The Lodgepole × Jack Pine Hybrid Zone in Alberta, Canada: A Stepping Stone for the Mountain Pine Beetle on its Journey East Across the Boreal Forest?

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Journal of Chemical Ecology ISSN 0098-0331 J Chem EcolDOI 10.1007/s10886-013-0334-8

The Lodgepole × Jack Pine Hybrid Zone inAlberta, Canada: A Stepping Stone for theMountain Pine Beetle on its Journey EastAcross the Boreal Forest?

Inka Lusebrink, Nadir Erbilgin & MayaL. Evenden

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The Lodgepole × Jack Pine Hybrid Zone in Alberta, Canada:A Stepping Stone for the Mountain Pine Beetle on its JourneyEast Across the Boreal Forest?

Inka Lusebrink & Nadir Erbilgin & Maya L. Evenden

Received: 11 July 2013 /Revised: 19 July 2013 /Accepted: 23 July 2013# Springer Science+Business Media New York 2013

Abstract Historical data show that outbreaks of the treekilling mountain pine beetle are often preceded by periodsof drought. Global climate change impacts drought frequencyand severity and is implicated in the range expansion of themountain pine beetle into formerly unsuitable habitats. Itsexpanded range has recently reached the lodgepole × jackpine hybrid zone in central Alberta, Canada, which could actas a transition from its historical lodgepole pine host to a jackpine host present in the boreal forest. This field study testedthe effects of water limitation on chemical defenses of maturetrees against mountain pine beetle-associated microorganismsand on beetle brood success in lodgepole × jack pine hybridtrees. Tree chemical defenses as measured by monoterpeneemission from tree boles and monoterpene concentration inneedles were greater in trees that experienced water deficitcompared to well-watered trees. Myrcene was identified asspecific defensive compound, since it significantly increasedupon inoculation with dead mountain pine beetles. Beetlesreared in bolts from trees that experienced water deficitemerged with a higher fat content, demonstrating for the firsttime experimentally that drought conditions benefit mountainpine beetles. Further, our study demonstrated that volatilechemical emission from tree boles and phloem chemistryplace the hybrid tree chemotype in-between lodgepole pine

and jack pine, which might facilitate the host shift fromlodgepole pine to jack pine.

Keywords Mountain pine beetle . Range expansion .

Drought . Tree defenses . Beetle condition

Introduction

Global climate change has allowed a vast number of plant andanimal species to extend their range into formerly unsuitablehabitats (Parmesan 1996, 2006; Stange and Ayres 2001), includ-ing the mountain pine beetle (hereafter MPB), Dendroctonusponderosae Hopkins (Coleoptera: Curculinoidae, Scolytinae)(Cudmore et al. 2010; Cullingham et al. 2011). The MPB isthe most destructive insect pest of pine forests in western NorthAmerica (Safranyik and Carroll 2006). During the most recentoutbreak, the MPB killed an estimated 18.1 million ha of mainlylodgepole pine (Pinus contorta Dougl. ex. Loud.) forests inBritish Columbia (BC, www.for.gov.bc.ca). The MPB is nativeto western North America, and its historical range within Canadais in central BC, west of the Rocky Mountains (Safranyik et al.2010). The unprecedented source population that established inBC in the late 1990’s promoted the dispersal of beetles eastwardinto the neighboring province of Alberta where MPB initiated amassive invasion of the north-eastern slopes of the RockyMountains in 2002 (Safranyik et al. 2010). In Alberta, MPBhas continued to expand its range eastward and attack maturepine trees in an area, where lodgepole pine and a closely relatedpine species, jack pine (Pinus banksiana Lamb), overlap andhybridize (Mirov 1956).Within this lodgepole × jack pine hybridzone, MPB has attacked both hybrid and genetically-pure jackpine trees (Cullingham et al. 2011). Jack pine is the major pinespecies in the boreal forest of Canada, and extends from northernAlberta to eastern Canada and to the Great Lakes Region of theUnited States. Despite the fact that jack pine represents a novel

I. Lusebrink :M. L. EvendenDepartment of Biological Sciences, University of Alberta, CW405Biological Science Building, Edmonton, Alberta, Canada T6G 2E9

N. ErbilginDepartment of Renewable Resources, University of Alberta, 4-42Earth Science Building, Edmonton, Alberta, Canada T6G 2E3

I. Lusebrink (*)Centre for Biological Sciences, University of Southampton,Life Sciences Building 85, Southampton, UK SO17 1BJe-mail: [email protected]: [email protected]

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host for MPB, it can support beetle colonization and develop-ment, as well as growth of theMPB-associated fungal symbionts(Cerezke 1995; Rice et al. 2007a).

The impact of global climate change on drought frequencyand severity (Breshears et al. 2009) may facilitate further rangeexpansion of MPB, as drought has been linked to eruptivepopulation dynamics of several insect species (Mattson andHaack 1987), including MPB (Alfaro et al. 2010). Drought issuspected to increase the susceptibility of forests to insectherbivores through a decrease in the expression of tree defenses(Thomson and Shrimpton 1984). One climate-suitability mod-el, originally developed in 1975 (Safranyik et al. 1975) andmodified in 2004 (Carroll et al. 2004), includes water deficit asa factor that benefits beetle fitness under the assumption thatwater deficit reduces tree resistance (Safranyik et al. 2010).However, the effect of drought-stressed host trees on the colo-nization and development of MPB has never been testedexperimentally.

Tree resistance in conifers to bark beetle attacks is basedprimarily on chemically-mediated tree defenses and can corre-late with monoterpene content of individual trees (Gollob 1980;Schiebe et al. 2011; Sturgeon 1979; Zhao et al. 2011).Monoterpenes are a major constituent of conifer resin alongwith sesquiterpenes and diterpenoid resin acids (Bohlmann2012; Keeling and Bohlmann 2006) that act as both physicaland chemical barriers against bark beetles. However, massattacks triggered by MPB aggregation pheromones and inocu-lation of phloem and xylem with its fungal symbionts canrapidly deplete host tree defenses (Boone et al. 2011; Raffaand Berryman 1983). Female MPB produce the aggregationpheromone that is needed for mass attack by hydroxylatingα-pinene, an abundant monoterpene in the Pinaceae, to trans-verbenol, which attracts both sexes of MPB (Blomquist et al.2010; Pitman et al. 1968). During the subsequent colonizationprocess, arriving males produce exo-brevicomin, which attractsmore females until the ideal attack density is reached, at whichtime both male and female MPB emit anti-aggregation phero-mones that prevent further colonization of the host tree(Rudinsky et al. 1974; Ryker and Libbey 1982). α-Pineneand other monoterpenes such as 3-carene, terpinolene, andmyrcene also synergize the response of MPB to its aggregationpheromones (Borden et al. 2008), thus MPB has evolved toexploit the primary chemical defenses of its host tree to its ownadvantage.

MPB is associated with several symbionts including thefungi Grosmannia clavigera , Ophiostoma montium , andLeptographium longiclavatum (Khadempour et al. 2012; Leeet al. 2005; Rice and Langor 2008; Six and Klepzig 2004),bacterial symbionts, and yeasts (Adams et al. 2008). The fungihelp the beetle deplete tree defenses (Reid et al. 1967; Rice et al.2007b), reduce sap flow (Yamaoka et al. 1990), and serve as anutrition source for MPB larvae (Adams and Six 2007; Bleikerand Six 2007) and teneral adults (Paine et al. 1997; Whitney

1971). Some of the bacteria might have the potential to helpMPBovercome tree defenses (Adams et al. 2013). Alternatively,bacteria and yeasts can affect the distribution of symbiotic fungiin the host tree, which subsequently might influence MPBfitness (Adams et al. 2008). Because of the close associationbetween MPB and its fungal symbionts, artificial inoculation oftrees with those fungi often is used to simulate beetle attack as aproxy to study tree defenses (Boone et al. 2011; Lieutier et al.2009; Wang et al. 2013).

Due to the proven impact of drought on host plant-herbivorous insect interactions, we further tested the hypothesisthat tree defense stimulation will vary between well-wateredtrees and those that experience water deficit. To test this hy-pothesis, we conducted a field experiment at the front of theeastward range expansion of MPB in Alberta, Canada withinthe lodgepole × jack pine hybrid zone with the followingobjectives: (1) to develop a chemical profile of volatile organiccompounds (VOCs; primarily monoterpenes) released from thebole of lodgepole × jack pine hybrids; (2) to evaluate variationof volatile chemical profiles of different water (water-deficit vs.well-watered) and biological treatments that stimulate tree de-fense; (3) to determine whether the monoterpene content ofphloem and needle tissue is affected by the water treatments;and (4) to assess whether water and biological treatmentsapplied to trees affect MPB brood success.

Methods and Materials

To understand the potential importance of water limitation totree defense response against invading organisms, weconducted a field study in the summer of 2009 at a site 25 kmnorth-west of Whitecourt, Alberta, Canada (54°13.595’ N,116°03.148’ W). In this area, the ranges of lodgepole and jackpine overlap, and the species hybridize (Mirov 1956). Fortymature putative lodgepole × jack pine hybrid trees with anaverage diameter at breast height (DBH) of 23.9 cm±2.52 SDwere selected to investigate our objectives.

Water Treatments Selected trees were randomly assigned toone of two water treatment groups: well-watered and water-deficit trees (N=20 for each treatment group). For a continuouswater supply, a slow release watering bag setup (treegator®,Spectrum Products Inc., Youngsville, NC, U.S.A.) with a watercapacity of 160 L was attached to the well-watered trees. Thebags were filled with water from the nearby Athabasca River ona biweekly-basis from 5 June–20 August, 2009. Soil at the baseof the water-deficit trees was covered with a tarpaulin (size:12’×14’ (3.66m×4.27m); G. HjukstromLimited, Surrey, B.C.,Canada) to limit ambient water supply. Soil water content(SWC) around each tree, as well as at three randomly selectedspots in the field site, was monitored using time domain reflec-tometry (TDR) (Hillel 1998). The apparent dielectric constant of

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the soil wasmeasured by using stainless steel probes at depths of30, 60, and 90 cm with a Tektronix 1502B (Beaverton, OR,USA) and related to water content using an empirical equationfor mineral soils (Robinson et al. 2003). Soil water content datafor trees that experienced the different water treatments wasanalyzed using a nested ANOVA with probe length (30, 60,90 cm) and time (0 day, 1 day, 2, 5, and 7 wk) during the seasonspecified as random factors. The differences in SWC betweenwater treatments were analyzed using subsequent pairwise com-parisons with Bonferroni correction. All statistical analyses wereconducted using SPSS 19.0 for Windows (IBM Corporation,Armonk, NY, USA), unless otherwise stated.

Biological Treatments Five weeks after the water treatmentswere initiated, five trees in both water treatment groups wereadditionally exposed to one of four biological treatments: (1) noinduction control; (2) mechanical wounding only; (3) mechan-ical wounding followed by fungal inoculation withGrosmanniaclavigera ; or (4) mechanical wounding followed by inoculationwith mashed beetles alternated with G. clavigera inoculation.Inoculation with live beetles was not permitted in this geograph-ic area at the time of the experiment. Thus, we used mashedbeetles and associated fungal symbionts to simulate MPB at-tacks. All trees, with the exception of the control trees, werewounded ten times with a cork borer (1 cm diam) evenly spacedaround the bole at breast height. Fungal inoculations wereconducted withG. clavigera by placing a malt extract agar plugwith active fungal mycelium into the wound with the myceliumfacing the sapwood. For the mashed beetle treatment, freshlyemerged MPBs were killed by freezing them over night at−20 °C the day before inoculation. This approach would leaveonly the cold-hardy symbionts of the MPB. Late instar larvae,the overwintering stage, canwithstandwinter temperatures closeto −40 °C (Safranyik and Carroll 2006), so their fungal symbi-onts also should be able to tolerate similar low temperatures.Grosmannia clavigera and Leptographium longiclavatum , re-sist freezing at −20 °C for a 3-mo period (Rice et al. 2008).Frozen beetles were tranported to the field site on ice, and20 MPBs per each inoculation point were mashed and placedin every other wound of designated trees; the remaining woundswere inoculated with G. clavigera . This allowed a direct com-parison between the resulting lesions caused by the two treat-ments within the same tree.

Volatile Collection and GC Analysis To determine tree chem-ical response to the water and biological treatments, we collect-ed VOCs emitted from the bole of each tree at the followingtime points: one day before application of biological treatments,4 d, 2, 3, 5, 7, and 9 wk post biological treatments. To enablevolatile collection, two strips of 1 cm thick foam material(Quilting foam, Fabricland, Edmonton, AB, Canada) wereattached to each tree: one 15 cm above and one 15 cm belowthe biological treatment application site on the tree bole. An

oven bag (LOOK®, 45×55 cm) made of inert material was cutopen and wrapped around the tree covering both pieces of foamand secured. An adsorbent tube (Porapak Q (OD 6 mm, length110 mm; absorbent: front layer 150 mg, back up layer 75 mg;separated by glass wool) SKC Inc., Pennsylvania, USA) wasinserted underneath the foam into the space covered by theoven bag and attached to a pump secured to the tree withVelcro. Bole volatiles were collected for 1 h at a flow rate of1 L/min before biological treatment application. Emission ratesof VOCs were greatly enhanced after biological treatmentapplications, and therefore, collection time was reduced to15 min. After VOC collection, the sorbent tubes were cappedand stored on ice and transferred to a freezer in the lab at−40 °Cbefore extraction. Porapak Q tubes were extracted with 1 mL ofdichloromethane (Sigma-Aldrich, St. Louis, MO, USA) spikedwith 0.01 % (v/v) tridecane (Sigma-Aldrich, St. Louis, MO,USA) as surrogate standard and subsequently stored at −40 °Cbefore GC analysis.

Extracted VOC samples (1 μl) were injected in an Agilent7890A Gas Chromatograph (Agilent Technologies, SantaClara, CA, USA) with an HP Innowax column (I.D.0.32 mm, length 30 m; Agilent Technologies), helium carriergas flow at 1.8 ml/min, temperature 50 °C for 2 min, increasedto 160 °C by 5 °C per min, and then ramped up to 250 °C by20 °C. Peaks were identified using the following standards:Borneol, pulegone, α-terpinene, γ-terpinene, α-terpineol(Sigma-Aldrich, St. Louis, MO, USA), camphor, (+)-3-carene, α-humulene, terpinolene, α- and β-thujone, (−)-α-pinene, (−)-β-pinene, (S )-(−)-limonene, sabinene hydrate,myrcene, (−)-camphene, p -cymene (Fluka, Sigma-Aldrich,Buchs, Switzerland), bornyl acetate, cis -ocimene (SAFCSupply Solutions, St. Louis, MO, USA), β-phellandrene(Glidco Inc., Jacksonville, FL, USA). Calibration with thesestandards allowed for analysis of quantitative differences ofvolatile samples at different time points among treatments.The total monoterpene emission was log(x+1) transformed tomeet the assumptions of normality. A two-way repeated mea-sures ANOVA was conducted to account for repeated mea-surements of volatile emission from the same trees over thecourse of the experiment. The last two time points wereomitted from the analysis because they did not meet theassumption of homogeneity, and transformation did not re-solve the matter. Therefore, the composition of monoterpeneemissions at all time points as impacted by water and biolog-ical treatments of mature trees was analyzed using a canonicalredundancy analysis (RDA) with the rdaTest package(Legendre and Durand 2010) in R (R Development CoreTeam 2012). RDA axes were tested for significance by per-mutations with the vegan package (Oksanen et al. 2010).Explanatory variables included water and biological treat-ments, as well as the following tree variables: DBH, phloemthickness, and age. Temperature and humidity from a nearbyEnvironment Canada weather station for the days of volatile

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collection also were used as explanatory variables. The quan-tities of all individual monoterpene released at all time pointswere the response variables.

Tissue Samples and GC/MS Analysis At the end of the exper-iment, 9 wk post biological treatment application, all experi-mental trees were harvested and lesion dimensions from inoc-ulated trees were recorded. Phloem tissue was sampled from thearea between two lesions, and needles were collected frommid-crown of the tree, shock frozen in liquid nitrogen, and trans-ferred onto dry ice before storage at −40 °C in the lab prior toextraction. Tissue was ground in liquid nitrogen, and 100 mg ofthe tissue was transferred to a 1.5 ml microcentrifuge tube andextracted as described in Lusebrink et al. (2011). Extracts weretransferred into amber GC vials (Agilent Technologies) andstored at −40 °C before Gas Chromatograph/Mass Spec-trometer (GC/MS) analysis.

Extracts of tissue samples (3 μl) were injected at a split ratioof 20:1 in an Agilent 7890A/5062C GC/MS (AgilentTechnologies; the MS was not available during VOC collec-tion) with an HP-Chiral-20B column (I.D. 0.25 mm, length30 m; Agilent Technologies), helium carrier gas flow at 1.1 ml/min, temperature 75 °C for 15 min, increased to 230 °C by5 °C. Calibration with the standards used in GC-analysis andadditionally: 4-Allylanisole (also named estragole), (+)-α-pi-nene, (+)-β-pinene, (R)-(+)-limonene (Fluka, Sigma-Aldrich,Buchs, Switzerland), and α-phellandrene (SAFC SupplySolutions, St. Louis, MO, USA) allowed the quantification oftissue chemical content, as well as the analysis of differences instereoisomer composition of the differently treated trees.

Total carbon (dry combustion) and total nitrogen (Dumascombustion method) of phloem samples were determined bythe Natural Resources Analytical Laboratory (Department ofRenewable Resources, University of Alberta, Edmonton,Canada) using an ECS 4010 Elemental Combustion SystemCHNS-O (Costech Analytical Technologies Inc., Valencia, CA,USA).

Lesion lengths from the variously inoculated trees werecompared using a two-way nested ANOVA with lesion type(fungal and MPB mash caused lesion) specified as a randomfactor nested within biological treatment. The total and indi-vidual amounts of monoterpenes extracted from phloem didnot fulfill the assumptions of normality or homogeneity evenafter transformation. Therefore, the data were analyzed usingnon-parametric Kruskal-Wallis followed by Mann–WhitneyU tests as a post-hoc procedure. Additionally, we conducted aprincipal component analysis (PCA) based on the ratios of theindividual monoterpenes. The phloem chemistry dataobtained in the current study were compared to those of purelodgepole and jack pine trees retrieved in the same way from arelated field study conducted in 2010 and not reported here.The quantities of monoterpenes extracted from needles metthe assumptions of anANOVA after log (x+1) transformation.

A MANOVAwas conducted on the needle data that includedindividual and total monoterpenes as response variables.

Total nitrogen and carbon content of the phloem from thevariously treated trees were compared with a two-way ANOVAfollowed by Tukey’s HSD as post-hoc test in order to determinethe differences among biological treatments.

Beetle Condition in Experimentally Manipulated Trees To testthe effect of water and biological treatments on the condition ofMPB, a 50 cm bolt was harvested from just above the inocu-lation site of each inoculated tree. Bolts were cut from untreatedcontrol trees at the same height. All bolts were transported tothe laboratory where both ends of each bolt were covered inparaffin wax to avoid desiccation. They were stored in awalk in growth chamber (22 °C, 50 % humidity,16:8 h L:D). Each bolt was inoculated with four pairs of livefemale and male MPB. One female MPB was introduced toeach of 4, 1.5 ml microcentrifuge tubes attached to the lowerportion of the bolt. When it had burrowed in, a male MPB wasadded. Beetles were replaced if introduction was not success-ful. Bolts inoculated with beetles were kept in the growthchamber for 4–5 wk to allow early instar development, andthen were subjected to a cold period at 4 °C and constantdarkness to emulate winter conditions. Following a 3 mo coldperiod, bolts were transferred into rearing bins in the growthchamber to allow the offspring of the mating pairs to completetheir development. Emerging adult beetles were measured forfresh weight, size (pronotum width and total body length) andsex, and then were killed and stored in a freezer. Dead beetleswere oven dried for 24 h at 60 °C before extraction withpetroleum ether using a 250 ml soxhlet apparatus to determinefat content as percentage of removed dry weight. Fat content ofemerged beetles was compared among treatments with a 2-way ANOVA followed by Tukey’s HSD as post-hoc test.After all beetles emerged, the number of larval galleries wasalso assessed for all bolts. Age of harvested trees was deter-mined by scanning a cross section harvested from the base ofeach tree on a flatbed scanner followed by analysis withWinDENDRO™ (Regent Instruments Inc., Quebec, Canada).

Results

Soil Water Content Soil water content differed significantlywith water treatment applied to trees (F (2,6.217)=11.674,P=0.008; Fig. 1), but not with soil depth (F (6, 36.010)=1.558,P=0.188). Soil water content was lower across all sample depthsin the soil surrounding water-deficit trees compared to well-watered trees; the latter did not differ from SWC in the rest ofthe field site.

Volatile Emission As compared to VOCs emitted from purespecies (Jost et al. 2008; Lusebrink et al. 2011; Pureswaran

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et al. 2004; Rhoades 1990), those emitted from the hybridtrees in the current study represent a mixture of pure lodgepoleand jack pine monoterpene profiles (Table 1).

Total monoterpene emission was influenced by both water(F (1,32)=5.250, P=0.029; Fig. 2a) and biological (F (3,32)=62.434, P <0.001; Fig. 2b) treatments. Post-hoc tests withBonferroni correction revealed that trees that experienced awater deficit released more total monoterpenes than well-watered trees (Fig. 2a). Trees that were inoculated with thefungus G. clavigera , MPB mash or were mechanicallywounded, emitted greater amounts of monoterpenes thanuntreated control trees (P <0.001). The emission of volatilesfrom trees inoculated with fungus at 10 points around the boledid not differ significantly from trees inoculated with fungusand MPB mash, each at 5 points around the bole. The level ofmonoterpene emission from mechanically wounded treeswithout inoculum was lower than fungal- inoculated trees(P <0.001) but similar to trees treated with MPB mash andfungus together (Fig. 2b). Emission of monoterpenes fromtrees also varied over time (F (3.026,96.828)=78.592, P <0.001),with a sharp increase in emission following biological

treatment applications (Fig. 2c). There was also an interactionbetween sampling time point and biological treatments(F (9.078,96.828)=14.041, P <0.001; Fig. 2d). Monoterpeneemission among treated trees was similar shortly after treat-ment application. The emission went down over time inMPB-mash/fungus-inoculated and wounded trees, whereas fungal-inoculated trees continued to emit monoterpenes at high levelsuntil the end of the 3rd week. Even in fungal-inoculated trees,the VOC emission sharply declined by the 5th week followinginoculation.

A canonical redundancy analysis (RDA) tested the influenceof water and biological treatments on monoterpene emissionsfrom the variously treated trees. The RDA triplot illustrates therelationship between the explanatory variables and the emissionof individual monoterpenes from mature hybrid pine trees(Fig. 3). The correlation triplot could be considered as onedimensional because the first axis of the RDA was significant(P=0.001) but the second axis was only marginally significant(P=0.092) and explained less than 1 % of the variation in theRDA. Emission of α-pinene, 3-carene, β-pinene, and β-phellandrene was correlated mainly with fungal inoculation

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Fig. 1 Soil water content (mean±95 % CI) at three different soil depths (30, 60, and 90 cm) over the time course of the experiment. Bars with non-overlapping error bars are significantly different from each other

Table 1 Percentages of selected monoterpenes as part of the entire monoterpene profile emitted or extracted from the boles of mature lodgepole, jackand lodgepole × jack pine trees or entire seedlings determined in this and earlier studies

Species α-Pinene β-Pinene 3-Carene Limonene β-Phellandrene Source Reference

Lodgepole pine 8 9 7 3 49 Bole VOCs Rhoades 1990

17 35 n.a. 6 30 Bole VOCs Jost et al. 2008

5 16 6 6 50 Bole extracts Pureswaran et al. 2004

7 23 7 4 27 Seedling VOCs Lusebrink et al. 2011

Jack pine 91 7 n.a. 1 1 Bole VOCs Jost et al. 2008

27 21 21 9 1 Seedling VOCs Lusebrink et al. 2011

Hybrids 80 10 n.a. 5 1 Bole VOCs Jost et al. 2008

46 8 17 3 16 Bole VOCs This study

21 7 11 9 42 Bole extracts This study

n.a. data not available

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and water deficit, as well as with high ambient humidity andtemperature.

Lesion Length Lesions caused by inoculation with MPB-mash were shorter than those caused by fungal inoculation(F (1,25)=16.246, P <0.001; Fig. 4).

Tissue Extracts One of the trees in the water deficit × fungalinoculation treatment was naturally attacked by MPB duringour experiment and was removed from the analysis.

The phloem chemical profile of the ten control trees consistedof: β -Phellandrene (45.7 %±6.68 SE), (+)-α-pinene (14.3 %±3.09 SE), (S)-(−)-limonene (9.4 %±4.47SE), 3-carene (9.3 %±2.05SE), (−)-α-pinene (5.6 %±1.55SE), (−)-β-pinene (5.0 %±0.95SE), myrcene (3.2 %±0.31SE), and 7.5 % other monoter-penes, which contributed less than 2.5 % each to the chemicalprofile. The separation factor (α=retention time2/retentiontime1) for α-pinene was α=1.03, for β-pinene, α=1.03, andfor limonene, α=1.02.

There was no difference in total monoterpene concentrationof extracted phloem tissue based onwater treatments of the tree.Further analyses were conducted on trees pooled from bothwater treatments, which showed that the biological treatments

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Fig. 2 Bole monoterpeneemission (mean ± SE) frommature lodgepole × jack pinehybrids as a result of different awater treatments b biologicaltreatments c sampling time pointsd interaction of sampling timepoint and biological treatments.Different lowercase lettersindicate a statistically significantdifference (P<0.05, repeatedmeasures ANOVA)

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Fig. 3 Canonical redundancy analysis (RDA) triplot (scaling 2) illustratingthe influence of water and biological treatments as well as tree characteris-tics [age, phloem thickness, and diameter at breast height (DBH)] andclimate variables (temperature and humidity) on volatile emission of indi-vidual monoterpenes in mature lodgepole × jack pine hybrids

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affected the amount of myrcene (H (3)=8.864, P=0.031;Fig. 5a) and 3-carene (H(3)=8.130, P=0.043; Fig. 5b) in thephloem. Phloem tissue from trees treated with the MPB-mash/fungus treatment contained significantly more myrcene thanmechanically-wounded and untreated control trees. 3-Carenewas higher in the phloem of MPB-mash/fungus-treated treesthan wounded trees but did not differ statistically from that

found in the phloem of control or fungal-inoculated trees.Fungal-inoculated trees had an intermediate amount of bothcompounds (Fig. 5a, b). Likewise, biological treatments causeda marginal increase in terpinolene and borneol in the phloem(H(3)=7.632, P=0.054 and H(3)=7.568, P=0.056).

Total monoterpenes occurred in higher concentrations in theneedles of trees that experienced a water deficit (F (1,37)=6.437,P=0.016, Fig. 5c). This differencewas caused by elevated levelsof major individual monoterpenes, such as (−)- and (+)-α-pinene(F =7.660, P =0.009 and F =6.126, P =0.018), myrcene(F=8.640, P=0.006), 3-carene (F=6.063, P=0.019), (−)-β-pinene (F =4.632, P=0.038), and bornyl acetate (F =9.113,P=0.005). The water treatment had no influence on the amountof β-phellandrene (P=0.237), the signature compound oflodgepole pine.

The principal component analysis (PCA) conducted on thephloem chemistry data separated lodgepole and jack pines(Fig. 5d). The hybrid trees analyzed in the current study, clus-tered between the two pure species. Some of the hybrid treesshowed more similarity with lodgepole pine than jack pine.

Elemental Analysis Total carbon content of phloem tissue fromthe variously treated trees was not influenced by either the wateror biological treatments. In contrast, total nitrogen was affected

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by the biological treatments (F (3,32)=5.693, P=0.003, Fig. 6)andwas higher in tissues from fungal-inoculated trees (P=0.003)than in control orMPBmash/fungus trees.Wounded trees had anintermediate level of nitrogen in the phloem (Fig. 6). The phloemof watered trees contained marginally higher amounts of N thantrees that experienced a water deficit (F (1,32)=3.573, P=0.068).

Beetle Condition in Experimentally Manipulated Trees Overall329 viable adult beetles, 210 females and 119 males, emergedfrom 39 bolts, 67 of them on the same day. Neither biologicalnor water treatment affected the number of beetles thatemerged, their fresh weight, size, or the number of larvalgalleries. However, the fat content of female beetles reared inbolts from the variously treated trees was affected by the watertreatment applied to standing trees (F (1,202)=11.185, P=0.001;Fig. 7). Female beetles, which are the pioneering sex in MPB,had a higher fat content when they emerged from water-deficittrees (23.86 %±8.47 SD) compared to the well-watered trees(19.53 %±9.60 SD). As expected, male beetles contained lessfat than females (15.86 %±7.79 SD), and their fat content wasnot influenced by biological or water treatments.

Discussion

The stem volatiles emitted from the experimental trees in thecurrent study represent a mixture of pure lodgepole and jackpine monoterpene profiles, although the chirality of these com-pounds was not measured (Jost et al. 2008; Lusebrink et al.2011; Pureswaran et al. 2004; Rhoades 1990). A recentgenotyping study of lodgepole, jack pine and their hybridsrevealed that the ancestry of hybrid trees in central Alberta isbiased towards lodgepole pine (Cullingham et al. 2012). Thephloem chemical composition of the majority of the hybrid

trees tested in this study appears closer to that of pure lodgepolethan to pure jack pine. MPB shares a long co-evolutionaryhistory with lodgepole pine (Kelley and Farrell 1998) and thus,has adapted to exploit the secondary chemistry of this hostspecies (Boone et al. 2011; Keeling and Bohlmann 2006).The most recent establishment of MPB in hybrid trees and afew jack pine trees within the hybrid zone (Cullingham et al.2011) might facilitate subsequent colonization of jack pine treesin the eastern boreal forest. The phloem chemistry of hybridtrees resembles a mixture of both pure species and may, there-fore, provide the perfect stepping stone to enable further rangeexpansion of MPB. Preference for chemical similarity during ahost shift occurs in several beetle-plant relationships (Becerra1997; Futuyma et al. 1995), including one other Dendroctonusspecies, D. valens (Erbilgin et al. 2007).

Drought conditions can influence insect-plant interactions(Mattson and Haack 1987) and may influence the rapidity ofrange expansion by MPB (Alfaro et al. 2010). Soil watercontent was significantly lower around water-deficit trees ascompared to well-watered trees. Water limitation and the bio-logical treatments of plant defense stimulation affected theemission of stem volatiles in the current field study on maturelodgepole × jack pine hybrids. Monoterpene emission from thestem of host trees is hypothesized to be more relevant for MPBhost-finding and colonization behavior than emission fromfoliage, since beetles mostly constrain their flight to the lowerbole of their pine hosts (Seybold et al. 2006). Water-deficit andfungal inoculation correlated with the emission ofα-pinene and3-carene from the stem of lodgepole × jack pine hybrid. Similarresults were found in a previous study with pine seedlings(Lusebrink et al. 2011). Both compounds act as kairomones(Borden et al. 2008) to the MPB through synergy with theaggregation pheromone, and may make the emitting host treemore attractive to aggregating beetles.

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Plants exposed to mild and moderate droughts are expectedto shift carbon allocation toward the production of secondarymetabolites, like monoterpenes (Monson et al. 1995). Thishypothesis is supported by the chemical analysis of the currentneedles in which needles from water-deficit trees contain ahigher monoterpene concentration than needles from well-watered trees. The results are in accordance with previousstudies that show that drought stress increases the concentrationof monoterpenes in the needles of several conifers, includingScots pine (Pinus sylvestris ; Turtola et al. 2003), Aleppo pine(Pinus halepensis ; Llusià and Peñuelas 1998), ponderosa pine(Pinus ponderosa ; Johnson et al. 1997), and Norway spruce(Picea abies ; Kainulainen et al. 1992). A study on loblolly pinein Louisiana (Lombardero et al. 2000) also concluded thatdrought is more likely to increase than decrease tree defenses.However, under severe drought conditions, carbon assimilation,and therefore the production (Herms and Mattson 1992) andemission (Blanch et al. 2007) of secondary metabolites, such asmonoterpenes, is predicted to decline.

The length of necrotic lesions in the phloem in response tofungal inoculation is a commonly usedmeasure of tree resistanceor fungal virulence (Krokene et al. 2008; Rice et al. 2007b).Resistant trees show a more efficient defense response thatrestricts fungal growth more swiftly inside shorter lesions thansusceptible trees (Krokene and Solheim 1998; Raffa andBerryman 1983), and longer lesions may indicate better perfor-mance of the fungus (Lieutier et al. 2004; Masuya et al. 2003;Rice et al. 2007b). Unlike water treatment, inoculation type hadan impact on lesion formation. In the MPB-mash/fungus treat-ment, inoculation with mashed MPB and G. clavigera werealternated around the bole of the same tree in order to comparedirectly the lesions created by inoculation with all cold hardyMPB-associated microorganisms (e.g., Adams et al. 2008; Riceet al. 2008) or byG. clavigera alone. MPB-mash causes smallerlesions than inoculation with G. clavigera, suggesting that thefungus alone is more virulent than the mash, most likely becauseof a higher inoculum load. Tree defense response was moreefficient against the MPB mash, and this response was specific.The monoterpene myrcene was evoked at significantly higherlevels in the mash treatment compared to controls, which indi-cates that myrcene is important for tree defense against microbesassociated with theMPB (Bonello and Blodgett 2003). 3-Carenealso was present in phloem surrounding lesions created byinoculation with the MPB-mash/fungus treatment, although notat significantly higher concentrations than in wounded trees. 3-Carene, in particular, might play a role in tree defense in maturelodgepole (Ott et al. 2011) and jack pines (Raffa and Smalley1995), as its concentration in the necrotic lesion tissue increasesupon inoculation with bark beetle-associated fungi. Interestingly,MPB uses both these defensive compounds: myrcene and 3-carene as kairomones for host location, since both synergizebeetle response to the aggregation pheromone trans-verbenol(Borden et al. 2008). Similarly, the pine shoot beetle, Tomicus

piniperda , identifies susceptible trees based on increased defen-sive compounds of its host tree (Byers et al. 1985).

Even though high monoterpene levels are toxic to manyherbivores (Langenheim 1994), the MPB has evolved toovercome these defenses through aggregation and mass attack(Pitman et al. 1968). The MPB-associated fungal symbiontshelp the beetle to detoxify phloem monoterpenes via fungalmetabolism, and also may use somemonoterpenes as a carbonsource (DiGuistini et al. 2011; Wang et al. 2013). The symbi-onts also benefit the MPB by enhancing phloem nutrition(Bleiker and Six 2007; Goodsman et al. 2012). The hyphaeof fungal symbionts of Dendroctonus species can increasenitrogen levels in brood galleries of bark beetle larvae throughredistribution of nitrogen from the sapwood and distant phlo-em (Ayres et al. 2000; Bleiker and Six 2007). In the currentstudy, phloem nitrogen levels were significantly higher infungal-inoculated trees compared to the control and MPB-mash/fungus treatments. Likewise, Entomocorticium sp.,one of the mycangial fungi associated with the southern pinebeetles,Dendroctonus frontalis , concentrates nitrogen aroundlarval galleries (Ayres et al. 2000). Consequently, D. frontalisassociated with Entomocorticium sp. develop into larger adultbeetles with higher fat content (Coppedge et al. 1995).

In this study, female beetles that emerged from bolts of treesthat received the water-deficit treatment had a higher fat contentthan beetles reared in bolts from well-watered trees. This is thefirst experimental evidence that MPB directly benefits fromwater-deficit conditions. Higher fat content in bark beetles isexpected to positively influence dispersal, colonization, andreproductive success (Graf et al. 2012). Atkins (1966, 1975)found that Douglas-fir beetles, Dendroctonus pseudotsugae ,behave differently depending on their fat content: 1) beetleswith a high fat content (above 20 %) have the tendency todisperse and respond less to host volatiles; 2) beetles withintermediate fat content (11–20%) respond immediately to hostvolatiles and are good flyers; and 3) beetles with low fat content(10 % and below) fail to fly. Female MPB from the water-deficit treatment have a fat content that is higher than 20 %,whereas the beetles from the well-watered treatment containless than 20 % fat. If there is a similar relationship between fatcontent and behavior in MPB, water-deficit would enhancedispersal and range expansion; but additional studies are neededto establish a link between fat content and MPB behavior. Barkbeetles can benefit from the effects of drought on host treesthrough: elevated nutrient levels, increased emission of plantvolatile attractants, reduced oleoresin exudation pressure, andimproved conditions for their symbionts (Mattson and Haack1987). Since we artificially introduced and reared beetles inbolts, volatile attractants and resin pressure can be excluded aspossible reasons for the link between high fat content andwater-deficit. Nitrogen levels were not the cause of this result,since phloem nitrogen levels are higher in well-watered than inwater-deficit trees. Other nutrients may play a role, and further

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research on the benefits of drought on bark beetle performanceis needed.

The range expansion of the MPB and the accompanyingcolonization of jack pine as a new host could alter beetlepheromone production and their ability to mass attack trees, astree-producedα-pinene is the precursor of theMPB aggregationpheromone trans-verbenol (Blomquist et al. 2010). The enan-tiomeric composition of terpenoid bark beetle pheromones de-pends on the stereochemistry of the precursor, the enantiomeric-specificity of the synthesizing enzymes, and enantiomeric-specific olfactory receptors (Byers 1989). In MPB (−)-trans-verbenol elicits a significantly higher response than (+)-trans-verbenol (Whitehead et al. 1989), for which the (−)-isomer ofα-pinene most likely acts as a precursor (Vaněk et al. 2005).Unfortunately, there are few studies on the chirality of chemicalprofiles of mature lodgepole or jack pine. Pureswaran et al.(2004) reported that the bole of mature lodgepole pine emits67.7 % of (−)-α-pinene. We found that the phloem of maturehybrids contains 36.8 % (−)-α-pinene and 63.2% (+)-α-pinene,which might have a negative impact on pheromone productionand attractiveness. Furthermore, jack pine occurs on extremelywell-drained, nutrient-poor soils (Kenkel et al. 1997; Vidacović1991), and the response to drought in jack pinemight differ fromthat observed in lodgepole × jack pine hybrids in the currentstudy. Further research on the effect of drought on tree defenseresponse of pure jack pines and the chemically-mediated inter-actions with MPB needs to be conducted.

Acknowledgments We acknowledge Adriana Arango, JeremiahBolstad, Janice Cooke, Christina Elliott, Matt Ferguson, Andrew Ho,Ed Hunt, Brad Jones, Jean Linsky, Boyd Mori, and William Sperlingfor their help before and during the field season;Miles Dyck for providingus with the TDR equipment; Celia Boone for sharingβ-phellandrene. Weparticularly acknowledge Adrianne Rice for providing fungal culture andknowledge; TimMcCready fromMillar Western Forest Products Ltd. forproviding a suitable field site; Jörg Bohlmann for comments on themanuscript. Funding for this research was provided through grants fromthe Government of Alberta through Genome Alberta, the Government ofBritish Columbia throughGenome BC and Genome Canada in support ofthe Tria 1 and Tria 2 projects (http://www.thetriaproject.ca) of whichMLE and NE are co-investigators.

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