering trial 5 (25 April 2007)

Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting information supplied bythe authors. Any queries (other than missing material) shouldbe directed to the New Phytologist Central Office.


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