Volatile organic compounds emitted from silver birch of different provenances across a latitudinal gradient in Finland

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Tree Physiology 00, 1–12doi:10.1093/treephys/tpv052

Volatile organic compounds emitted from silver birch of different provenances across a latitudinal gradient in Finland

Mengistu M. Maja1,3, Anne Kasurinen1, Toini Holopainen1, Sari Kontunen-Soppela2, Elina Oksanen2 and Jarmo K. Holopainen1

1Department of Environmental Science, University of Eastern Finland, PO Box 127, Kuopio, Finland; 2Department of Biology, University of Eastern Finland, PO Box 111, Joensuu, Finland; 3Corresponding author ([email protected])

Received December 21, 2014; accepted May 11, 2015; handling Editor Jörg-Peter Schnitzler

Climate warming is having an impact on distribution, acclimation and defence capability of plants. We compared the emission rate and composition of volatile organic compounds (VOCs) from silver birch (Betula pendula (Roth)) provenances along a latitu-dinal gradient in a common garden experiment over the years 2012 and 2013. Micropropagated silver birch saplings from three provenances were acquired along a gradient of 7° latitude and planted at central (Joensuu 62°N) and northern (Kolari 67°N) sites. We collected VOCs emitted by shoots and assessed levels of herbivore damage of three genotypes of each provenance on three occasions at the central site and four occasions at the northern site. In 2012, trees of all provenances growing at the cen-tral site had higher total VOC emission rates than the same provenances growing at the northern site; in 2013 the reverse was true, thus indicating a variable effect of latitude. Trees of the southern provenance had lower VOC emission rates than trees of the central and northern provenances during both sampling years. However, northward or southward translocation itself had no significant effect on the total VOC emission rates, and no clear effect on insect herbivore damage. When VOC blend composition was studied, trees of all provenances usually emitted more green leaf volatiles at the northern site and more sesquiterpenes at the central site. The monoterpene composition of emissions from trees of the central provenance was distinct from that of the other provenances. In summary, provenance translocation did not have a clear effect in the short-term on VOC emissions and herbivory was not usually intense at the lower latitude. Our data did not support the hypothesis that trees growing at lower latitudes would experience more intense herbivory, and therefore allocate resources to chemical defence in the form of inducible VOC emissions.

Keywords: climate change, common garden, insect herbivory, latitudinal translocation, silver birch, VOC emission

Introduction

Recent climate warming has triggered changes in physiology, dis-tribution and phenology of insect species ( Parmesan et al. 1999, Parmesan and Yohe 2003). These changes facilitate outbreaks and northward expansion of herbivorous insects that can cause widespread defoliation of boreal forest trees ( Neuvonen et al. 1999, Volney and Fleming 2000, Jepsen et al. 2008). Damage caused by herbivores ( Paré and Tumlinson 1999) and extreme abiotic stress ( Peñuelas and Staudt 2010) can alter the quality and quantity of plant-emitted volatile organic compounds (VOCs).

Emission rates of monoterpenes (MTs) ( Laothawornkitkul et al. 2009), (E)-4,8-dimethyl-1,3,7-nonatriene [(E)-DMNT], sesqui-terpenes (SQTs) and green leaf volatiles (GLVs) ( Paré and Tumlinson 1997) are known to vary as a result of insect herbivory or exposure to elevated temperature in a range of plant species.

Silver birch (Betula pendula Roth) constitutes a significant component of the boreal forest biome and its proportion of the overall forest area is predicted to increase by 10–20% in Finland and other Scandinavian countries by the year 2100 ( Kellomäki et al. 2001, 2008). This species is under intense pressure from

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aphids and pole-ward migrating geometrid moth species, out-breaks of which are highly sensitive to climate ( Neuvonen et al. 2005, Hagen et al. 2007, Jepsen et al. 2008). Recent studies have revealed that silver birch emits a diverse array of VOCs in response to herbivory ( Vuorinen et al. 2007, Blande et al. 2010, Maja et al. 2014) and high temperature ( Eka et al. 2010, Ibrahim et al. 2010) and VOCs emitted by silver birch shoots in response to abiotic stress like warming and ozone vary among different genotypes ( Hartikainen et al. 2012). However, little is known about how transplanting genotypes of different silver birch provenances along a latitudinal gradient affects levels of herbivore damage and VOC emissions of trees in the face of a changing climate.

Plant VOC emissions vary at the regional scale depending on the type and composition of the plant community ( Geron et al. 2000, Staudt et al. 2004). In the future, changes in tem-perature-sensitive biochemical processes and longer growing seasons ( Myneni et al. 1997) along with a sufficient level of precipitation are expected to stimulate biomass production of forests ( Kellomäki et al. 2001, Kirilenko and Sedjo 2007). Fur-thermore, changes in local conditions affect the physiological state of plants leading to differences in photosynthesis, growth rate, chlorophyll content and other biochemical processes vital for VOC emission. Common garden experiments along the latitu-dinal gradients offer a powerful tool to assess the effects of a warming climate, including changes in the dynamics of coloniza-tion by herbivorous insects, on northern silver birch populations ( Nooten et al. 2014). In such experiments, translocation of plant provenances from higher latitudes towards lower latitudes mim-ics a shift to future warmer climatic conditions.

The aim of this study was to assess the effect of translocation of silver birch provenances along a latitudinal gradient of 7° in Finland on the emission rates and composition of VOCs under field conditions. We also assessed the relationship between natu-ral herbivore damage of trees selected for VOC collection and the VOC emission rates during the two growing seasons. Plants also emit VOCs from belowground parts to defend themselves against biotic agents and facilitate interaction with other organisms ( Wenke et al. 2010). Since VOC emission rates from the rhizo-sphere (roots and microbial community) may also be altered by changes in environmental conditions, we studied differences in VOC emission from the rhizospheres of the trees at the central site. Our hypotheses were (i) that the quality and quantity of VOC emissions would differ between experimental sites and among silver birch provenances so that northward translocation of southern (Loppi 60°N) and central (Vehmersalmi 62°N) prove-nances would result in reduced VOC emissions while southward translocation of northern (Kittilä 67°N) provenances would result in increased VOC emissions and (ii) that there would be greater herbivore stress at lower latitudes than at higher latitudes result-ing in a greater induction of VOC emissions at lower latitudes and a change in composition of the blend might also happen.

Materials and methods

Common garden experiment: plant material and experimental design

Field plantations of silver birch saplings were established at three sites for a broad assessment of climate adaptation and interac-tions with insects ( Heimonen et al. 2014). The sites were in southern (Tuusula 60°21.5′N, 25°00.2′E), central (Joensuu 62°36′N, 29°45′E) and northern (Kolari 67°19′N, 23°46′E) Finland (Figure 1). This study, assessing VOC emissions, was only part of the broad climate adaptation and insect interaction assessment and was conducted only at central and northern sites. The sites differ in temperature, photoperiodic rhythm and soil characteristics (Tables 1 and 2). The central site is located in a botanical garden, with no mature trees in the surroundings. The northern site is an abandoned field surrounded by silver and downy birches as well as aspen, and the soil is relatively rich in nutrients. The average annual temperature sum, calculated as the sum of daily mean temperatures above +5 °C, varies between 1300 and 1500 at the southern site, between 1100 and 1300 at central site and between 700 and 800 at northern site ( Finnish Meteorological Institute 2015).

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Figure 1. Map showing provenance origins (filled circles, see text for abbreviations) and experimental sites (filled squares) where the experiment was established, Southern, Tuusula; Central, Joensuu; Northern site, Kolari.

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The birch saplings were cloned from dormant branches acquired from six natural Finnish forest populations in winter 2009. The six forests are located at Loppi (60°36′N, 24°25′E), Punkaharju (61°48′N, 29°19′E), Vehmersalmi (62°45′N, 28°10′E), Posio (65°53′N, 27°39′E), Rovaniemi (66°27′N, 25°14′E) and Kittilä (67°44′N, 24°50′E), traversing the latitudinal cline from 60° to 67° in Finland. Plantlets were replicated in vitro with a standard tissue culture method ( Ryynänen 1996), grown on ver-miculite at high humidity for 2 weeks and transferred into plastic trays filled with fertilized peat. Plantlets in plastic trays were transferred to a greenhouse and acclimated to outdoor condi-tions before being transferred to the southern, central and north-ern sites in the summer of 2010. At each site the saplings were planted into five blocks (randomized block design), each block containing two replicates of each genotype. Altogether, there were 260 plantlets at each site with a 1.2 m distance between plantlets. More information on the experimental setup is given in Heimonen et al. (2014).

Collection of VOCs

In this study, only three provenances (Loppi = LO, Vehmer-salmi = VE and Kittilä = KI) were used to represent the latitudinal cline of Finland (Figure 1). It was not possible to collect VOC samples from all provenances for logistical reasons. Volatile organic compound collections were made at two sites, Kolari and Joensuu, from three genotypes per provenance and by using

one tree per genotype per provenance from each block. Thus, 3 provenances × 3 genotypes × 5 blocks = 45 trees from each site were sampled in total.

Volatile organic compounds emitted by shoots were sampled from branches using a portable volatile collection system ( Himanen et al. 2010) during the summers of 2012 and 2013. In 2012, VOCs were sampled on two occasions (2–4 July 2012 and 6–7 August 2012) at the northern site and once (23–24 July 2012) at the central site. In 2013, VOCs were sampled twice at both sites (11–12 June 2013 and 19–20 August 2013 at the central site, and 2–3 July 2013 and 4–5 August 2013 at the northern site). For VOC collection, a 30–40 cm length of side branch at the top of the canopy was enclosed in an oven-cleaned (1 h, at 120 °C) polyethylene terephthalate (PET) bag (size, 25 × 55 cm). Teflon® air inlet tubes were inserted next to the branch and the bag opening was tied around both branch and inlet tube with wire. Inlet air was filtered through a charcoal filter and a MnO2 scrubber and was pumped into the bags at a flow rate of 600 ml min−1 for 10 min to flush the bags. The change of airflow before collection might affect photosynthesis, but we had to compromise to get enough replicates in two mea-surement days and this procedure was the same for all the sam-ples. After flushing, stainless steel tubes filled with 150 mg Tenax TA adsorbent (Supelco, Bellefonte, PA, USA) were inserted into the PET bags through holes in the bag corners and secured in position with wire. The air inlet flow rate was reduced to 300 ml min−1 and a sampling line was attached to the Tenax TA filled tube. Air was drawn through the tube for 20 min at a rate of ∼200 ml min−1 with a vacuum pump (Thomas 5002 12V DC). Sample tubes were sealed with Teflon-coated brass caps immediately after collection, kept in a cold box during transpor-tation and stored in a refrigerator until analysis. Temperature, relative humidity and photosynthetically active radiation were monitored during VOC sampling as background data for calcula-tions (Hobo Micro Station, Onset Computer Corporation, Bourne, MA, USA). Since the same branches of the selected trees were used throughout the two growing seasons, VOC samplings were performed in a non-destructive way.

Volatile organic compounds in the rhizosphere soil were sam-pled twice at the Joensuu site (on the 26–27 July 2012 and

VOC emissions of silver birch across a latitudinal gradient 3

Table 1. Soil characteristics of the study sites. Soil samples were taken from each of the five blocks per site and the numbers represent range or median (source: Heimonen et al. 2014).

Central site (Joensuu)

Northern site (Kolari)

Soil type Fine sandy till Sandy tillOrganic matter content (%) 3–6 6–12pH 6.0–6.1 4.9–5.5Calcium (mg l−1) 870 380Phosphorus (mg l−1) 8.5 20.8Potassium (mg l−1) 46 59Magnesium (mg l−1) 100 29Sulphur (mg l−1) 4.4 8.7Soluble nitrogen (kg ha−1) 11.8 7.6

Table 2. Temperature sum (GDD5, i.e., growth degree days over 5 °C) and photoperiod of different VOC measurement rounds in 2012 and 2013 at the two study sites (source: Finnish Meteorological Institute). Mean temperature and photosynthetically active radiation for measurement days were calculated from Hobo data.

Year Study site Measurement date

Temperature sum until measurement date

Photoperiod (measurement date)

Temperature (°C)

PAR (µ mol m−2 s−1) (measurement dates)

2012 Central 23 July 1064 18 h 22 min 23.3 738.2North 6 August 903 18 h 25 min 21.3 831.0

2013 Central—first collection 11 June 519 19 h 49 min 16.7 533.0Central—second collection 19 August 1679 15 h 51 min 30.6 859.3North—first collection 2 July 673 24 h 00 min 22.6 658.2North—second collection 4 August 1136 18 h 45 min 27.4 689.4


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16–17 July 2013). For these measurements, the rhizosphere zone of the same trees considered for collection of VOCs emit-ted by shoots were used (N = 45, n = 5 trees per genotype per provenance). One week before the first samples were taken in 2012, the root zones of the trees were weeded, and plastic col-lars (diameter 9 cm) were inserted carefully into the soil to an approximate depth of 2 cm. In order to estimate the background emissions from the soil, collars were established at two separate rootless sites in the same field ∼3 m away from the birch trees. Each collar was covered with a mesh cloth to prevent accumula-tion of litter on the soil surface, and if needed, the soil was weeded again approximately an hour before the second mea-surement in 2013. Collection of VOCs from the rhizosphere soil was done by fastening pre-cleaned PET bags to the collars with rubber bands. Air inlet holes were cut at the bag corners and tubes were inserted through the holes and fastened in position with wire. After fixing the PET bag in position, a similar VOC col-lection technique to that used for collection of VOCs emitted from shoots was employed, but with a collection time of 1 h.

Gas chromatography-mass spectrometry analysis

All VOC samples were analysed by gas chromatography-mass spectrometry (GCMS) (GC type 6890, MSD 5973: Hewlett Packard, Wilmington, DE, USA) as described in Blande et al. (2010). Identification of compounds was made by comparing the mass spectra with those of compounds in the Wiley library and with authentic external standards. The emission rates of MTs and SQTs that were not in the external standard were calcu-lated using α-pinene and trans-β-caryophyllene as reference compounds, respectively. In order to calculate the VOC emission rates of shoots in nanograms of compound per square metre of leaf material per second (ng m−2 s−1), leaf area was determined for the shoots used in the VOC collections. Immediately after VOC collection, leaves of the enclosed shoot were photographed with millimetre marked paper as a background for scaling pur-poses. The ImageJ program (version 1.46r) was used to calcu-late the leaf areas from the photos. Emission rates of VOCs from the rhizosphere were quantified in nanograms per square metre of soil surface area per second (ng m−2 s−1). Volatile organic compound emission rates from all shoots were adjusted to a standard temperature of 30 °C with a β value of 0.09 K−1 for MTs and 0.18 K−1 for SQTs ( Guenther et al. 1993, Duhl et al. 2008, Mäntylä et al. 2008), and any emissions found in blank samples were subtracted from the emission collected both from shoots and rhizosphere to determine the actual emission.

Herbivory assessment and plant growth

Natural insect herbivory was assessed in leaves of the branches used in the VOC sampling at both central and northern sites. Immediately after VOC collection, the leaves were photographed with millimetre marked paper as a background for scaling. An estimation of herbivore damage was made by counting the

number of damaged leaves as well as the total number of leaves enclosed in PET bags. We opted to focus only on the number of damaged leaves due to various types of herbivory (sap sucking, leaf mining and galling) in the field, which made calculation of damaged area difficult. In addition to herbivory, plant height mea-surements were made at the end of the growing season both in 2012 and 2013.

Statistical analyses

Volatile organic compounds emitted from shoots (total amount of MTs, GLVs, SQTs, all VOCs and VOC composition), herbivore damage and plant height growth data were tested in linear mixed models ANOVA (LMM ANOVA) where the fixed factors were experimental site and provenance, and the random factors were tree identity and the nested term genotype (site × provenance). The VOC collections could not be conducted simultaneously at the two sites (Kolari and Joensuu), so the two collection dates closest to each other at the different sites were considered a single measurement. The first VOC collection at the Kolari site in 2012 has no comparative collection performed at the Joensuu site so the samples were used to assess provenance effect. Dif-ferences in VOC composition were tested by principal compo-nent analysis (PCA, SIMCA-P 11.5; Umetrics AB, Umeå, Sweden) in order to obtain scores that were then used as testable vari-ables in LMM ANOVA. In PCA, before extraction of loadings and scores, data were centred and unit variance scaling was per-formed. Since rhizosphere VOCs were only measured in one site, data were tested with an LMM ANOVA design, where the only fixed factor was provenance, and tree identity and the nested term genotype (provenance) were used as random factors. The differences between means were considered to be statistically significant at P < 0.05 and marginally statistically significant at P < 0.1. A natural logarithmic transformation was performed for the VOC data before tests as the data were not normally distrib-uted. Pearson’s correlation test was run to determine the rela-tionship between the level of herbivore damage and VOC emission rates during each measurement at both sites. Pearson’s correlation test was also run to show the relationship between plant height and VOC emission rates. Linear mixed models ANOVA and correlation tests were performed with IBM SPSS Statistics 19 for Windows (International Business Machines Corp., Armonk, NY, USA).

Results

VOC emission rates

In the second measurement of 2012, shoots of all provenances growing at the central site emitted more MTs, SQTs and total VOCs than those growing at the northern site (significant site main effect, Figure 2a and b, Table 3). In 2013, trees at the northern site emitted significantly more GLVs and total VOCs than those growing at the central site during both measurements,

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but MT and SQT emissions were more variable (Figure 2c–f, Table 3). In the first measurement of 2013, MT emissions were higher at the central site than at the northern site, but there was

no clear site difference for SQT emissions. However, in the last measurement, both MT and SQT emissions were significantly higher at the northern site than at the central site (Figure 2c–f,


Figure 2. Mean (±SE) standardized VOC emission rates (ng m−2 s−1) of total MTs, SQTs, total GLVs and total VOCs from B. pendula of three prove-nances at both sites: (a) 2012 central site, (b) 2012 northern site, (c) first collection of 2013 at central site, (d) first collection of 2013 at northern site, (e) second collection of 2013 at central site, (f) second collection of 2013 at northern site. LO, Loppi (southern provenance); VE, Vehmersalmi (central provenance); KI, Kittilä (northern provenance).


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Table 3). A significant main effect of provenance was also seen for the emission rates of total VOCs (Table 3) as the southern provenance (Loppi) emitted less VOCs than the other two prov-enances (Vehmersalmi and Kittilä) in the three latter measure-ments across 2012 and 2013. The total VOC emissions from shoots of the Vehmersalmi and Kittilä provenances followed a similar trend to the total MT emissions, as these provenances emitted MTs during most of the VOC measurements (prove-nance main effects P < 0.05 in the second collection of 2013, and P < 0.1 in 2012 and first collection of 2013 (Table 3, Figure 2)).

The rates of VOC emission from the rhizosphere were low (ranging from 0.00 to 0.09 ng m−2 s−1 among the provenances in 2012 and 0.00–0.53 ng m−2 s−1 in 2013), and there were no statistically significant differences between the provenances on any of the compounds (data not shown). In 2012, only two GLVs (cis-3-hexenol and 1-hexanol) and four SQTs (longifolene,

aromadendrene, α-farnesene and α-humulene) were emitted from the rhizosphere at a concentration level that our analytical detection system was able to quantify. In 2013, two MTs (sabi-nene and limonene), six SQTs (α-copaene, longifolene, trans- β-caryophyllene, aromadendrene, α-farnesene and α-humulene) and two GLVs (cis-3-hexenol and cis-3-hexenyl acetate) were detected from samples of the rhizosphere.

Composition of the VOC blend emitted by shoots

The relative contribution of different compound groups to the blend of VOCs emitted by shoots varied over the two growing seasons with total GLVs accounting for 73, 94 and 21% of the total VOC emission at the northern site in 2012, and the first and second collections of 2013, respectively. At the central site, the proportion of total GLVs in the overall VOC blend was lower than at the northern site, accounting for only 16, 26 and 43% of the total emission for the respective measurements. In contrast, terpenoids (MTs and SQTs) were emitted in larger pro-portions at the central site especially in 2012 and the first col-lection of 2013, accounting for 84 and 74% of the total VOCs, respectively.

In the first collection of 2012, done in Kolari only, the compo-sition of the MT and SQT + GLV components of the VOC blend emitted by shoots did not statistically differ between the prove-nances (Table 4). However, PCA combined with LMM ANOVA showed that there was a statistically significant (P < 0.05) main effect of experimental site in the second measurement of 2012 as provenances at the central site emitted a more diverse blend of MTs than their counterparts at the northern site (Table 4, Figure 3a, PC1 for the measurement in 2012 explained ∼51% of data variation). The Vehmersalmi provenance emitted more MTs such as β-ocimene, neo allo-ocimene, limonene and (E)-DMNT at the central site in particular, but there was no sta-tistically significant site × provenance interaction. Similarly, in the first collection of 2013 (Figure 3b, Table 4), marginally significant site and statistically significant main effects of prov-enance were detected for MT composition. In this collection, the Vehmersalmi provenance was only separated from the other

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Table 3. Linear mixed model results (P-values) of total emission rates for main effects (experimental site and provenance) and their interaction effects on VOCs and herbivory: MTs, monoterpenes; SQTs, sesquiter-penes; GLVs, green leaf volatiles. P-values <0.1 are shown in bold font.

Collections Total MTs

Total SQTs

Total GLVs

Total VOCs

Herbivory

2012 (Kolari only) Provenance 0.628 0.177 0.433 0.524 0.533

2012 Site 0.013 0.000 0.470 0.001 0.140 Provenance 0.092 0.429 0.120 0.004 0.124 Site × provenance 0.523 0.260 0.766 0.261 0.062

2013 (1) Site 0.027 0.186 0.000 0.001 0.000 Provenance 0.060 0.490 0.014 0.013 0.700 Site × provenance 0.260 0.635 0.147 0.509 0.315

2013 (2) Site 0.000 0.000 0.000 0.000 0.080 Provenance 0.034 0.374 0.138 0.005 0.955 Site × provenance 0.264 0.397 0.288 0.529 0.345

Table 4. Linear mixed model results of VOC blend composition emitted by shoots (MTs and SQTs + GLVs) over two growing seasons. P-values for main effects (experimental site and provenance) and their interaction effect (experimental site × provenance origin) are given. P-values <0.1 are shown in bold font.

P values 2012 (Kolari only) 2012 2013 (1) 2013 (2)

PC1 PC2 PC1 PC2 PC1 PC2 PC1 PC2

MTs Site n/a n/a 0.043 0.967 0.092 0.547 0.000 0.000 Provenance 0.775 0.832 0.142 0.979 0.043 0.527 0.001 0.011 Site × provenance n/a n/a 0.388 0.288 0.165 0.463 0.024 0.079

SQTs + GLVs Site n/a n/a 0.000 0.072 0.000 0.714 0.000 0.731 Provenance 0.185 0.153 0.604 0.817 0.080 0.701 0.820 0.021 Site × provenance n/a n/a 0.406 0.991 0.372 0.304 0.850 0.024


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provenances at the central site because it emitted more MTs such as α-pinene, γ-terpinene, α-terpinolene and β-myrcene. In the second collection of 2013 (Figures 3c and 4c), MT

emissions showed a statistically significant interaction effect of experimental site × provenance (P < 0.05; Table 4, PC1 explained 56% of data variation) due to trees of the Vehmersalmi


Figure 3. Principal component analysis biplot diagrams showing the loading plot of shoot MTs (MT compound loadings indicated by multiplication symbols and names of compounds) superimposed on the score plot of three provenances at two sites: (a) 2012, (b) first collection of 2013, (c) second collection of 2013. KI, Kittilä; VE, Vehmersalmi; LO, Loppi; C, central site (Joensuu); N, northern site (Kolari).


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provenance emitting more MTs such as β-myrcene, cineole and camphene at the central site. Moreover, there was a marginally significant interaction effect of experimental site × provenance (P < 0.1; Table 4, PC2 explained 17% of data variation) due to trees from the Kolari provenance emitting more MTs

such as limonene, β-pinene and neo allo-ocimene at the central site.

In the second measurement of 2012, a statistically significant (P < 0.05) main effect of experimental site was detected on the composition of the blend of SQTs + GLVs (Table 4, Figure 4a,

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Figure 4. Principal component analysis biplot diagrams showing the loading plot of shoot SQTs + GLVs (SQT and GLV compound loadings indicated by multiplication symbols and names of compounds) superimposed on the score plot of three provenances at two sites: (a) 2012, (b) first collection of 2013, (c) second collection of 2013. KI, Kittilä; VE, Vehmersalmi; LO, Loppi; C, central site (Joensuu); N, northern site (Kolari).


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PC1 explained ∼33% of the data variation). This effect was due to trees of all provenances at the central site emitting more SQTs than northern site-based trees, while at the northern site trees emitted more GLVs than their counterparts at the central site. In the first measurement of 2013, the composition of the blend of SQT + GLV was significantly affected by site, due to GLVs such as cis-3-hexenol, cis-3-hexenyl acetate and cis-3-hexenyl butyrate being emitted more by trees growing at the northern site than those growing at the central site (Figure 4b). There was also a marginally significant main effect of provenance on the composi-tion of the blend of SQT + GLV emitted in 2013, due to trees of the Kittilä and Vehmersalmi provenances emitting a more diverse blend of SQTs and GLVs than trees of the Loppi provenance. However, in the last measurement of 2013, the composition of the blend of SQT + GLVs (site × provenance interaction, P < 0.05; Table 4, PC2 explained 18% of data variation) was only influenced by provenance at the northern site, due to trees of the Loppi and Vehmersalmi provenances emitting more GLVs than trees of the Kittilä provenance. At the central site, SQT + GLV composition was similar among the different provenances.

Herbivore damage and plant growth

There was a marginally significant interaction effect of experi-mental site × provenance (P < 0.1) on the percentage of leaves damaged by herbivores during the sampling period of 2012 (Figure 5, Table 3). In 2012, trees of the Kittilä and Vehmer-salmi provenances had similar levels of herbivore damage at both sites, with damage level generally being highest for trees of the Kittilä provenance. Trees of the Loppi provenance had vary-ing degrees of herbivore damage across the latitudinal gradient with more herbivore damage occurring at the northern site than the central site. In the first measurement of 2013, trees of all provenances had significantly more herbivore damaged leaves at the northern site than at the central site (site main effect, Table 3, Figure 5). During the second measurement of 2013, there was a marginally significant main effect of site due to all provenances having slightly more herbivore damage at the cen-tral site, but the difference in herbivore damage between the two sites was clearer for the Loppi provenance. Trees of all prove-nances grew taller at the central site than at the northern site during both assessment years (Table 5). There was also a sta-tistically significant main effect of provenance due to trees of both Loppi and Vehmersalmi provenances being taller than trees of the Kittilä provenance during both years. In 2013, northward translocation of Loppi and Vehmersalmi provenances signifi-cantly reduced height growth, whereas Kittilä provenance trees were unaffected by southward translocation.

Effect of herbivore damage and plant growth on VOC emission

A Pearson correlation test showed that there was a weak negative correlation between herbivore damage level and the emission

rates of total MTs (Pearson correlation coefficient (r) = −0.302, n = 45, P < 0.05) and total GLVs (r = −0.316, n = 43, P < 0.05) in the second collection of 2012 at the central site and the first col-lection of 2013 at the northern site, respectively. Total VOC emis-sion showed a weak positive correlation with herbivore damage at the central site for the first collection of 2012 (r = 0.365, n = 44, P < 0.05) and a weak negative correlation at the northern site in the first collection of 2013 (r = −0.317, n = 43, P < 0.05). Simi-larly, Pearson’s correlation tests between plant height growth and VOC emission rates for the last measurement of 2013 showed that


Figure 5. Mean (±SE) percentage of damaged leaves on the branches enclosed in PET bags during the VOC sampling that had been damaged by herbivores.

Table 5. Mean ± SE of final plant height of silver birch saplings at the end of each growing season at Joensuu and Kolari sites during 2012–13. C, central Joensuu site; N, northern Kolari site; 60 LO, Loppi provenance; 62 VE, Vehmersalmi provenance; 67 KI, Kittilä provenance). P-values < 0.05 are shown in bold font.

Plant height (cm) (2012)

Plant height (cm) (2013)

Site C 191.4 ± 7.0 319.3 ± 11.6 N 147.3 ± 5.3 244.4 ± 8.2Provenance 60 LO 192.3 ± 8.4 320.8 ± 15.1 62 VE 179.9 ± 8.4 298.1 ± 12.4 67 KI 135.8 ± 4.8 226.7 ± 7.5Site × provenance C-LO 230.6 ± 6.9 391.9 ± 10.2 N-LO 154.0 ± 6.2 249.7 ± 11.4 C-VE 203.9 ± 7.2 334.7 ± 9.9 N-VE 156.0 ± 11.5 261.5 ± 17.6 C-KI 139.7 ± 6.1 231.3 ± 10.9 N-KI 131.8 ± 7.5 222.1 ± 10.5P-values 2012 2013Site 0.001 0.000Provenance 0.002 0.001Site × provenance 0.107 0.027


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there was a negative correlation between plant height growth and emission rates of total GLVs (r = −0.341, n = 45, P < 0.001) and a moderate negative correlation between plant height growth and emissions of total SQTs (r = −0.502, n = 45, P < 0.05).

Discussion

Our results indicate that southward or northward translocation of silver birch provenances along a latitudinal gradient had no sig-nificant effect at least in the short-term on the temperature- standardized emission rates of VOCs. Thus the results did not support our first hypothesis of translocation-induced changes in VOC emission rates. However, VOC blend composition was sig-nificantly affected by translocation in the second collection of 2013 when all provenances emitted more GLVs at the northern site than at the central site. If this was indicating an overall higher herbivore stress in plants growing at higher latitudes, then the result was totally opposite to our second hypothesis. Indeed, the proportion of leaves was more damaged in the northern site dur-ing the first sampling in 2013. The central provenance emitted more total VOCs, whereas the southern provenance had the low-est total VOC emission rates at both sites during the sampling period, contrasting with the widely accepted view that plants of lower latitudes are better defended against biotic stress.

Experimental site and provenance affected VOC emissions

According to the existing literature, plants experience more biotic stress and therefore develop more defence traits in low-latitude ecosystems ( Coley and Aide 1991, Schemske et al. 2009, but see Moles 2013). However, our results did not show consistently higher VOC emission at the central site throughout the sampling period. There was large variation in VOC emission rates between the two sites with emission rates being higher at the central site in 2012 and at the northern site in 2013. This variation in VOC emission between the two sites could be driven by differences in many environmental variables including accumulated tempera-ture sum, light level, photoperiod and edaphic factors (Tables 1 and 2) ( Loivamäki et al. 2007, Winter and Rostás 2010). Poten-tial adjustment of biological processes that lead to VOC synthesis could have been driven by short-term variability in light and tem-perature at both experimental sites. These adjustments can drive changes in VOC substrate pools, whereas the long-term acclima-tion responses arise from selection pressure on genes that encode terminal enzymes involved in VOC synthesis ( Li and Sharkey 2013). We observed an increase in GLV emissions with increasing latitude as all provenances had higher emissions at the northern site than the central site. This trend contrasts with a previously observed decrease in the emission rates of GLVs from milkweed with increasing latitude ( Wason et al. 2013).

Studies of tree provenances belonging to different species have shown a geographical pattern for MT concentration and emission rates ( Manninen et al. 2002, Kivimäenpää et al. 2012). For

example, MT-storing Scots pine (Pinus sylvestris L.) trees of a southern provenance emitted higher rates of MTs than trees of a central and northern origin ( Kivimäenpää et al. 2012). This con-trasts with our current finding in which the southernmost (Loppi) provenance had the lowest VOC emission rates of the provenances studied. Trees of the Loppi provenance were the tallest at both sites during both experimental years (Table 5), which suggests that more resources might be allocated to growth than to VOC emission in trees of this provenance, whereas the trade-off between height growth and VOC emission was not clear for trees of other provenances. The variation in VOC emission among trees of the different provenances could also be attributed to biogeo-graphical diversity ( Loreto et al. 2009), degree of herbivore dam-age, plant part under herbivore attack ( Niinemets et al. 2013), type and developmental stage of herbivore species as well as growing conditions ( Takabayashi et al. 1994, Geervliet et al. 1997).

In general, long-distance latitudinal transfer has a negative impact on trees ( Ovaska et al. 2005, Viherä-Aarnio and Velling 2008, but see also Rousi et al. 2012), enhancing the suscepti-bility of trees to various environmental stresses. Both biotic and abiotic factors could differentially impact growth, chemical com-position and herbivore damage intensity and thus contribute to variation in the concentration and composition of VOC blends emitted by shoots. In this study, northward or southward transfer of different silver birch provenances along a latitudinal gradient of 2–7° did not cause statistically significant changes in VOC emission rates throughout the sampling period. However, in the last collection of 2013, VOC blend composition showed changes along a latitudinal gradient with trees of the Kittilä prov-enance emitting more compounds at the central site and trees of the Loppi and Vehmersalmi provenances emitting more com-pounds at the northern site. The effects of tree provenance translocation and herbivore damage were not reflected in the emission rate or blend composition of rhizosphere VOCs. Vola-tile organic compounds emitted from the rhizosphere were at comparable rates to those from the rhizospheres of silver birch reported in Maja et al. (2014), but in this study GLVs were also detected in samples, probably due to damage of root tissues while preparing the site and setting up the collection lines.

Impact of herbivore damage on VOC emission

We found greater emission of GLVs, such as cis-3-hexenol, cis-3-hexenyl acetate and cis-3-hexenyl butyrate, at the northern site, which could be partly explained by greater herbivore dam-age at the northern site as these compounds are usually induced by herbivore feeding ( Paré and Tumlinson 1999, Holopainen 2004, Laothawornkitkul et al. 2009). However, we found posi-tive correlations only between total VOC emissions and herbiv-ory during the first measurement of 2012 at the central site. The VOC emission responses to herbivore damage are likely affected by growth conditions and availability of resources ( Niinemets et al. 2013). It should also be noted that some of the damage

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might have developed over a long period of time so that the responses of damage were not necessarily reflected in the VOC emission rates during our sampling. Green leaf volatiles from birch foliage are typically emitted immediately after damage to plant tissues ( Maja et al. 2014). Green leaf volatiles may also be released when cell membranes are damaged during insect ovi-position ( Rodriguez-Saona et al. 2002), by wind or mechani-cally ( Copolovici et al. 2011).

There was a separate herbivory assessment conducted by Heimonen et al. (2014, 2015) on trees in the same experimen-tal sites during the summers of 2011 and 2012. They esti-mated the mean leaf area consumed by herbivores for each tree and calculated the means for trees of the different provenances and genotypes. In their assessment, herbivory increased down the latitudinal gradient in 2011, which is in line with the widely accepted view, whereas in 2012 there was no latitudinal gradi-ent effect. The herbivore damage assessment presented here was conducted directly on the branches enclosed inside the bags with a view of better explaining the variation in VOC emis-sions, whereas the other damage assessment was conducted on leaves of the leading shoot and the upper branches, which may explain the differences.

This field study has some limitations including the difficul-ties of controlling factors such as wind and cloudy conditions during measurement which might have an impact on the emis-sion rates. We were unable to measure VOCs from the south-ern site because there was high plant mortality due to a fungal disease, which would have increased the latitudinal range other-wise. Moreover, our VOC collection has some constraints includ-ing mechanical induction of VOCs while fixing the lines and changes in air flow rates between flushing the bags and sampling VOCs. Finally, more frequent monitoring of VOC emission would have given us more confidence in verifying our hypotheses.

Conclusions

The results of this study suggest that emission rates of VOCs from silver birch provenances were not affected by translocation, at least in the short-term, but emission rates differed between the two experimental sites. Herbivore damage did not decrease along the latitudinal gradient as predicted and had no clear effect on VOC emission during the experimental period. An important exception is that plant translocation affected VOC blend during the last collection in 2013. We recommend further studies of larger latitudinal gradients and longer monitoring peri-ods to see clear emission responses to biotic and abiotic envi-ronmental factors along latitudinal gradients.

Acknowledgments

Thanks are due to the staff of Natural Resource Institute of Finland (Luke) in Haapastensyrjä and H. Hakulinen at the UEF

for micropropagation and producing the plantlets. We also thank all the staff of Luke in Kolari and Botanical Garden in Joensuu for maintaining the plants in the field. The authors thank Dr James Blande for his constructive comments and editing the language.

Conflict of interest

None declared.

Funding

This research was supported by the strategic funding of the University of Eastern Finland, project number 931050.

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Volatile organic compounds emitted from silver birch of different provenances across a latitudinal gradient in Finland