Drought predisposes pi~non–juniper woodlands to insect attacksand mortality

Monica L. Gaylord1, Thomas E. Kolb1, William T. Pockman2, Jennifer A. Plaut2, Enrico A.Yepez2,3,

Alison K. Macalady4, Robert E. Pangle2 and Nate G. McDowell5

1School of Forestry, Northern Arizona University, Flagstaff, AZ 86011, USA; 2Department of Biology, MSC03 2020, 1 University of New Mexico, Albuquerque, NM 87131-0001, USA;

3Departamento de Ciencias del Agua y del Medio Ambiente, Instituto Tecnol�ogico de Sonora, Ciudad Obreg�on M�exico, 85000, M�exico; 4School of Geography and Development and

Laboratory of Tree-Ring Research, University of Arizona, Tucson, AZ 85721, USA; 5Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA

Author for correspondence:Monica L. Gaylord

Tel: +1 928 523 3079Email: Monica.Gaylord@nau.edu

Received: 2 November 2012

Accepted: 7 January 2013

New Phytologist (2013)doi: 10.1111/nph.12174

Key words: climate change, drought, hostdefense, insect resistance, juniper, pi~non,resin.

Summary

� To test the hypothesis that drought predisposes trees to insect attacks, we quantified the

effects of water availability on insect attacks, tree resistance mechanisms, and mortality of

mature pi~non pine (Pinus edulis) and one-seed juniper (Juniperus monosperma) using an

experimental drought study in New Mexico, USA.� The study had four replicated treatments (409 40m plot/replicate): removal of 45% of

ambient annual precipitation (H2O�); irrigation to produce 125% of ambient annual precipi-

tation (H2O+); a drought control (C) to quantify the impact of the drought infrastructure; and

ambient precipitation (A).� Pi~non began dying 1 yr after drought initiation, with higher mortality in the H2O� treat-

ment relative to other treatments. Beetles (bark/twig) were present in 92% of dead trees.

Resin duct density and area were more strongly affected by treatments and more strongly

associated with pi~non mortality than direct measurements of resin flow. For juniper, treat-

ments had no effect on insect resistance or attacks, but needle browning was highest in the

H2O� treatment.� Our results provide strong evidence that � 1 yr of severe drought predisposes pi~non to

insect attacks and increases mortality, whereas 3 yr of the same drought causes partial canopy

loss in juniper.

Introduction

A positive relationship between drought and insect attacks ontrees is widely accepted (e.g. Mattson & Haack, 1987). Much ofthe evidence for this positive relationship is based on correlativeobservations of insect activity and tree mortality after drought.Experimental manipulations of water availability to mature treesthat include measurements of insect defense mechanisms and treemortality are rare. Furthermore, drought stress in trees may havea negative or no effect on insect populations and effects often varyby insect feeding guild, damage agent status (i.e. primary or sec-ondary), and intensity and duration of drought (Larsson, 1989;Huberty & Denno, 2004; Jactel et al., 2012). An improvedunderstanding of drought impacts on tree resistance to insectattacks is critical because climate models forecast more frequentand severe droughts in the future across many subtropical regions(Seager et al., 2007), and forests globally appear susceptible toincreases in mortality during drought (e.g. Allen et al., 2010).Here, we report the first experimental evidence regarding the roleof drought in insect resistance mechanisms, insect attacks, andmortality of mature trees of the two dominant trees of woodlands

of the southwestern USA, pi~non pine (Pinus edulis) and one-seedjuniper (Juniperus monosperma).

Bark beetles (Coleoptera: Curculionidae), which include themost lethal insects to trees in western forests (Raffa et al., 2008),often attack and kill drought-stressed trees (Huberty & Denno,2004; Jactel et al., 2012). Drought has well-established physio-logical impacts on trees, including compromised hydraulic func-tion, reduced carbohydrate production, and possible disruptionof carbohydrate supply to sinks (McDowell et al., 2008;McDowell & Sevanto, 2010; Sala et al., 2010; Ryan, 2011).Reduced carbon uptake as a result of stomatal closure anddiminished transport to sinks could diminish tree bark beetledefense by reducing carbon available for resin production(McDowell et al., 2008; Sala et al., 2010), which is acknowledgedas the primary defense against bark beetles (Berryman, 1972;Christiansen et al., 1987; Herms & Mattson, 1992; Strom et al.,2002; Franceschi et al., 2005). Furthermore, the growth-differen-tiation-balance hypothesis (GDBH) predicts that moderatelywater-stressed trees may actually be more resistant to bark beetleattacks because moderate water stress constrains growth sinksmore than defensive compound sinks, leading to a relative

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increase in resin production (Loomis, 1932; Lorio, 1986; Herms& Mattson, 1992; Dunn & Lorio, 1993; Stamp, 2003). Theframework also predicts that long-term, severe droughts eventu-ally reduce defense because chronic depletion of carbohydratesreduces carbon availability for resin production (McDowell et al.,2011). Similarly, reduced phloem transport during droughtcould reduce carbon available for resin production (Dunn &Lorio, 1992; Sala et al., 2010). Despite substantial research oncarbon allocation within trees (e.g. Litton et al., 2007), the roleof drought in resin defense of trees is poorly understood.

Mortality of pi~non was unusually high throughout the south-western USA during a severe drought in 2000–2003 (Breshearset al., 2005; Shaw et al., 2005; Kleinman et al., 2012). In 2002,Arizona and New Mexico had one of the driest and warmest yearson record and by 2003 c. 774 711 ha of pi~non woodlands hadevidence of bark beetle activity (United States Department ofAgriculture, aerial surveys (USDA 2002–2010); Kleinman et al.,2012). Regional (Arizona, Utah, Colorado and Nevada) pi~nonmortality increased at least two-fold during this drought (Shawet al., 2005; Williams et al., 2012) and certain areas had > 90%mortality (Breshears et al., 2005; Floyd et al., 2009). Mortalitywas lower for juniper than for pi~non during this drought (Shawet al., 2005; Floyd et al., 2009; Koepke et al., 2010).

Juniper and pi~non have been useful model organisms for stud-ies of tree mortality during drought because they often growtogether, but their different survival and hydraulic strategies arerepresentative of a broad range of species in semi-arid regionsglobally (Mueller et al., 2005a; McDowell et al., 2008; Adamset al., 2009; Koepke et al., 2010). Pi~non is more isohydric thanjuniper (McDowell et al., 2008; Plaut et al., 2012) and minimizesxylem cavitation during drought via stomatal closure to reducewater loss. Juniper, by contrast, can withstand negative xylempressures with little cavitation during drought and continuescarbon assimilation (Linton et al., 1998).

Trees can be attacked by many pathogens and guilds of herbi-vores. The most lethal or abundant herbivores can determinewhich components of tree defense are most crucial to tree sur-vival. The most lethal insect to pi~non in recent years is Ipsconfusus (LeConte), the pi~non ips bark beetle, which generallyattacks stressed or recently dead trees (Wood, 1982b; Rogers,1995; Raffa et al., 2008). In contrast to pi~non, little is knownabout the insect guild of junipers. Insects that can damage juni-pers include bark beetles and wood borers (Coleoptera: Bupresti-dae & Cerambycidae) (Furniss & Carolin, 1977; Itami & Craig,1989), but data are scarce regarding the amount of tree mortalityattributable to these agents.

Our study evaluated the impacts of water availability on insectattacks, resin defenses, canopy condition, and mortality ofco-occurring pi~non and one-seed juniper over 3 yr via a large-scale precipitation manipulation experiment performed in situ onmature trees. Based on differences in mortality during nonexperi-mental drought (Shaw et al., 2005; Koepke et al., 2010) andphysiological differences in carbon uptake and water relationsbetween these species (e.g. McDowell et al., 2008), we hypothe-sized that 3 yr of experimental drought would decrease resindefense and increase bark beetle attacks and mortality of pi~non,

but have little impact on juniper. Moreover, we compared treat-ment effects on the carbon isotope ratio (d13C) of leaf sugars andbole resin of pi~non to assess the role of recent photosynthate inresin synthesis. Finally, we examined the relationship betweenresin volume and other parameters of defense/vigor in pi~non tounderstand possible tradeoffs.

Materials and Methods

Study site

Our study site was located in a mature pi~non–juniper woodland(Pinus edulis Engelm. and Juniperus monosperma (Engelm.) Sarg.)� 100 km south of Albuquerque, New Mexico in the Los PinosMountains in the Sevilleta National Wildlife Refuge and theSevilleta Long-Term Ecological Research Area (34°23′11″N,106°31′46″W; elevation 1911 m). Mean temperature (20-yr)ranges from a minimum of �3.3°C (December) to maximum31°C (July). Mean precipitation (20-yr) at the site is 362.7 mm(Sevilleta Long-Term Ecological Monitoring, 2011), over half ofwhich arrives as late-summer rain. Pi~non and juniper basal area(at 30 cm stem height) averaged 20.0 m2 ha�1 across the studysite, with pi~non basal area comprising 2.9 m2 ha�1 of this total(Pangle et al., 2012). Pangle et al. (2012) and Plaut et al. (2012)provide more detailed site information, including a photo of thestudy site, information on the block design, assignment oftreatments to plots, and criteria for determining plot size.

The experimental design consisted of four treatments and threeblocks dispersed across� 25 ha. Installation of treatment infra-structure was completed in August 2007 and consisted of a waterremoval treatment (H2O�), a water addition treatment (H2O+),an ambient treatment (A), and an additional control (C), whichwas designed to assess effects of the plexiglass covers used in theH2O� plots. Replicates of each treatment were assigned to409 40 m plots that contained several mature pi~nons and juni-pers. In the H2O� and the C treatments, 29 parallel plexiglassgutters were attached to metal poles at� 1 m above ground leveland covered� 45% of ground area of plots. In the H2O� plotsthese gutters intercepted precipitation and transported it off-plotto collection troughs. In the C plots, the gutters were inverted andprecipitation dripped to the ground. The gutters increased averagemaximum soil and ground-level air temperature by 1–4°C duringthe growing season (Pangle et al., 2012). Water was applied to theH2O+ plots from May to October using 16 equally spaced sprin-kler nozzles placed 2–3 m higher than mean tree height. Precipita-tion amounts in the H2O+ plots averaged 125% of those in theambient plots and average tree predawn water potentials (Ψpd)were significantly higher in the H2O+ treatment than in theH2O� treatment for both species in all post-treatment study years(Supporting Information Tables S1, S2). More details on siteinfrastructure and methods for physiological measurements areavailable in Plaut et al. (2012) and Pangle et al. (2012).

At the beginning of the study (2007), five trees of each specieswere identified per plot as target trees. Target trees were all cen-trally located on plots and their stem diameters at a height of30 cm ranged from 9.0 to 40.9 cm for pi~non and from 9.0 to

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74.8 cm for juniper (Pangle et al., 2012; Plaut et al., 2012). Therewas no significant difference in tree diameter among treatments(P = 0.26 for both pi~non and juniper).

We present methods separately for pi~non and juniper becausedifferent measurements were performed on each species. Specifi-cally, our measurement of insect resistance mechanisms on juni-per was limited to bole resin because our preliminary workshowed little resin was exuded from clipped twigs of juniper, andjuniper increment cores could not be reliably cross-dated formeasurement of sapwood resin ducts (Esper, 2000; Bra€uning,2001; Sass-Klaassen et al., 2008).

Pi~non resistance and insect measurements

Pi~non bole resin We measured pi~non resin flow from bolewounds once before the onset of treatments (3 July 2007) and onfive subsequent dates (18 August 2008, 24 May 2009, 1 August2009, 2 June 2010, and 16 August 2010) using methods firstdescribed by Lorio (1993). Briefly, we removed most bark from asection of the bole at � 1 m above the ground to create a smoothsurface. We used a 1-cm-diameter arch punch to remove a cylin-der of the remaining bark and phloem. Funnels were placedunder the wound (one per tree), and resin was collected in 15-mltubes for 24 h and volume recorded to the nearest 0.5 ml. For the2007, 2008 and May 2009 measurements, we collected resinfrom a subset of the previously described target trees (two or threetrees/plot; n = 8–9 trees/treatment). By August 2009, pi~nonmortality in the H2O� treatment reduced sample size for thistreatment to between three and five trees total.

Pi~non twig resin We collected twig resin on the same pi~nonsused for bole resin sampling between 12:00 and 16:00 h oncebefore the onset of treatments (3 July 2007) and on sevensubsequent dates (6 September 2007, 19 June 2008, 18 August2008, 24–26 May 2009, 31 July 2009 to 1 August 2009, 1–3June 2010, and 16–17 August 2010). When, because of timeconstraints, twig resin collection was spread over several consecu-tive days, we structured our sampling so that complete treatmentblocks would be sampled on the same day. We collected twigresin using methods described by Mopper et al. (1991). Briefly,we clipped one terminal twig segment from two, opposite aspectsof each tree directly behind the previous year’s needles (May,June and early July sample dates) or current-year growth (lateJuly, August and September sample dates). Aspect varied bygrowth form of the tree. After 10 min we used a toothpick toscrape the resin bubble that formed on the proximal side of thecut branch into a pre-weighed microcentrifuge tube. Werecorded resin flow as the difference between the initial mass (g)of the vial and toothpick and the mass after collection.

Pi~non resin ducts We used an increment borer (5 mm diame-ter) to sample sapwood from the target trees initially identified in2007 for resin collection (n = 8–9 per treatment). One core wasextracted from the lower bole (0.5 m above ground) of each treein August 2010. Xylem resin duct characteristics have been asso-ciated with pine survival during drought and bark beetle

outbreaks (Kane & Kolb, 2010; Kl€ay, 2011). We measured resinducts using methods modified from Kane & Kolb (2010).Briefly, cores were allowed to air-dry for 1 wk before mountingon wood blocks. We then sanded the cores using progressivelyhigher grit sand paper (120–400). We scanned each core to createa high-resolution image (1200 dpi), which was analyzed withWINDENDRO software (Regent Instruments Inc, 2009) to assignannual ring boundaries and measure ring widths. We assignedcalendar years to the last 10 yr of growth before the onset of treat-ments (1996–2007) via identification of narrow and wide markeryears from a previously constructed master chronology for thesite. Visual cross-dating was confirmed using the statistical soft-ware COFECHA (Holmes, 1983; Grissino-Mayer, 2001). We didnot include post-treatment years in our cross-dating correlationbecause the treatments probably altered the annual precipitationsignals on which cross-dating was based. Next, we imported thecore images into the public domain image-processing programIMAGE J (Rasband, 1997-2011), and we measured resin duct char-acteristics for each year from 2003 to 2010, or until year of death,along a 2-mm-wide core section. Resin duct characteristics aredescribed in detail by Kane & Kolb, (2010) and Kl€ay (2011) andincluded production (number of ducts), average duct area (mm2),resin duct density (number of ducts per area of ring (mm2)), andper cent area (mm2 of resin ducts mm�2 of growth). For twotrees in the H2O� treatments, a missing ring was noted betweentreatment onset and year of death. Because we were interested inyearly allocation to growth and defense, we assigned values ofzero for tree growth and resin duct production and area to themissing rings. We assumed growth declined as water stressincreased and assigned the missing ring to the most recent yearbefore death, or the end of our study. Because of low samplenumbers in the H2O� treatment in 2009 and 2010 (six andthree trees for 2009 and 2010, respectively), a sensitivity analysisto confirm our assumption was not possible.

Pi~non insect attacks and tree condition We surveyed the fivetarget pi~nons on each plot (n = 15 per treatment) once before theonset of treatments (2 July 2007) for canopy condition, mortal-ity, and insect attacks. We surveyed the trees once or twice ineach post-treatment year (19–20 June 2008, 23–26 May 2009,30 July to 2 August 2009, 1–5 June 2010, and 16–18 August2010). Once a tree was recorded as dead (defined as 100% needlebrowning), it was dropped from subsequent surveys. Sample sizesfor the post-treatment surveys ranged between five and 17 treesper treatment because of tree mortality and the addition ofreplacement survey trees. For each survey we recorded the pres-ence or absence of new (since previous survey) twig beetle attacks(as evidenced by needle browning, feeding activity, or presence oftwig beetles) and bark beetle attacks (presence/absence based onentrance holes, galleries, or presence of beetles), and classifiedtrees as live or dead. Bark beetle attacks were further quantifiedby counting successful attacks (defined as entrance holes with vis-ible frass) or unsuccessful attacks (resin only) on the bottom� 1.5 m of the main bole. Bark beetles were excavated from gal-leries of a subset of trees and were identified to species based onmorphological characteristics (Chansler, 1964; Wood, 1982b),

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and comparison with voucher specimens from the Rocky Moun-tain Research Station, Flagstaff, Arizona. Twig beetles were iden-tified to species and voucher specimens preserved by Jim LaBonte(Oregon Department of Agriculture, Salem, OR, USA). Otherinsects noted during pi~non surveys (damage or actual insect)included the pi~non stem-boring moth (Dioryctria albovittella,Lepidoptera: Pyralidae), aphids (Hemiptera: Aphididae), pi~nonneedle scale (Matsucoccus acalyptus Herbert, Hemiptera: Marga-rodidae), pi~non spindle gall midge (Pinyonia edulicola, Diptera:Cecidomyiidae), pitch moth (Synanthedon spp., Lepidoptera: Se-siidae), and pi~non sawfly (Hymenoptera: Diprionidae). Data onthese species were not analyzed because of low presence and pre-sumed low tree impact.

Pi~non resin and leaf carbon isotope ratios

To better understand the source of carbon used for resin produc-tion, we compared the d13C of pi~non bole resin and recentlyassimilated leaf sugars among treatments. Leaf sugar isotopes wereanalyzed because they reflect carbon isotope discrimination duringrecent photosynthesis and thus would indicate whether the watertreatments had an immediate impact on leaf gas exchange. In aridsystems, a higher (less negative) d13C generally indicates lowerstomatal conductance and photosynthesis (Ehleringer, 1991).

Bole resin was collected from pi~non as described above forpinon bole resin collection and frozen at��2°C in sealed tubesuntil processed for isotope analysis. We were unable to analyzesome samples because of the small volume collected. For 2007,2008, and May 2009, we measured bole resin d13C on seven tonine trees per treatment. For the remaining dates (August 2009,2010, and June 2010), sample sizes were between eight and ninetrees for the C, A and H2O+ treatments, but reduced to three tofive trees for the H2O� treatment. Between 1.5 and 3.0 mg ofresin was weighed into small tin capsules and analyzed for d13Cin a Costech ECS 4010 elemental analyzer (Costech AnalyticalTechnologies, Valencia, CA, USA) coupled with a ThermoElectron Delta Plus Advantage mass spectrometer (Bremen,Germany) operated in continuous flow at the Colorado PlateauStable Isotope Laboratory, Northern Arizona University, Flag-staff, AZ, USA. Isotope ratios are expressed relative to ViennaStandard Peedee Belemnite. A total of 202 resin samples wererun and overall precision for d13C was 0.070/00 (n = 60).

We sampled soluble sugars from new, healthy foliage from fivetrees per treatment in one sample block (n = 20) on the south sideof the tree in the afternoon on 13 dates between October 2006and August 2008. Trees sampled for resin (as described above)were a subset of these same trees within the respective sampleblock. Samples were collected in the afternoon, 6–8 h aftersunrise. To extract carbohydrates from leaf samples, we used pro-cedures described by Brugnoli et al. (1988) and adapted to pi~nonby West et al. (2007). Briefly, we clipped leaf tissues into coinenvelopes which were immediately buried in dry ice (CO2) in acooler and kept frozen for transportation. In the laboratory thesesamples were kept frozen at �80°C until analysis. Before analy-sis, samples were freeze-dried and then 150 mg of sample wasground in a ball mill and placed in a 50-ml centrifuge tube with

35 ml of deionized water. The tube was boiled in a water bath for30 min, cooled, and then centrifuged for 15 min at 12 100 g. Wecollected the supernatant and passed it through a C-18 Sep-Pakcartridge (Waters, Milford, MA, USA) to remove large organicmolecules. Next, we passed the sample through an ion-exchangecolumn, consisting of Dowex-50 and Dowex-1 (Sigma Aldrich)and then filtered with 45 lM Acrodiscs (Pall Corp., PortWashington, NY, USA). We then evaporated the sample andweighed 2 mg of the remaining sugars into tin capsules for stableisotope analysis. Carbohydrate extracts were analyzed at theDepartment of Earth and Planetary Sciences, University of NewMexico in a Costech elemental analyzer (EA 1108) coupled witha Delta Plus Mass Spectrometer. Isotope ratios are expressedrelative to Vienna Standard Peedee Belemnite with an overallprecision for d13C of 0.030/00 (n = 261).

Juniper resistance and insect measurements

Juniper bole resin For juniper, we measured bole resin pro-duced after wounding using methods similar to those describedfor pi~non, except that resin was collected using pre-weighedtoothpicks and vials because of the low flow. Briefly, on each datewe wounded a subset of the target trees (two or three junipers perplot; eight to nine trees per treatment; one wound per tree) to theface of the xylem and, after 24 h, scraped resin from the face ofthe xylem using a pre-weighed toothpick into a pre-weighedmicrocentrifuge tube. We recorded resin flow as the differencebetween the initial weight of the vial and toothpick and theweight after collection.

Juniper insect attacks and tree condition For juniper werecorded needle browning (% of total canopy) at each survey dateon the five target trees per plot (n = 15 trees per treatment). Juni-per mortality was recorded when 100% of needles had browned.Insect attack data on juniper, such as branch flagging from juni-per twig pruners and Buprestid and Cerambycid emergenceholes, were not analyzed because of the low number of emergenceholes and, in the case of twig pruners, because it was difficult toconclusively determine if twig dieback was from twig pruners orwater stress in the H2O� treatment.

Statistical analysis

We evaluated treatment impact on juniper and pi~non bole resin,pi~non twig resin, resin duct parameters, bole resin d13C, and leafsugar d13C using separate repeated measures MANCOVA foreach characteristic with individual trees as the experimental unit,date as the repeated factor, and treatment as the independent var-iable. Blocks were not formally included as a factor for all analy-ses in order to increase power. Values were rank-transformed forjuniper and pi~non bole resin, pi~non twig resin, and resin ductdensity to correct for normality of residuals as indicated by theShapiro–Wilk W test. In the case of juniper bole resin, valueswere still not normal, and thus P values close to zero should betreated with caution. Pretreatment values were a covariate foreach respective variable to account for any pretreatment

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differences. For pi~non, because of tree mortality and the use ofreplacement trees, the pretreatment value assigned to each treefor twig and bole resin and bole resin d13C was the respective pre-treatment plot average. For pi~non leaf sugar d13C and resin ductparameters, the average pretreatment value (seven measurementsbetween October 2006 and August 2007 for pi~non leaf sugard13C; years 2003–2007 for resin duct parameters) of each indi-vidual tree was the covariate. The covariate for juniper bole resinwas the single pretreatment value of each tree. We used Student’st-tests with Bonferroni-adjusted alpha levels for post hoc compari-sons of treatment and date means.

We used a chi-squared analysis with a null hypothesis of evendistribution among treatments at the end of the study (2010) toanalyze the effect of treatment on cumulative twig beetle attacks(presence/absence) and cumulative pi~non mortality. We used anANOVA to analyze the effect of treatment on cumulative pi~nonbark beetle attacks (number of successful attacks/unit bark area).

We used simple linear regression to evaluate the relationships(1) between pi~non tree diameter and bark beetle attacks (densityand presence/absence); (2) between pi~non twig resin and treediameter growth; and (3) between pi~non bole resin volume andthe following variables: twig resin volume, tree diameter, treediameter growth, and all measured resin duct parameters. Wealso examined the relationship between pi~non bole resin and allpredictor variables using multiple regression; however, this didnot result in a better model fit. We used ANOVA to analyze thedifference in resin volume and resin duct parameters (mean val-ues of 2007 to either 2010 or the last measurement before mor-tality/attack for both resin volume and resin ducts) betweenattacked and unattacked pi~non trees. We also used ANOVA toanalyze the difference in Ψpd between attacked and unattackedpi~non trees both before and after attacks. The Ψpd measurementswere taken 3 or 4 wk before, and within 2 wk after bark beetleattacks were first recorded on trees.

The effect of treatment on juniper needle browning was ana-lyzed with repeated measures MANOVA with date, treatmentand their interaction as independent variables. Cumulative needlebrowning was calculated using the pretreatment year of 2007 asthe base year of 100% live crown and subsequent percentageswere subtracted from this initial total. Needle browning wassquare root-transformed to correct for normality and heterosce-dascity as indicated by the Shapiro–Wilk W test. Juniper mortal-ity was not analyzed because only one juniper (H2O� treatment)had died by the end of the study in 2010. All statistics were per-formed using JMP 9 (SAS Institute Inc, 2010). For ease of inter-pretation we present untransformed data in figures and tables.

Results

Pi~non insect resistance mechanisms

Pi~non bole resin flow was not significantly affected by the interac-tion of treatment and date (Wilks’ lambda, P = 0.49; Table S3) ortreatment (P = 0.84). Date had a significant effect (P = 0.0380)(Fig. 1), as did the covariate pretreatment resin flow (P = 0.0017).For pi~non twig resin flow, the interaction of treatment and date

was significant (Wilks’ lambda, P = 0.0210; Table S3), and thuseach date was analyzed separately. Treatments had a significantinfluence on pi~non twig resin on two dates (May 2009,F ratio = 4.56, df = 3, P = 0.0096; August 2009, F ratio = 7.64,df = 3, P = 0.0007), when trees in the H2O� (May and August)and H2O+ (August) treatments had significantly lower resin flowthan trees in the A treatment (Fig. 2). The pretreatment co-variate was significant for August 2009 (F ratio = 5.11, df = 1,P = 0.0321).

Pi~non resin duct production, area, density, and per cent areawere all significantly affected by the treatment and time interac-tion (Wilks’ lambda, P � 0.0149; Table S4), and thus each yearwas analyzed separately using ANOVA. Treatment had no signif-icant effect on any resin duct characteristic in 2008 or 2009(Table S5; Fig. 3a–d). In 2010, resin duct production, area, den-sity, and per cent area were consistently lowest in the H2O�treatment, and significant differences among treatments occurredfor production, density, and per cent area. The pretreatmentcovariate was significant (P � 0.0181) for some years/variables.

Pi~non insect attacks and tree mortality

By August 2010, treatment significantly (v2 = 10.37, df = 3,P = 0.0157) impacted attacks by twig beetles (Pityophthorusopaculus LeConte) on pi~non, with the highest percentage ofattacks occurring in the H2O� treatment (Fig. 4a). Bark beetle(Ips confusus (LeConte)) attack densities were significantly higher(F ratio = 3.85, df = 3, P = 0.0134) in the H2O� treatment thanin the other treatments (Fig. 4b). Treatment significantly affectedpi~non mortality (v2 = 28.04, df = 3, P < 0.0001), which was

Fig. 1 Mean� SE of resin volume collected over a 24-h period afterphloem wounding of pi~non boles at the Sevilleta, NM field site by date andtreatment (H2O+, water addition plots; H2O�, water removal plots; C,cover control; A, ambient; n = 30–35 on each date). Treatments wereinitiated in August 2007 as indicated by the arrow on the graph.MANCOVA, with plot-mean pretreatment values as the covariate,showed no significant effect of the treatment and date interaction (Wilks’lambda, P = 0.49) or treatment (P = 0.84). Date had a significant effect(P = 0.0380). Dates labeled with different letters are significantly differentbased on Student’s t-test with Bonferroni-corrected a = 0.005.

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greatest in the H2O� treatment (Fig. 4c). At the end of our mea-surements in 2010, pi~non mortality was c. 70% in the H2O�treatment and 10% or less in the other treatments.

Pi~non stem diameter had no linear relationship with densityof bark beetle attacks (P = 0.28, R2 = 0.02, n = 59) and nologistic relationship with presence/absence of bark beetle attacks(P = 0.21, v2 = 1.57, n = 59). There was no linear (P = 0.24,R2 = 0.01, n = 145) or nonlinear (P � 0.40 for quadratic andcubic) relationship between pi~non twig resin and bole resin.Similarly, no other relationship was found between pi~non boleresin volume and tree diameter, or resin volume and measuredresin duct parameters (P � 0.0543 for all; n = 171–179)(Fig. S1a,c–f). Bole resin had a weakly significant (P = 0.0479,R2 = 0.03, n = 180) quadratic relationship with tree diametergrowth (Fig. S1b). There was no relationship (linear, quadratic,or cubic; P � 0.68; n = 176) between stem diameter growthand twig resin flow in pi~non (Fig. S2).

Average resin duct size during the experiment was significantly(P = 0.0022; Table S6) smaller (37%) in pi~non trees that diedthan in those that survived, whereas there was no significant dif-ference in any other resin duct parameter (production, density orper cent area), bole resin, or twig resin between trees that survivedand those that died (Table S6). In addition, there was no signifi-cant difference in Ψpd either pre- or post-attack between attackedand unattacked trees (Table S7).

Fig. 2 Mean� SE weight of pi~non twig resin flow collected 10min aftertwigs were cut at the Sevilleta, NM field site, by date and treatment (H2O+, water addition plots; H2O�, water removal plots; C, cover control; A,ambient; n = 30–36 on each date). Treatments were initiated in August2007 as indicated by the arrow on the graph. MANCOVA, with plot-mean, pretreatment values as the covariate, showed a significant inter-action of treatment and date for post-treatment twig resin flow (Wilks’lambda, P = 0.0210), and thus each date was analyzed separately.Treatment was significant on two dates (May 2009, P = 0.0096; andAugust 2009, P = 0.0007). Treatments within dates not labeled by thesame letter are significantly different as determined by Student’s t-testswith Bonferroni-corrected a = 0.0083.

(a) (b)

(c) (d)

Fig. 3 Mean� SE of pi~non resin ductcharacteristics from increment cores (n =28–35) collected from the lower 0.5m of themain bole of trees at Sevilleta, NM. Char-acteristics are: (a) production (number ofducts per 2-mm width of ring), (b) averageduct area (mm2), (c) density (number ofducts per area of ring mm�2), and (d) percent area (mm2 of duct area mm�2 of ringarea). Results are presented by year andtreatment (H2O+, water addition plots;H2O�, water removal plots; C, cover control;A, ambient). Treatments were initiated inAugust 2007 as indicated by arrows ongraphs. MANCOVA, with individual treepretreatment values (average of years 2003–2007) as the covariate, indicated that therewas a significant treatment and yearinteraction for each characteristic (Wilks’lambda, P � 0.0149, for all parameters), andthus each year was analyzed separately. In2008 and 2009, there was no effect oftreatment on resin duct characteristics. In2010, treatment significantly affectedproduction, density, and per cent area, asindicated by * on graphs, but did not affectduct area. Treatments in 2010 labeled withdifferent letters are significantly different asdetermined by Student’s t-tests withBonferroni-corrected a = 0.0083.

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Pi~non resin and leaf sugar carbon isotope ratios

The d13C of pi~non bole resin averaged �24.0� 0.04& (SE)over all treatments and dates and was not significantly affected bythe interaction of treatment and date (Wilks’ lambda, P = 0.53),treatment (P = 0.94), or date (P = 0.97) (Table S3; Fig. 5a).Pi~non leaf sugar d13C averaged �23.6� 0.06 & over all treat-ments and dates and was not significantly affected by the treat-ment and date interaction (Wilks’ lambda, P = 0.09) or date(P = 0.80) (Table S3). Leaf sugar d13C in the H2O+ treatmentwas consistently lower (P = 0.0263) than in other treatmentsthroughout the first growing season (2008) after the onset oftreatments (Fig. 5b).

Juniper insect resistance mechanisms and canopy condition

Juniper bole resin flow was low for most measurements (< 0.01 g)and was not significantly affected by the treatment and date

interaction (Wilks’ lambda, P = 0.12) or treatment (P = 0.84).The covariate pretreatment resin flow (P = 0.0003) and date(P = 0.0012) had significant effects (Table S3; Fig. S3).

Juniper cumulative needle browning was significantly influ-enced by the treatment and date interaction (approx. F = 6.37,Numerator df = 9, Denominator df = 131.6, Wilks’ lambda,P < 0.0001); therefore, each date was analyzed individually usingANOVA (Table S8). Treatment did not significantly affect nee-dle browning in 2008 (P = 0.18); however, in 2009 and 2010junipers in the H2O� treatment had significantly more needlebrowning (P < 0.0001, for both) than trees in other treatments(Fig. 6). By the end of the study in 2010, � 50% of juniper

(a)

(b)

(c)

Fig. 4 For the post-treatment study period (2008–2010): (a) cumulativeper cent of pi~nons attacked by twig beetles (Pityophthorus opaculusLeConte) by treatment, (b) mean� SE density of successful bark beetleattacks (successful attacks per m2 of pi~non bark) of bark beetles (Ipsconfusus (LeConte)), and (c) cumulative pi~non tree mortality by treatment(n = 69 for all graphs). Treatment significantly affected cumulative treeattacks by twig beetles (P = 0.0157; chi-squared), density of bark beetleattacks (P = 0.0134; ANOVA), and cumulative tree mortality (P < 0.0001;chi-squared). Significant differences in bark beetle attack densities bytreatment, based on Bonferroni-corrected a = 0.0083, are indicated bydifferent letters on the graph. Treatments are: H2O+, water addition plots;H2O�, water removal plots; C, cover control; A, ambient.

(a)

(b)

Fig. 5 Mean� SE carbon isotope ratio (d13C) of (a) pi~non bole resin(n = 28–32/date) and (b) leaf sugars (n = 12–20/date) collected at theSevilleta, NM field site by date and treatment (H2O+, water addition plots;H2O�, water removal plots; C, cover control; A, ambient). Treatmentswere initiated in August 2007 as indicated by arrows on graphs.MANCOVA, with pretreatment values (plot average for bole resin d13C;average of seven measurements between October 2006 and August 2007for leaf sugar d13C) as the covariate, indicated no significant interaction oftreatment and date (Wilks’ lambda, P = 0.53), and no significant effect oftreatment (P = 0.94) or date (P = 0.97) on tree bole resin d13C. For leafsugar d13C there was a significant treatment effect on post-treatment d13C(P = 0.0263), but no effect of the treatment and date interaction (Wilks’lambda, P = 0.09) or date (P = 0.80). Trees in the H2O+ treatment hadsignificantly lower d13C leaf sugar than the other treatments post-treatment, as determined by Student’s t-tests with Bonferroni-correcteda = 0.0083.

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foliage was brown in the H2O� treatment compared with15–20% in the other treatments.

Discussion

Our study provides the first experimental evidence that droughtpredisposes mature pi~nons to bark beetle attacks and improvesunderstanding of mechanisms of drought impacts on insect resis-tance of pi~nons. One or 2 yr of drought with 45% reduction inambient precipitation increased bark and twig beetle attacks andtree mortality for pi~non. In addition, we found that junipers werestressed, but not killed, by 3 yr of drought.

Interestingly, experimental drought had variable impacts onthe insect resistance characteristics we measured on pi~non. Basedon previous reports of reduced photosynthesis of pi~non duringdrought (McDowell et al., 2008; Breshears et al., 2009) and evi-dence of 7 months of near-zero gas exchange of pi~non in thedrought treatments at our study site (Plaut et al., 2012), wehypothesized that pi~nons in the H2O� treatment would be car-bon limited and thus have lower resin flows in response towounding than in other treatments. Support for this hypothesisdiffered for bole and twig resin. For bole resin, the H2O� treat-ment had little impact on flow. We should note that, similar toother studies of southwestern pines (Gaylord et al., 2007, 2011),pi~non bole resin flow was extremely variable in our study (coeffi-cient of variation = 127%), and thus a larger sample size wouldbe needed to detect treatment effects. In addition, our samplingmethodology did not allow us to examine within-tree variation ofbole resin flow.

Our finding of no treatment effect on pi~non bole resin d13Cin combination with lower d13C for leaf sugars in the H2O+treatment suggests a lag between drought impacts on carbonassimilation and resin synthesis, because most bole resin wasconstitutive and formed from previously assimilated carbon. Alarger difference in d13C between pre- and post-treatment valuesof leaf sugars (� 2.5&) than resin (� 0.6&) provides additionalsupport for this interpretation. Our interpretation that pine boleresin is largely constitutive and formed from old assimilateshas been previously reported for seedlings (Lewinsohn et al.,1991; Gu�erard et al., 2007), but this is the first report formature trees.

The results for twig resin, that is, less flow in both the H2O�and the H2O+ treatments than in other treatments on somedates, were more consistent with our hypothesis and with predic-tions of the GDBH framework of a bell-shaped relationshipbetween carbon-based defense and drought stress (Herms &Mattson, 1992; Stamp, 2003). The bell-shaped relationship isexpected because severely water stressed trees, such as in theH2O� treatment, are not able to supply photosynthate for resinproduction, whereas well-hydrated trees, such as in the H2O+treatment, preferentially allocate photosynthate to growth ratherthan defense. This tradeoff results in the moderately water-stressed trees (the A and C treatments in our study, which experi-ence seasonal water stress from the ambient precipitation cycle)having the greatest resin production and intermediate growth.Contrary to our prediction, however, we found no relationshipbetween twig resin volume and tree radial growth (Fig. S2). Inter-estingly, we found a weakly significant, bell-shaped relationshipbetween bole resin volume and tree radial growth (Fig. S1b). Inconclusion, our findings of no relationship between twig resinflow and bole resin flow, and differing treatment effects on resinflow from the bole and twigs, strongly suggest that resin synthesisand flow are compartmentalized within pi~non trees, and argueagainst attempts to infer the defense capability of an entire treefrom resin flow measured on a single location or tissue.

The greater bark and twig beetle attacks on pi~nons in theH2O� treatment than in other treatments may be explained bytreatment effects on tree characteristics other than resin flow. Forinstance, I. confusus, the attacking bark beetle in our study, mayselect hosts via olfactory or auditory cues from drought-stressedtrees (Kimmerer & Kozlowski, 1982; Wood, 1982a; Tyree et al.,1984; Mattson & Haack, 1987; Stumpf & Johnson, 1987;Tadege et al., 1999; Seybold et al., 2006). Furthermore, resinquality (e.g. terpenes and crystallization rate) or production ofinduced defenses, both of which could be impacted by tree waterstress, may be more important than constitutive resin to beetleattack success (Raffa & Berryman, 1982; Lieutier, 2002). Theimportance of induced defenses for pi~non, which could includethe production of additional resin after constitutive resin hasbeen drained by initial attacks, is not known, although studies ofanother southwestern pine (Pinus ponderosa) have shown littleinduction of resin flow by traumatic bole wounding or inocula-tion with beetle-vectored fungi (Wallin et al., 2003; Gaylordet al., 2011). Moreover, other studies have reported a positiveassociation between sapwood resin duct abundance and pine

Fig. 6 Mean� SE cumulative needle browning on juniper trees at theSevilleta, NM field site by date and treatment (H2O+, water addition plots;H2O�, water removal plots; C, cover control; A, ambient; n = 60/date).Treatments were initiated in August 2007 as indicated by the arrow on thegraph. MANOVA showed that there was a significant treatment and dateinteraction (Wilks’ lambda, P < 0.0001), and therefore each date wasanalyzed individually. Treatment was not significant in 2008 (P = 0.13),but was significant in 2009 and 2010 (P < 0.0001 for both, as indicated by* on graph). Treatments within each date labeled with different letters aresignificantly different as determined by Student’s t-tests with Bonferroni-corrected a = 0.0083.

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survival during drought and bark beetle attacks (Kane & Kolb,2010; Kl€ay, 2011; A. K. Macalady, unpublished data). Our studyfound that trees that died had smaller resin ducts than those thatsurvived, and most duct parameters were reduced by the H2O�treatment by the third year, but not earlier (Fig. 3). In contrast toother studies (Blanche et al., 1992), our study did not find a sig-nificant relationship between constitutive resin flow and resinduct parameters (Fig. S1c-f).

We measured pi~non twig resin because of previous reportsthat twig resin is important for tree resistance to stem-boringmoths (Whitham & Mopper, 1985; Mopper et al., 1991; Brownet al., 2001; Mueller et al., 2005b). Tree damage from stemmoths was minor in our study (ranging from 0 to 5% foliageimpacted on a tree for any year); however, twig beetle attackswere common in dying pi~nons and may have contributed tomortality. To our knowledge, no studies have assessed the effectof twig resin on twig beetle performance or host selection. Wefound no difference in twig resin flow between trees that wereattacked by twig beetles and those that were not (P = 0.32). Thisresult suggests little functional importance of twig resin flow totree resistance to twig beetles, although more research is neededon this topic.

Extremes of water availability in our study had no effect onjuniper resin flow, which is consistent with current understandingthat juniper maintains gas exchange during drought (e.g.McDowell et al., 2008; Plaut et al., 2012). We also found noevidence of bark beetles in junipers, suggesting that susceptibilityto insect attacks was unchanged by water availability. Damageassociated with the juniper twig pruner (twig dieback) occurredat the beginning of our study before the onset of treatments, butthis damage did not increase during the study and few junipersdied over the 3 yr of the study, consistent with reports that thisCerambycid is generally not associated with tree death (Furniss& Carolin, 1977). In addition, there was some evidence ofBuprestids on dead junipers, as indicated by ovoid as opposed tocircular exit holes; however, no western cedar borers (a Buprestidassociated with juniper mortality) were collected in Lindgren fun-nel traps (Lindgren, 1983), which we deployed at the study sitein 2010 for monitoring purposes (n = 4, two with I. confusus lure(cis-verbenol, 50/50 ipsdienol and 50/50 ipsenol), and two witha general wood borer lure (ethanol ultra high release (UHR) andalpha pinene gel UHR)) (Contech Enterprise, Victoria, BC,Canada). Although we know little about juniper resistance toinsect attacks, our study and others (e.g. Floyd et al., 2009) haveconcluded that insect damage is not a strong contributor tojuniper decline during drought.

Greater needle browning of junipers in the H2O� treatmentthan in other treatments over 3 yr clearly shows sublethal impactsof drought on juniper. Our finding of drought-induced canopyloss in juniper, but not whole-plant mortality, is consistent withprevious nonexperimental field observations in the southwesternUSA after severe drought in 2002 (Koepke et al., 2010). Whileour results show that mature one-seed juniper can survive at least3 yr with 45% of ambient precipitation, the intensity and dura-tion of drought required for substantial juniper mortality are notyet known.

Implications

Climate predictions for the southwestern USA suggest thatdroughts will be more frequent and severe as a consequence ofglobal warming (e.g. Seager et al., 2007). Our results providestrong experimental evidence that 1 or more years of droughtwith 45% less than ambient precipitation will kill many pi~nons,and 3 yr of the same drought treatment will cause substantialcanopy loss of one-seed juniper and suggest increased mortalityin the future. Differences in mortality among species could leadto fundamental ecosystem shifts in dominant plants of wood-lands and also changes in associated communities dependent onthese plants (Allen & Breshears, 1998, 2007; Brown et al.,2001). Furthermore, biotic agent impacts may lag drought,which may provide opportunities for future investigators toexamine the correlation between droughts and bark beetle out-breaks more fully (e.g. Williams et al., 2012). Although ourstudy cannot address whether bark beetles were the ultimateagent of tree mortality during drought, our study shows thatdrought predisposes pi~non trees to bark beetle attacks. Further-more, increased temperatures, which occur often with drought inthe southwestern USA, could increase bark beetle populationsand tree mortality via increases in insect reproductive rate andgeographic range (Ayres & Lombardero, 2000; Tran et al., 2007;Bentz et al., 2010). Increased availability of susceptible hosts forbark beetle attacks during warming and drought could lead toincreased bark beetle outbreaks and further landscape-scale treemortality.

Acknowledgements

Thanks to K. Barrett, J. Hockersmith, M. McKinney, N. Gehres,and J. Kane for assistance with laboratory and field work. Thanksto the Northern Arizona University Statistical Consulting laband Chonggang Xu (Los Alamos National Labs) for assistancewith statistical analyses. We also thank Matt Ayres and two anon-ymous reviewers for valuable comments. Support for this researchwas provided by grants from the National Institute of ClimaticChange Research (NICCR), Department of Energy (DOE)Terrestrial Carbon Program, and Drought Impacts on RegionalEcosystems Network (DIREnet via NSF). The precipitationmanipulation experiment was funded by grants to N.G.McDowell (LANL) and W.T. Pockman (UNM) by the Office ofScience (BER), US DOE.

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Supporting Information

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

Fig. S1 Scatter plots for the relationships between resin volumeand pi~non tree size, growth, and resin duct parameters.

Fig. S2 Scatter plot for the relationship between tree-ring growthand twig resin volume for pi~non pine trees at the Sevilleta fieldsite.

Fig. S3 Mean (SE) weight of juniper resin flow collected afterphloem wounding on the main tree bole over a 24-h period atthe Sevilleta, NM field site.

Table S1 Total precipitation, including supplemental irrigation,by treatment for plots during post-treatment years 2008–2010,at the Sevilleta, NM field site

Table S2 Mean� SE predawn water potential (MPa) for juniperand pi~non trees at the Sevilleta, NM field site during the growingseason (May–September) in post-treatment years 2008–2010

Table S3 P value, df terms and F-test values for MANCOVA forthe effects of treatment, date, and their interaction on pi~non boleresin, pi~non twig resin, carbon isotope ratio (d13C) of pi~non boleresin, d13C of leaf sugars, and juniper bole resin for trees at theSevilleta, NM field site

Table S4 P value, df terms and approximate F-test values forMANCOVA for the effect of the interaction of treatment anddate on pi~non resin duct characteristics from increment cores col-lected at the Sevilleta, NM field site

Table S5 P value, error df, and F ratio from ANCOVA for theeffect of treatments and the pretreatment covariate on pi~non resin

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duct characteristics from increment cores (n = 28–35) collected atthe Sevilleta field site

Table S6 Comparison (ANOVA) of measured variables forpi~non pine trees at the Sevilleta, NM field site successfullyattacked or unattacked by pi~non ips bark beetles (Ips confusus)during the study years (2008–2010)

Table S7 Comparison of predawn water potential measurements(Ψpd) for pi~non pine trees successfully attacked and unattackedby pi~non ips bark beetles (Ips confusus) at the Sevilleta, NM fieldsite during the study years (2008–2010)

Table S8 Results of ANOVA for the effect of treatment on nee-dle browning for juniper trees at the Sevilleta field site in NewMexico, USA (n = 60 yr�1) for post-treatment years 2008–2010

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

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