Abstract

In the present experiment, the single and combined effects of elevated temperature and ozone (O3) on four silver birch genotypes (gt12, gt14, gt15 and gt25) were studied in an open-air field exposure design. Above- and below-ground biomass accumulation, stem growth and soil respiration were measured in 2008. In addition, a 13C-labelling experiment was conducted with gt15 trees. After the second exposure season, elevated temperature increased silver birch above- and below-ground growth and soil respiration rates. However, some of these variables showed that the temperature effect was modified by tree genotype and prevailing O3 level. For instance, in gt14 soil respiration was increased in elevated temperature alone (T) and in elevated O3 and elevated temperature in combination (O3 + T) treatments, but in other genotypes O3 either partly (gt12) or totally nullified (gt25) temperature effects on soil respiration, or acted synergistically with temperature (gt15). Before leaf abscission, all genotypes had the largest leaf biomass in T and O3 + T treatments, whereas at the end of the season temperature effects on leaf biomass depended on the prevailing O3 level. Temperature increase thus delayed and O3 accelerated leaf senescence, and in combination treatment O3 reduced the temperature effect. Photosynthetic : non-photosynthetic tissue ratios (P : nP ratios) showed that elevated temperature increased foliage biomass relative to woody mass, particularly in gt14 and gt12, whereas O3 and O3 + T decreased it most clearly in gt25. O3-caused stem growth reductions were clearest in the fastest-growing gt14 and gt25, whereas mycorrhizal root growth and sporocarp production increased under O3 in all genotypes. A labelling experiment showed that temperature increased tree total biomass and hence 13C fixation in the foliage and roots and also label return was highest under elevated temperature. Ozone seemed to change tree 13C allocation, as it decreased foliar 13C excess amount, simultaneously increasing 13C excess obtained from the soil. The present results suggest that warming has potential to increase silver birch growth and hence carbon (C) accumulation in tree biomass, but the final magnitude of this C sink strength is partly counteracted by temperature-induced increase in soil respiration rates and simultaneous O3 stress. Silver birch populations' response to climate change will also largely depend on their genotype composition.

Introduction

Anthropogenic activities are increasing greenhouse gas concentrations as well as altering the reflective properties of the earth's surface and hence accelerating global climate change (IPCC 2007). Average global air temperature has already increased by 0.74 °C from the preindustrial level and, based on IPCC projections, global mean temperatures are predicted to further increase 1.1–6.4 °C by the end of this century (IPCC 2007). According to the temperature projections for Finland (Jylhä et al. 2004, Tietäväinen et al. 2010), the increase in the annual mean temperature during the next 30 years will continue to be the same (0.6 °C per decade) as the observed warming during the last 30 years (0.7 °C per decade), and this warming is likely to be the strongest during winter months. Simultaneously with climate warming, background tropospheric ozone (O3) levels will continue to rise (Vingarzan 2004, The Royal Society 2008), and by 2100 global ground-level O3 concentrations are projected to increase from the current ∼40 ppb level by 40–60% (Meehl et al. 2007, Wittig et al. 2009). Tropospheric O3 concentrations in the Nordic countries are relatively low compared with ambient concentrations, e.g., in central and southern Europe (Laurila et al. 2004, Klumpp et al. 2006, Lindskog et al. 2007). However, longer summer days and a cooler and more humid climate in northern Europe compared with southern Europe promote O3 uptake by foliage, and therefore northern forests might be at increasing risk of negative O3 effects even in mild O3 stress (Karlsson et al. 2005, Matyssek et al. 2007). Tropospheric O3 is thus the most harmful phytotoxic air pollutant at the moment (Grantz et al. 2006, Matyssek et al. 2007), and also the greenhouse gas with the third strongest radiative forcing on climate (IPCC 2007). There is also a feedback between these two climate factors, as increasing tropospheric O3 level is further accelerating global climate warming (Meehl et al. 2007) and vice versa, warmer surface air temperatures will contribute to the increase in tropospheric O3 levels (Zeng et al. 2008).

Boreal forest is the second largest biome in the world and represents a significant terrestrial carbon (C) sink (Dixon et al. 1994, Janssens et al. 2003, Sitch et al. 2007), but responses of boreal forest trees to simultaneous warming and increases in tropospheric O3 concentrations are still uncertain. Tree growth in boreal regions is considered to be temperature limited (Lahti et al. 2005, Briceno-Elizondo et al. 2006), so it has been widely assumed that boreal forest trees might at least initially benefit from climate warming. However, temperature response will depend on other co-occurring environmental factors, how close trees are to their thermal optimum for growth, intensity and duration of temperature exposure, and tree species (Way and Oren 2010). In a recent meta-analysis, general though not universal trends were that increasing temperature stimulated stem height growth more than that of stem diameter, and more biomass was allocated to leaves relative to roots, causing also a reduction in root : shoot ratios (R : S ratios) (Way and Oren 2010). Warming can either directly (e.g., changing soil moisture conditions) or indirectly via altered C assimilation and allocation affect the growth patterns of roots and mycorrhizal fungi. In one published study on ectomycorrhizal fungi soil warming changed the dominance of an ectomycorrhizal community by altering the relative proportions of morphotypes (Rygiewicz et al. 2000), but there is no knowledge with regard to what happens when the warming is employed on the whole tree–soil system. A warmer growing seasons have been also suggested to change boreal forests from a C sink into a C source (Goulden et al. 1998) by enhancing CO2 efflux from soil (Rustad et al. 2001). Most soil-warming experiments have indeed shown an initial increase in soil respiration rates, but later this warming effect can be reduced when the pool of readily decomposable soil organic matter is exhausted (Melillo et al. 2002, Eliasson et al. 2005, Hartley et al. 2007, Bronson et al. 2008). However, there is also a link between tree physiology, C allocation and CO2 released via surface soil respiration (Högberg et al. 2001, Sampson et al. 2007), and therefore warming climate effects on soil respiration should be also assessed with air + soil warming experiments in which the whole system is warmed instead of using traditional soil-only warming designs.

Ozone effects on forest trees have been widely studied, and on the basis of these studies O3 stress is known to usually reduce tree growth and productivity (Cooley and Manning 1987, Oksanen et al. 2009, Wittig et al. 2009). Ozone stress effects on growth can be mediated through reduced C assimilation, impaired phloem loading, accelerated leaf senescence, and increased ­metabolic costs related to leaf repair and defence processes (Andersen 2003, Riikonen et al. 2004). Thus, the primary site for O3 action is in the foliage, whereas roots, mycorrhizas and soil respiration are affected through potential O3-induced changes in tree C allocation. Several studies also indicate that root growth might be affected first before any clear reductions in above-ground growth are seen (Andersen 2003, Yamaji et al. 2003, Oksanen et al. 2009). According to recent meta-analyses (Grantz et al. 2006, Wittig et al. 2009), a general, but highly variable trend was that also tree R : S ratios decreased due to O3 stress. Similarly, soil respiration responses to O3 stress have been more variable and previous studies report either no significant changes, decreased or increased soil CO2 emissions (Andersen 2003). However, even small O3 effects on growth or physiology in perennial species can cause large cumulative effects over time under chronic O3 exposures (Oksanen 2003, Oksanen et al. 2007).

In recent years, C isotopes as in situ tracers have become more common tools in ecological studies (Simard et al. 1997, Subke et al. 2009). During photosynthesis, plants discriminate against 13CO2 in favour of 12CO2 and as a consequence, their biomass becomes more 13C-depleted than atmospheric air (Farquhar et al. 1989, Fry 2006). Any climate factor that affects stomatal conductance and/or the rate of C assimilation may also change the 13C signature of plants. However, in studies using the natural abundance of C isotopes the fate of recently fixed C is difficult to trace due to relatively small differences in isotopic signatures between the plant or above- and below-ground components. Therefore, pulse-chase labelling experiments using highly enriched 13C are needed to give a more precise picture of possible climate-induced changes in fluxes of recently fixed C from leaves to roots and the return of assimilated C, as CO2 from soil to air. In tracer studies, discrimination against 13C is generally ignored.

In the present experiment, we used silver birch (Betula pendula Roth.) as an experimental tree material. Silver birch is ecologically and economically important tree species in northern Europe. In Finland, this species is growing in its northern distribution limit and already experiencing climate warming. As a fast-growing species, silver birch may benefit to some extent from warming (Peltola and Kellomäki 2005), but on the other hand it is considered to be an O3-sensitive tree species, though its sensitivity to O3 widely varies among genotypes (Oksanen et al. 2007, 2009). Recently, Riikonen et al. (2009) also showed that mild O3 stress reduced the ability of one silver birch genotype to utilize the warmer growth environment by increasing the stomatal limitation for photosynthesis during the second exposure season.

We used a pot experiment, as a model system, to explore interactive effects of ozone and temperature on C allocation in silver birch trees. For all four birch genotypes tree and fungal growth as well as soil respiration measurements were performed, and in one genotype these measurements were also complemented by 13C labelling. Ozone fumigation was conducted in a free-air exposure system (Karnosky et al. 2007, Häikiö et al. 2007) and warming exposure (air + soil warming) was realized by using infrared heaters above the canopy (Wan et al. 2002, Kimball 2005, Hartikainen et al. 2009). Our first main hypothesis was that O3 would reduce and temperature would increase tree growth and that in combination their effects on growth may be counteractive. However, in O3 exposure root growth might be first affected, whereas in temperature treatments above-ground growth responses may be the most prominent, and as an end result R : S ratios could be decreased in both treatments. We also hypothesized that the effects of elevated temperature and ozone on soil respiration (root respiration + microbial respiration) could be opposite. Temperature increase may stimulate soil respiration rates, if soil moisture conditions remain favourable, and if tree productivity and thereby availability of labile C compounds for the decomposers increase, whereas elevated O3 may decrease tree C assimilation and productivity, and thereby also soil respiration rates. Based on previous knowledge, mycorrhizal root growth might be also affected by warming and ozone treatments, but these responses can be complex and highly variable. In addition, we assumed that silver birch genotypes might differ in their O3 and temperature responses.

Materials and methods

Open-air exposure experiment: tree material, experimental design and exposures

Four silver birch clones (gt12, gt14, gt15 and gt25) were micropropagated for this open-air exposure experiment at the University of Eastern Finland. The clones used in this study were randomly selected from a naturally regenerated mixed birch stand (Laitinen et al. 2000), and thus represent a natural variation within this wild Finnish birch population. At the end of May 2007, 320 silver birch trees were planted in 10-l pots containing fertilized sphagnum peat (Kekkilä Finnpeat) and quartz sand (2 : 1, v : v), and then the potted trees were evenly distributed among eight experimental plots (Ø 10 m) at the Ruohoniemi open-air exposure site (62°53′N, 27°37′E, 80 m a.s.l.) in Kuopio. Peat:sand mixture used in the pots (hereafter the term ‘soil’ is used for this mixture) was not sterilized before tree planting, and neither there was any artificial microbial inoculation added to it.

This open-air exposure system consisted of four ambient and four elevated O3 plots, and each plot was further divided into two infrared-heated and two ambient temperature subplots (Hartikainen et al. 2009). This arrangement resulted in four different exposure treatments: (i) control (C) = ambient O3 + ambient air temperature, (ii) elevated O3 alone (O3), (iii) elevated temperature alone (T) and (iv) elevated O3 and elevated temperature in combination (O3 + T, n = 4 per each treatment). Thus, in each subplot (1.1 × 1.4 m), there were five trees per genotype in pots submerged into the soil of the experimental site (i.e., 10 trees per subplot). All experimental trees were placed in two adjacent rows in each subplot and the distance between these two rows was ∼0.2 m (this small gap was left in the middle of the subplot). In addition, extra saplings of various genotypes were placed around the experimental trees to protect them from the possible side effects of wind. During the experiment, all trees were watered when soil moisture in pots fell below 10 vol% and fertilized with Kekkilä Superex (19.4% N, 5.3% P, 20% K, 0.2% Mg), resulting in a total amount of 33 kg N ha−1 year−1 in 2007 and 72 kg N ha−1 year−1 in 2008.

Both O3 and temperature exposures were run during the growing seasons 2007–08. Ozone generation and fumigation (see Häikiö et al. 2007, Hartikainen et al. 2009 for more detailed description) in plots was kept on from 4 June to 22 October 2007 and from 2 May to 30 September 2008, while subplots were heated from 5 June to 22 October 2007 and from 2 May to 30 September 2008 (i.e., in 2008 both exposures were started at the same time as leaf opening). Warming treatment (24 h day−1) was realized by installing one infrared heater (Model Comfortintra CIR 105–220, 230–400 V, Frico AB, Partille, Sweden) ∼70 cm above the canopy in the middle of each heated subplot. In ambient temperature subplots, a wooden bar of the same size, shape and colour as the infrared heater was installed similarly above the canopy to mimic the shading effect of a heater. Both heaters and wooden bars were lifted during the growing season to keep the distance between the heater/bar and the canopy constant. Temperature control for the whole system was thus based on canopy air temperature measurements. The target value for air temperature elevation was +1 °C, but in 2007 and 2008 air temperature elevation was on average +0.8 °C (Table 1). Leaf temperatures were ∼ + 0.5 °C higher than the measured air temperatures (Riikonen et al. 2009), and soil temperature was increased on average by +1 °C in 2007 and by +0.6 °C in 2008 (Table 1). In both years the warmed subplots had also lower average soil moisture content than the ambient temperature subplots (Table 1).

Table 1.

Average O3 concentrations (14 h day−1) and AOT40 (accumulated over a threshold of 40 ppb) values in ambient and elevated O3 plots, and average air and soil temperatures (24 h day−1), air humidity, and soil moisture in ambient and elevated temperature subplots in 2007 and 2008. Year 2007 data are calculated between June and October, and year 2008 data between May and September. Values are means or means ± SD (n = 4 for O3 plot data and n = 8 for temperature subplot data, except for 2007 soil moisture data where values are monthly averages of one subplot only).

 O3 (ppb)
 
AOT40 (ppm h)
 
Air temperature (°C)
 
Soil temperature (°C)
 
Air humidity (%)
 
Soil moisture (vol%)
 
2007 Ambient O3 Elevated O3 Ambient O3 Elevated O3 Ambient temperature Elevated temperature Ambient temperature Elevated temperature Ambient temperature Elevated temperature Ambient temperature Elevated temperature 
June 27.1 ± 0.9 36.9 ± 1.1 0.04 1.8 15.6 ± 0.7 16.7 ± 0.8 14.2 ± 1.1 17.8 ± 1.0 71.0 ± 3.2 66.3 ± 2.3 – – 
July 23.7 ± 0.7 28.7 ± 2.5 0.04 2.7 17.5 ± 0.4 18.6 ± 0.4 15.7 ± 1.3 16.4 ± 1.0 80.0 ± 2.2 75.1 ± 1.5 38.3 26.9 
August 26.2 ± 0.8 32.3 ± 3.3 0.13 4.3 17.1 ± 0.4 18.0 ± 0.3 15.0 ± 1.4 15.2 ± 0.9 79.4 ± 4.1 74.1 ± 1.3 37.1 23.6 
September 19.4 ± 0.4 21.3 ± 1.2 0.14 4.5 9.7 ± 0.2 10.3 ± 0.2 8.6 ± 1.5 8.8 ± 0.8 86.7 ± 1.8 83.1 ± 1.0 41.7 29.5 
October 20.4 ± 0.6 21.1 ± 1.0 0.14 4.9 6.0 ± 0.2 6.5 ± 0.8 5.3 ± 1.6 5.9 ± 0.8 86.1 ± 1.9 82.5 ± 1.1 45.4 33.8 
Whole season 23.4 ± 0.7 28.1 ± 1.8 0.14 4.9 13.2 ± 0.4 14.0 ± 0.5 11.8 ± 1.4 12.8 ± 0.9 80.6 ± 2.6 76.2 ± 1.4 40.5 28.3 
2008 
May 35.0 ± 0.2 45.3 ± 1.9 1.0 4.6 10.5 ± 1.8 11.3 ± 1.9 8.3 ± 0.7 9.3 ± 0.8 62.5 ± 1.6 59.9 ± 1.6 39.8 ± 4.0 30.4 ± 4.0 
June 28.8 ± 0.2 41.5 ± 1.1 1.5 8.3 14.3 ± 0.2 15.3 ± 0.3 12.7 ± 0.9 13.4 ± 1.0 70.9 ± 1.2 66.5 ± 1.7 42.5 ± 6.6 33.3 ± 6.2 
July 20.5 ± 0.5 27.2 ± 0.6 1.6 8.8 16.3 ± 0.3 17.3 ± 0.4 15.0 ± 0.9 15.5 ± 0.8 76.6 ± 1.0 72.0 ± 1.4 43.1 ± 9.5 31.7 ± 7.9 
August 17.5 ± 0.4 23.1 ± 0.7 1.6 8.9 14.0 ± 0.3 14.7 ± 0.3 13.3 ± 0.7 13.7 ± 0.8 82.8 ± 0.4 80.0 ± 1.2 44.4 ± 7.0 37.6 ± 4.6 
September 17.2 ± 0.4 22.8 ± 0.9 1.6 9.0 8.2 ± 0.4 8.9 ± 0.4 8.6 ± 1.1 9.3 ± 1.4 83.0 ± 0.8 80.0 ± 1.9 43.1 ± 9.6 30.3 ± 7.7 
Whole season 23.8 ± 0.3 32.0 ± 1.0 1.6 9.0 12.7 ± 0.6 13.5 ± 0.7 11.6 ± 0.9 12.2 ± 1.0 75.2 ± 1.0 71.7 ± 1.6 42.6 ± 7.1 32.7 ± 5.6 
 O3 (ppb)
 
AOT40 (ppm h)
 
Air temperature (°C)
 
Soil temperature (°C)
 
Air humidity (%)
 
Soil moisture (vol%)
 
2007 Ambient O3 Elevated O3 Ambient O3 Elevated O3 Ambient temperature Elevated temperature Ambient temperature Elevated temperature Ambient temperature Elevated temperature Ambient temperature Elevated temperature 
June 27.1 ± 0.9 36.9 ± 1.1 0.04 1.8 15.6 ± 0.7 16.7 ± 0.8 14.2 ± 1.1 17.8 ± 1.0 71.0 ± 3.2 66.3 ± 2.3 – – 
July 23.7 ± 0.7 28.7 ± 2.5 0.04 2.7 17.5 ± 0.4 18.6 ± 0.4 15.7 ± 1.3 16.4 ± 1.0 80.0 ± 2.2 75.1 ± 1.5 38.3 26.9 
August 26.2 ± 0.8 32.3 ± 3.3 0.13 4.3 17.1 ± 0.4 18.0 ± 0.3 15.0 ± 1.4 15.2 ± 0.9 79.4 ± 4.1 74.1 ± 1.3 37.1 23.6 
September 19.4 ± 0.4 21.3 ± 1.2 0.14 4.5 9.7 ± 0.2 10.3 ± 0.2 8.6 ± 1.5 8.8 ± 0.8 86.7 ± 1.8 83.1 ± 1.0 41.7 29.5 
October 20.4 ± 0.6 21.1 ± 1.0 0.14 4.9 6.0 ± 0.2 6.5 ± 0.8 5.3 ± 1.6 5.9 ± 0.8 86.1 ± 1.9 82.5 ± 1.1 45.4 33.8 
Whole season 23.4 ± 0.7 28.1 ± 1.8 0.14 4.9 13.2 ± 0.4 14.0 ± 0.5 11.8 ± 1.4 12.8 ± 0.9 80.6 ± 2.6 76.2 ± 1.4 40.5 28.3 
2008 
May 35.0 ± 0.2 45.3 ± 1.9 1.0 4.6 10.5 ± 1.8 11.3 ± 1.9 8.3 ± 0.7 9.3 ± 0.8 62.5 ± 1.6 59.9 ± 1.6 39.8 ± 4.0 30.4 ± 4.0 
June 28.8 ± 0.2 41.5 ± 1.1 1.5 8.3 14.3 ± 0.2 15.3 ± 0.3 12.7 ± 0.9 13.4 ± 1.0 70.9 ± 1.2 66.5 ± 1.7 42.5 ± 6.6 33.3 ± 6.2 
July 20.5 ± 0.5 27.2 ± 0.6 1.6 8.8 16.3 ± 0.3 17.3 ± 0.4 15.0 ± 0.9 15.5 ± 0.8 76.6 ± 1.0 72.0 ± 1.4 43.1 ± 9.5 31.7 ± 7.9 
August 17.5 ± 0.4 23.1 ± 0.7 1.6 8.9 14.0 ± 0.3 14.7 ± 0.3 13.3 ± 0.7 13.7 ± 0.8 82.8 ± 0.4 80.0 ± 1.2 44.4 ± 7.0 37.6 ± 4.6 
September 17.2 ± 0.4 22.8 ± 0.9 1.6 9.0 8.2 ± 0.4 8.9 ± 0.4 8.6 ± 1.1 9.3 ± 1.4 83.0 ± 0.8 80.0 ± 1.9 43.1 ± 9.6 30.3 ± 7.7 
Whole season 23.8 ± 0.3 32.0 ± 1.0 1.6 9.0 12.7 ± 0.6 13.5 ± 0.7 11.6 ± 0.9 12.2 ± 1.0 75.2 ± 1.0 71.7 ± 1.6 42.6 ± 7.1 32.7 ± 5.6 

AOT40 values are calculated by summing the hourly values >40 ppb to achieve ppm h.

The target level for O3 exposure was 1.5 times the ambient O3, but in 2007–08 O3 exposure reached only 1.2–1.3 times the ambient levels (the monthly and whole season AOT40 values are given in Table 1). The O3 fumigation was run 14 h day−1 (from 08:00 to 22:00 h) during each study period, 7 days a week, except during very low wind velocities, or if the ambient O3 concentration was <10 ppb (these low O3 concentrations occurred during high precipitation conditions, e.g., in early morning hours and during rain). Each plot was also continuously monitored for wind speed and direction, and each subplot for relative humidity (Table 1).

Tree and fungal growth plus soil CO2 respiration measurements of all genotypes

In mid-August (Harvest 1), the first set of experimental trees (n = 64 trees) were harvested in order to obtain tree biomass data before the leaf abscission period was started. Genotype 15 trees harvested in mid-August were also the same ones that were used in the pulse-chase labelling experiment (see below). Final harvest of trees was performed at the end of September (Harvest 2). In Harvest 2, leaves and stems of 96 trees were harvested, while roots were collected only from 64 trees. Leaves, stems and washed roots that were not used in the microscopy and ergosterol analyses were weighed after drying at +60 °C for a week. Stem height and diameter growth were measured from May to September 2008, but here we only report the Harvest 2 results. In addition, root : shoot and photosynthetic : non-photosynthetic tissue ratios (R : S and P : nP ratios) were calculated for both harvest times as R : S ratio = root biomass/(stem + leaf biomass) and P : nP ratio = leaf biomass/(stem + root biomass).

In May 2008, 64 trees were selected for the soil respiration measurements (two trees per genotype per plot). Three weeks before the soil CO2 efflux measurements started, one soil collar (Ø 10 cm) per pot was inserted at a depth of 2 cm in the soil. From June to August, soil respiration was measured seven times with a soil respiration chamber (Licor 6400-09) attached to a portable infrared gas analyser (IRGA) (Licor 6400XT, Licor Inc., Lincoln, NE, USA). At the same time as soil CO2 efflux measurements a soil temperature probe attached to a Licor 6400XT was inserted at a depth of 7 cm and soil temperature was automatically recorded in the console. The soil moisture content (vol%) next to collars was measured with a Theta-Probe soil moisture sensor (Delta-T Devices Ltd, Cambridge, UK).

The washed root samples (labelled gt15 samples from Harvest 1 and 64 samples from Harvest 2) for short root analyses were kept frozen (−20 °C). In microscopical ­examination, the total numbers of mycorrhizal and non-mycorrhizal short roots were determined from 0.5 m long, randomly collected fine root (<0.5–1 mm in diameter) samples. The fine root ramification index was calculated by dividing the total number of all short roots by the length of the root sample, and the results are expressed as the number of short root tips per 1 m of fine root length. Total mycorrhizal infection percentage is the total number of all mycorrhizal short roots divided by the total number of all short roots × 100. Root ergosterol samples were collected simultaneously with microscopy samples, and stored in a deep-freezer (−80 °C) until extraction. Extraction and analysis of root ergosterol samples followed the method of Nylund and Wallander (1992) with minor modifications (Markkola 1996, Kasurinen et al. 2001). During the 2008 season, sporocarp production was pronounced and therefore it could be measured from all the treatments and genotypes. Sporocarps were harvested in total three times from August to the beginning of September (from labelled gt15 trees only one sporocarp harvest was conducted during this monitoring period). Each time after the collection, sporocarps were gently cleaned from debris and oven-dried (+60 °C) to a constant weight. Sporocarp production is expressed as cumulative sporocarp mass (dry weights of three harvests combined) for the final harvest. All sporocarps belong to Laccaria laccata (Scop.) Cooke, which is considered to be a mycorrhizal fungal species (Smith and Read 2008).

13C pulse-chase labelling experiment with gt15

In 2008, a 13C-CO2 pulse-chase experiment was carried out on all the above treatments using only gt15 trees (a labelling experiment had to be limited to one randomly selected genotype due to high labelling gas costs). In late May 2008, 16 trees were selected for the labelling experiment (n = 4 trees per treatment), and at the same time soil collars (Ø 10 cm) for soil respiration measurements were inserted in each pot (one collar per pot at a depth of 2 cm in the soil). On 17 June 2008 pre-labelling samples for 13C analyses were collected from all trees. Solid samples for bulk 13C analyses were collected from foliage, root and soil. For the measurement of 13C derived from recently fixed C compounds and released via soil respiration, we used closed chambers (volume 0.8 l) from which air was sampled four times during an 8 min incubation period (i.e., 0.4–1 ml depending on CO2 concentration) with a 1 ml gas-tight syringe (Hamilton Co., Reno, NV, USA). Each CO2 sample (four samples per measurement point) was injected into its own, previously evacuated and N2-flushed CO2-free glass vial (12 ml Exetainer vials, capped with airtight rubber septa, Labco Ltd., High Wycombe, UK). By collecting multiple samples over a certain time period, we were thus able to construct a Keeling plot intercept for both pre-labelling and labelling samples to determine the δ13C value of soil CO2 efflux. The regression coefficient (R2) of the linear relationship between the 13C signal of CO2 in the chamber and 1/[CO2] was always >0.80, giving us high confidence in this intercept. Simultaneously with this 13C sampling, soil CO2 efflux was measured from a soil collar with soil respiration chamber (Licor 6009) attached to a portable IRGA (Licor 6400XT, Licor Inc.).

Labelling was conducted on two consecutive days (25 and 26 June 2008) and during daylight hours only. On each labelling day there were trees from each of the four treatments. During 13C-CO2 labelling with 99 atom% 13C-CO2 gas (Cambridge Isotope Laboratories, Inc., Andover, MA, USA), each individual tree was covered with a clear plastic bag (average bag volume 248 l). Bags were secured tightly at the base of the tree to minimize labelling gas leakage to the soil, surrounding air and trees. Labelling gas was released from a lecture bottle via plastic tubes connected to the bags, and gas was mixed in the air inside the bags with two fans. Before giving the first pulse, the prevailing CO2 level of air inside the bags was allowed to drop down below the ambient CO2 concentration (380 ppm), and when the level was stabilized to 100–150 ppm level (levels inside the bag were monitored with a Licor 6100 IRGA the whole time), the first gas pulse was given to the trees. Although IRGA measurements are known to be less sensitive to 13C than to 12C (McDermitt et al. 1993, Lee et al. 2006) and thus the actual 13C-CO2 concentrations in the chamber were probably underestimated by ∼1/3 (McDermitt et al. 1993), we nonetheless observed clear decreases and increases in CO2 concentrations before and after the pulses (i.e., the IRGA system used here was not completely insensitive to 13C). Hence, when the CO2 level inside the bag reached 800–1000 ppm according to the IRGA, the pulse was stopped, and when the levels inside the bag reached 400–500 ppm, a new pulse was released into the bags, and in this way the 13C-CO2 gas levels were increased above the ambient air CO2 for the whole time of the labelling (during 2.5 h labelling each tree received six short gas pulses). Immediately after labelling, 2–3 leaves per tree were collected for bulk 13C analyses, and 13C signatures of soil CO2 efflux as well as soil CO2 efflux rates were measured. Pulse-chase periods for the 13C signature in soil CO2 efflux and solid (leaves, fine roots and soil) samples were 21 and 49 days, respectively, and O3 and warming exposures were not interrupted during any stage of the labelling or pulse-chase period. On the 49th day of the pulse-chase period, all labelled trees were harvested, and their root, stem and leaf dry weights were measured.

All CO2 samples were analysed on the same day as they were collected, whereas solid samples were first oven-dried to a constant weight (+60 °C for 2 days) and stored in a dark room (+20 °C) until ball milling and 13C analyses. Carbon dioxide samples were analysed for δ13C with a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer (IRMS) coupled with a Trace GC and Precon unit, and δ13C of solid samples were determined by an elemental analyser (EA) coupled with a ThermoFinnigan Delta Plus XP IRMS. For solid sample analyses 2–3 mg of dry sample was weighed into small tin cups.

All 13C data were converted from δ13C values to 13C atom% (unlabelled samples) and 13C atom% excess (labelled samples) values. The following equation was used to convert δ 13C values to abundance (atom%) values: 13C atom% = [Rsample/(Rsample + 1)] × 100. Then the 13C atom% excess for labelled samples was calculated: 13C atom% excess = 13C atom% of labelled samples − 13C atom% of unlabelled samples. The 13C atom% and 13C atom% excess values were also multiplied by the mass of C measured in solid samples or soil CO2 efflux in order to obtain total 13C amount values in these compartments (these are expressed as 13C g m−2 or mg m−2 day−1). Only labelled data (excess 13C data) were used in the statistical analyses.

Statistical analyses

The experimental design of the labelling experiment (13C excess data of solid and soil CO2 samples) and of all other repeated measurements (e.g., soil respiration measurements from all genotypes) was a split-split-plot design in which O3 was the completely randomized whole-plot treatment, temperature was the split-plot treatment and sampling time was the split-split-plot treatment. Biomass accumulation measurements were non-repeated measurements and therefore, we used O3, temperature and genotype as fixed factors and plot identity as a random factor in the analysis of variance (ANOVA) model. All ANOVA tests were performed using linear mixed model ANOVA (SPSS 17.0 for Windows, Chicago, IL, USA) and using either split-split-plot or split-plot means.

The relationship between the soil respiration, soil temperature, soil moisture and tree growth at the Harvest 2 was studied with stepwise linear regression. In the labelling experiment (gt15 trees only), the relationship between the treatments, tree and fungal biomass and growth data and 13C data were studied with principal component analysis (PCA). Before the actual test, data were centred and unit variance scaling was ­performed. Original data dimensions were thus 16 objects (i.e., trees) × 17 variables. In the PCA, only solid-sample 13C data collected on the 49th post-labelling day and soil respiration 13C data collected on the 21st post-labelling day were included, whereas all tree biomass and growth data as well as mycorrhizal infection and sporocarp biomass data were from Harvest 1. Soil respiration rates were overall growth season averages measured from the labelled trees. Principal component analysis was performed with SIMCA-P 11.5 (Umetrics AB, Umeå, Sweden), and extracted scores were further tested with the linear mixed model ANOVA using O3 and temperature as fixed factors and plot identity as a random factor. In addition to basic labelled data testing, partial 13C budgets were also calculated on the basis of 13C excess amounts in solid samples collected on the 12th and 49th post-labelling days. The idea was to see whether the allocation of excess 13C to different compartments were changed due to the treatments over the chase period (overall relative 13C proportion data were tested with linear mixed model ANOVA as above). Before ANOVA, all data and residuals were checked for normality and when necessary the values were transformed to satisfy the assumptions of the test. Differences were considered statistically significant when P  < 0.05, and marginally statistically significant when P < 0.10.

Results

Biomass accumulation and stem growth

Temperature effects on tree biomass accumulation were clear in Harvest 1, but later some of these temperature effects were either modified by O3 level or genotype in Harvest 2 (Tables 2 and 3). In short, leaf, stem, root and thereby total tree biomasses all were clearly increased due to elevated temperature (temperature main effect) in Harvest 1 (Tables 2 and 3). In the first harvest, temperature-induced increases for leaf, stem, root and total tree biomasses (trees averaged over genotype and O3 levels) were 86, 72, 49 and 63%, respectively. Root : shoot ratios were not significantly affected by any of the treatments (Table 2). P : nP ratios were increased in gt12, gt14 and gt25 due to temperature treatments, whereas in gt15 temperature treatments decreased P : nP ratio (significant genotype × temperature interaction, Table 2).

Table 2.

Genotype, O3 and temperature main and interaction effects on silver birch above- and below-ground compartments, total tree biomass and allocation response parameters in mid-August (Harvest 1) and at the end of September (Harvest 2) in 2008. Bolded values in the table indicate statistically significant (P < 0.05) or marginally statistically significant (P < 0.1) P values.

P values Leaf Stem Root Total tree R : S ratio P : nP ratio 
Harvest 1       
Genotype 0.495  < 0.0005 0.032 0.001 0.150  < 0.0005 
O3 0.579 0.365 0.201 0.206 0.412 0.571 
Temperature  < 0.0005  < 0.0005  < 0.0005  < 0.0005 0.172 0.185 
Genotype × O3 0.627 0.597 0.448 0.860 0.181 0.356 
Genotype × temperature 0.455 0.501 0.241 0.160 0.264 0.007 
O3 × temperature 0.505 0.906 0.702 0.885 0.914 0.163 
Genotype × O3 × temperature 0.626 0.310 0.797 0.767 0.278 0.523 
Harvest 2       
Genotype 0.002  < 0.0005 0.995 0.500 0.542 0.002 
O3 0.037 0.169 0.899 0.425 0.385  < 0.0005 
Temperature  < 0.0005  < 0.0005  < 0.0005  < 0.0005 0.835 0.004 
Genotype × O3 0.106 0.042 0.782 0.305 0.810 0.370 
Genotype × temperature 0.507 0.694 0.080 0.428 0.260 0.113 
O3 × temperature 0.030 0.372 0.614 0.853 0.642 0.072 
Genotype × O3 × temperature 0.303 0.250 0.312 0.687 0.106 0.058 
P values Leaf Stem Root Total tree R : S ratio P : nP ratio 
Harvest 1       
Genotype 0.495  < 0.0005 0.032 0.001 0.150  < 0.0005 
O3 0.579 0.365 0.201 0.206 0.412 0.571 
Temperature  < 0.0005  < 0.0005  < 0.0005  < 0.0005 0.172 0.185 
Genotype × O3 0.627 0.597 0.448 0.860 0.181 0.356 
Genotype × temperature 0.455 0.501 0.241 0.160 0.264 0.007 
O3 × temperature 0.505 0.906 0.702 0.885 0.914 0.163 
Genotype × O3 × temperature 0.626 0.310 0.797 0.767 0.278 0.523 
Harvest 2       
Genotype 0.002  < 0.0005 0.995 0.500 0.542 0.002 
O3 0.037 0.169 0.899 0.425 0.385  < 0.0005 
Temperature  < 0.0005  < 0.0005  < 0.0005  < 0.0005 0.835 0.004 
Genotype × O3 0.106 0.042 0.782 0.305 0.810 0.370 
Genotype × temperature 0.507 0.694 0.080 0.428 0.260 0.113 
O3 × temperature 0.030 0.372 0.614 0.853 0.642 0.072 
Genotype × O3 × temperature 0.303 0.250 0.312 0.687 0.106 0.058 

R : S ratio, root : shoot ratio; P : nP ratio, photosynthetic : non-photosynthetic tissue ratio.

Table 3.

Leaf, stem, root and total tree biomasses for genotypes 12, 14, 15 and 25 in mid-August (Harvest 1) and at the end of September (Harvest 2) 2008. Values are means ± SE (units are g dry weight).

Harvest 1 O3 O3 + T Harvest 2 O3 O3 + T 
Gt12 Gt12 
Leaf (g) 17.1 ± 2.7 19.5 ± 1.5 32.2 ± 0.9 31.7 ± 1.2 Leaf (g) 7.4 ± 2.0 5.8 ± 0.6 16.7 ± 2.4 12.2 ± 1.8 
Stem (g) 47.3 ± 6.6 57.1 ± 3.3 87.0 ± 5.3 81.3 ± 2.5 Stem (g) 55.9 ± 8.4 54.0 ± 0.8 104.8 ± 8.4 88.0 ± 4.7 
Root (g) 104.1 ± 27.4 65.2 ± 9.5 100.1 ± 15.5 82.0 ± 15.5 Root (g) 105.3 ± 19.5 68.2 ± 7.7 172.7 ± 14.1 189.4 ± 33.8 
Total tree (g) 168.5 ± 32.8 141.8 ± 11.7 219.3 ± 17.0 195.0 ± 16.3 Total tree (g) 168.6 ± 26.0 128.0 ± 9.4 294.2 ± 17.5 289.6 ± 33.2 
Gt14 Gt14 
Leaf (g) 20.2 ± 2.5 17.2 ± 0.9 37.6 ± 2.5 41.0 ± 8.1 Leaf (g) 11.1 ± 2.1 8.2 ± 1.5 25.9 ± 4.4 14.2 ± 2.3 
Stem (g) 55.7 ± 4.7 49.5 ± 2.5 101.6 ± 11.5 102.3 ± 14.2 Stem (g) 75.4 ± 14.6 63.1 ± 4.7 135.1 ± 17.7 101.0 ± 8.6 
Root (g) 77.8 ± 34.8 47.4 ± 5.38 133.1 ± 23.6 82.9 ± 7.9 Root (g) 144.3 ± 43.2 106.7 ± 36.5 127.3 ± 32.2 154.5 ± 24.9 
Total tree (g) 153.6 ± 38.6 114.1 ± 7.6 272.3 ± 29.8 226.2 ± 28.7 Total tree (g) 230.8 ± 51.5 178.0 ± 45.2 288.3 ± 67.3 269.7 ± 20.2 
Gt151 Gt15 
Leaf (g) 21.2 ± 2.8 18.5 ± 0.7 35.7 ± 4.5 31.3 ± 1.2 Leaf (g) 7.5 ± 1.2 5.6 ± 0.2 15.1 ± 2.7 13.8 ± 1.8 
Stem (g) 42.2 ± 6.8 37.7 ± 1.0 86.3 ± 9.5 69.0 ± 3.2 Stem (g) 55.6 ± 10.1 50.5 ± 2.1 87.3 ± 7.2 98.2 ± 4.7 
Root (g) 34.3 ± 10.3 40.3 ± 8.2 95.2 ± 26.7 81.0 ± 24.1 Root (g) 78.8 ± 16.5 111.4 ± 22.8 169.8 ± 24.7 172.1 ± 27.3 
Total tree (g) 97.7 ± 16.6 96.5 ± 7.1 217.3 ± 39.0 181.3 ± 24.9 Total tree (g) 141.9 ± 29.5 167.5 ± 25.6 272.2 ± 41.6 284.1 ± 33.2 
Gt25 Gt25 
Leaf (g) 22.9 ± 5.8 14.5 ± 1.9 36.2 ± 8.5 35.5 ± 3.9 Leaf (g) 9.3 ± 2.0 5.9 ± 0.7 18.3 ± 1.6 10.5 ± 0.8 
Stem (g) 81.9 ± 14.3 56.7 ± 8.2 103.4 ± 20.2 103.7 ± 8.5 Stem (g) 75.6 ± 8.2 57.2 ± 3.9 115.5 ± 11.0 92.8 ± 3.5 
Root (g) 89.6 ± 1.4 77.5 ± 12.6 121.5 ± 29.1 103.1 ± 8.5 Root (g) 86.7 ± 11.5 94.9 ± 22.9 196.3 ± 29.3 170.6 ± 16.5 
Total tree (g) 194.4 ± 18.7 148.7 ± 6.2 261.1 ± 37.7 242.3 ± 12.9 Total tree (g) 171.6 ± 26.5 158.0 ± 25.9 330.1 ± 32.8 273.9 ± 22.0 
Harvest 1 O3 O3 + T Harvest 2 O3 O3 + T 
Gt12 Gt12 
Leaf (g) 17.1 ± 2.7 19.5 ± 1.5 32.2 ± 0.9 31.7 ± 1.2 Leaf (g) 7.4 ± 2.0 5.8 ± 0.6 16.7 ± 2.4 12.2 ± 1.8 
Stem (g) 47.3 ± 6.6 57.1 ± 3.3 87.0 ± 5.3 81.3 ± 2.5 Stem (g) 55.9 ± 8.4 54.0 ± 0.8 104.8 ± 8.4 88.0 ± 4.7 
Root (g) 104.1 ± 27.4 65.2 ± 9.5 100.1 ± 15.5 82.0 ± 15.5 Root (g) 105.3 ± 19.5 68.2 ± 7.7 172.7 ± 14.1 189.4 ± 33.8 
Total tree (g) 168.5 ± 32.8 141.8 ± 11.7 219.3 ± 17.0 195.0 ± 16.3 Total tree (g) 168.6 ± 26.0 128.0 ± 9.4 294.2 ± 17.5 289.6 ± 33.2 
Gt14 Gt14 
Leaf (g) 20.2 ± 2.5 17.2 ± 0.9 37.6 ± 2.5 41.0 ± 8.1 Leaf (g) 11.1 ± 2.1 8.2 ± 1.5 25.9 ± 4.4 14.2 ± 2.3 
Stem (g) 55.7 ± 4.7 49.5 ± 2.5 101.6 ± 11.5 102.3 ± 14.2 Stem (g) 75.4 ± 14.6 63.1 ± 4.7 135.1 ± 17.7 101.0 ± 8.6 
Root (g) 77.8 ± 34.8 47.4 ± 5.38 133.1 ± 23.6 82.9 ± 7.9 Root (g) 144.3 ± 43.2 106.7 ± 36.5 127.3 ± 32.2 154.5 ± 24.9 
Total tree (g) 153.6 ± 38.6 114.1 ± 7.6 272.3 ± 29.8 226.2 ± 28.7 Total tree (g) 230.8 ± 51.5 178.0 ± 45.2 288.3 ± 67.3 269.7 ± 20.2 
Gt151 Gt15 
Leaf (g) 21.2 ± 2.8 18.5 ± 0.7 35.7 ± 4.5 31.3 ± 1.2 Leaf (g) 7.5 ± 1.2 5.6 ± 0.2 15.1 ± 2.7 13.8 ± 1.8 
Stem (g) 42.2 ± 6.8 37.7 ± 1.0 86.3 ± 9.5 69.0 ± 3.2 Stem (g) 55.6 ± 10.1 50.5 ± 2.1 87.3 ± 7.2 98.2 ± 4.7 
Root (g) 34.3 ± 10.3 40.3 ± 8.2 95.2 ± 26.7 81.0 ± 24.1 Root (g) 78.8 ± 16.5 111.4 ± 22.8 169.8 ± 24.7 172.1 ± 27.3 
Total tree (g) 97.7 ± 16.6 96.5 ± 7.1 217.3 ± 39.0 181.3 ± 24.9 Total tree (g) 141.9 ± 29.5 167.5 ± 25.6 272.2 ± 41.6 284.1 ± 33.2 
Gt25 Gt25 
Leaf (g) 22.9 ± 5.8 14.5 ± 1.9 36.2 ± 8.5 35.5 ± 3.9 Leaf (g) 9.3 ± 2.0 5.9 ± 0.7 18.3 ± 1.6 10.5 ± 0.8 
Stem (g) 81.9 ± 14.3 56.7 ± 8.2 103.4 ± 20.2 103.7 ± 8.5 Stem (g) 75.6 ± 8.2 57.2 ± 3.9 115.5 ± 11.0 92.8 ± 3.5 
Root (g) 89.6 ± 1.4 77.5 ± 12.6 121.5 ± 29.1 103.1 ± 8.5 Root (g) 86.7 ± 11.5 94.9 ± 22.9 196.3 ± 29.3 170.6 ± 16.5 
Total tree (g) 194.4 ± 18.7 148.7 ± 6.2 261.1 ± 37.7 242.3 ± 12.9 Total tree (g) 171.6 ± 26.5 158.0 ± 25.9 330.1 ± 32.8 273.9 ± 22.0 

C, control, O3, elevated O3 alone, T, elevated temperature alone, O3 + T, elevated O3 and elevated temperature in combination (n = 3–4 per treatment).

1Gt15 trees shown in Harvest 1 are the same ones used in the pulse-chase labelling experiment.

Since leaf abscission had already started before the final harvest, leaf biomass data collected in Harvest 2 reflects possible treatment effects on leaf senescence and abscission. There was a statistically significant O3 × temperature interaction in leaf biomass data at the final harvest (Table 2). Largest leaf biomass was observed in elevated temperature trees (+116% increase), whereas elevated O3 alone reduced leaf biomass (−27% reduction), and in combination treatment elevated O3 partly cancelled temperature effects (+44% increase) on leaf biomass (Table 3). Stem (68% increase, trees averaged over genotype and O3 levels) and total tree (76% increase, trees averaged over genotype and O3 levels) biomasses were clearly bigger in temperature-treated subplots (temperature main effect, Tables 2 and 3). In harvest 2, stem biomass was also affected by elevated O3, but the magnitude and direction of this O3 effect depended on the genotype (genotype × O3 interaction effect, Table 2). In gt14 and gt25, stem biomass was reduced due to O3 in both ambient and elevated temperature, whereas in gt12 and gt15 O3 effects on stem biomass were negligible under ambient temperature, and in combination treatment gt15 stem biomass accumulation was enhanced in most of the treatments (Table 3). Root biomass was affected by elevated temperature, but in Harvest 2 this temperature effect depended also on the genotype (genotype × temperature effect, Table 2). In gt14, temperature increased root biomass only slightly in combination treatment, whereas in other genotypes the temperature effect was clearer and seen in both ambient and elevated O3 (Table 3). Root : shoot ratios were not clearly changed in any of the exposure treatments, whereas P : nP ratios showed a marginally statistically significant genotype × O3 × temperature interaction (Table 2). In short, in gt12 and gt14 trees only temperature caused an increase in P : nP ratios (30 and 94% increases, respectively), whereas in other treatment trees P : nP ratios were similar to those of C trees. Interestingly, in gt15 and gt25 temperature effect on P : nP ratios were negligible, whereas O3 treatment reduced this ratio (38 and 32% reductions, respectively). However, in combination treatment also these two genotypes differed, as the gt15 P : nP ratio was only slightly decreased (9%), while the gt25 P : nP ratios were clearly decreased (30% decrease in O3 + T trees) like in O3 trees.

Only Harvest 2 stem height and diameter results are reported here. Temperature treatments enhanced stem height and diameter growth by 32 and 18%, respectively (temperature main effect, Figure 1a and b). In stem height data, there was a marginally significant genotype × O3 interaction effect (Figure 1a) showing that O3 reduced height growth in gt14 and gt25, but in gt12 a negative O3 effect was seen in the combination treatment only, and in gt15 height growth was actually slightly increased in O3 and O3 + T treatments.

Figure 1.

(a) Stem height and (b) base diameter growth of four silver birch genotypes exposed to ambient and elevated levels of O3 and temperature at the end of September (Harvest 2) 2008. Values are means ± SE. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Figure 1.

(a) Stem height and (b) base diameter growth of four silver birch genotypes exposed to ambient and elevated levels of O3 and temperature at the end of September (Harvest 2) 2008. Values are means ± SE. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Soil respiration, mycorrhizal roots and fungal responses

Overall soil CO2 efflux rates showed that temperature effects on soil respiration depended partly on genotype and prevailing O3 level (Figure 2a). Both gt12 and gt14 showed a clear temperature-induced increase in soil respiration under ambient and elevated O3, but in gt12 the temperature effect on soil respiration was also partly reduced due to O3 in the combination treatment. In other genotypes significant temperature-induced increases were either observed only under ambient (gt25) or elevated O3 (gt15). The temporal pattern of the O3 effect on soil respiration was inconsistent, as soil respiration response to O3 varied from 5% increase to 17% decrease over the growing season 2008 (marginally significant time × O3 interaction, Figure 2b). Stepwise linear regression analyses (Table 4) showed that ­short-term day-to-day variation in soil CO2 efflux rates was mainly related to soil temperature and soil moisture changes as almost 60% of day-to-day soil respiration variation was explained by these two factors. When the overall soil CO2 efflux was compared with abiotic and biotic factors, variation in soil respiration rates was significantly related to soil moisture conditions and total tree biomass indicating that soil CO2 efflux was not merely controlled by soil abiotic factors.

Figure 2.

(a) Overall and (b) temporal patterns of soil CO2 efflux beneath four silver birch genotypes during the growing season 2008. Values are means ± SE. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Figure 2.

(a) Overall and (b) temporal patterns of soil CO2 efflux beneath four silver birch genotypes during the growing season 2008. Values are means ± SE. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Table 4.

Stepwise linear regression (forward selection) comparing the relationship of soil CO2 efflux to soil temperature (7 cm) and soil moisture (values used in the test were daily means of treatments averaged over genotypes and from seven measurement days, n = 112). Similar stepwise linear regression was conducted to compare overall soil CO2 efflux to soil temperature, soil moisture and final harvest biomass and stem growth data (n = 16, daily treatment means averaged over the whole season and genotypes). The significance value of F-statistics shows how well the independent variable, e.g., soil temperature, explains the variation in the dependent variable (soil CO2 efflux). The t-statistics show the relative importance of each independent variable in the model. If the t-test value is < − 2 or > + 2, then the independent variable is considered to be an important explanatory factor in the model (bolded values in the table indicate statistically significant F- and t-test values).

Dependent variable Model  r r2 F P value 
Day-to-day variation 
Soil CO2 efflux (n = 112) Soil temperature and soil moisture  0.776 0.595 82.685  < 0.0005 
Independent variable t P value     
Soil temperature 8.678  < 0.0005     
Soil moisture  − 8.503  < 0.0005     
Overall variation 
Soil CO2 efflux (n = 16) Soil moisture and total tree biomass  0.920 0.823 35.828  < 0.0005 
Independent variable t P value     
Soil moisture  − 3.159 0.008     
Total tree biomass 2.744 0.017     
Mycorrhizal infection % −1.329 0.209     
Soil temperature −0.745 0.471     
Root ergosterol 0.662 0.520     
Final stem height 0.609 0.554     
Final stem biomass 0.531 0.605     
Final root biomass 0.307 0.764     
Final stem diameter 0.208 0.839     
Final leaf biomass 0.188 0.854     
Dependent variable Model  r r2 F P value 
Day-to-day variation 
Soil CO2 efflux (n = 112) Soil temperature and soil moisture  0.776 0.595 82.685  < 0.0005 
Independent variable t P value     
Soil temperature 8.678  < 0.0005     
Soil moisture  − 8.503  < 0.0005     
Overall variation 
Soil CO2 efflux (n = 16) Soil moisture and total tree biomass  0.920 0.823 35.828  < 0.0005 
Independent variable t P value     
Soil moisture  − 3.159 0.008     
Total tree biomass 2.744 0.017     
Mycorrhizal infection % −1.329 0.209     
Soil temperature −0.745 0.471     
Root ergosterol 0.662 0.520     
Final stem height 0.609 0.554     
Final stem biomass 0.531 0.605     
Final root biomass 0.307 0.764     
Final stem diameter 0.208 0.839     
Final leaf biomass 0.188 0.854     

Although trees were growing in artificial peat : sand mixture and in the pots, root mycorrhization levels were high (Table 5). Mycorrhizal infection levels and sporocarp production were both stimulated due to O3 stress (Table 5) in the end of the experiment (Harvest 2). Both temperature and O3 treatments increased root ergosterol concentrations, but only the temperature effect became statistically significant (Table 5). Although the whole root system growth was increased in most of the temperature-treated trees (Tables 2 and 3), the fine root ramification index decreased in the elevated temperature treatments (Table 5).

Table 5.

Ergosterol concentrations, total fine root ramification indexes, total mycorrhizal infection percentages of short roots and cumulative biomass of sporocarps at the end of September 2008 (Harvest 2). Values are means ± SE pooled over the genotypes. Genotype, O3, and temperature main and interaction effects on these variables are also shown below. Bolded values in the table indicate statistically significant (P < 0.05) or marginally statistically significant (P < 0.1) P values.

Treatment Ergosterol (μg g−1Total ramification index (root tips per 1 m) Total mycorrhizal infection (%) Sporocarps (g dry weight) 
150.3 ± 10.6 1433 ± 107 81.7 ± 2.3 35.8 ± 14.8 
O3 186.1 ± 12.5 1621 ± 91 89.4 ± 1.2 271.6 ± 48.3 
182.3 ± 11.9 1280 ± 62 80.1 ± 2.6 97.1 ± 37.3 
O3 + T 202.3 ± 21.2 1429 ± 91 84.6 ± 1.9 321.5 ± 101.6 
P values     
Genotype 0.690 0.131 0.064 0.714 
O3 0.234 0.166 0.003 0.062 
Temperature 0.082 0.053 0.113 0.331 
Genotype × O3 0.334 0.353 0.109 0.861 
Genotype × temperature 0.388 0.587 0.732 0.685 
O3 × temperature 0.564 0.819 0.427 0.920 
Genotype × O3 × temperature 0.131 0.747 0.766 0.519 
Treatment Ergosterol (μg g−1Total ramification index (root tips per 1 m) Total mycorrhizal infection (%) Sporocarps (g dry weight) 
150.3 ± 10.6 1433 ± 107 81.7 ± 2.3 35.8 ± 14.8 
O3 186.1 ± 12.5 1621 ± 91 89.4 ± 1.2 271.6 ± 48.3 
182.3 ± 11.9 1280 ± 62 80.1 ± 2.6 97.1 ± 37.3 
O3 + T 202.3 ± 21.2 1429 ± 91 84.6 ± 1.9 321.5 ± 101.6 
P values     
Genotype 0.690 0.131 0.064 0.714 
O3 0.234 0.166 0.003 0.062 
Temperature 0.082 0.053 0.113 0.331 
Genotype × O3 0.334 0.353 0.109 0.861 
Genotype × temperature 0.388 0.587 0.732 0.685 
O3 × temperature 0.564 0.819 0.427 0.920 
Genotype × O3 × temperature 0.131 0.747 0.766 0.519 

C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n  = 4 per treatment).

Genotype differences

Genotype 14 had the highest P : nP ratio at the final harvest (Harvest 2), indicating that this genotype had slower leaf senescence and abscission in general. Based on stem biomass, gt25 and gt14 were the largest and gt12 and gt15 the smallest trees, and this trend was seen in both harvests (Tables 2 and 3). Interestingly, in Harvest 1 root and total tree biomasses of gt15 were the smallest when compared with fast-growing gt14 and gt25, but in Harvest 2 these genotype differences had disappeared (Tables 2 and 3). In harvest 2, gt12 and gt15 had somewhat lower total mycorrhizal infection levels than gt14 and gt25 (Table 5).

Treatment effects on 13C signal and 13C allocation

Natural abundance of 13C in leaves and roots ranged from 1.0729 to 1.0740 atom% and from 1.0723 to 1.0732 atom%, respectively. In soil, the natural abundance range was from 1.0772 to 1.0776 atom%, and respiration was from 1.0742 to 1.0795 atom%. Hence, before labelling plant material was slightly more 13C-depleted than soil samples and soil respiration. Immediately after labelling (Day 0), the 13C enrichment was 2.1–2.5 atom% excess in leaves in all treatments, and thereafter varied from 0.2 to 0.7 atom% excess over the rest of the chase period. Fine root samples were collected only on two post-labelling days (on Days 12 and 49 after labelling), and also they showed clear 13C enrichment (atom% excess values ranged from over 0.2 to over 0.3 over the chase period). Soil respiration 13C abundance values varied from 0.1 to 2.1 atom% excess over the 21-day chase period. The highest enrichment was observed 2 days after labelling, whereas in general the first appearance of 13C started already on 0 day just a few hours after labelling. Although foliage, fine root and soil respiration enrichments were high, there were no clear treatment effects on these 13C atom% excess values over the whole pulse-chase period. Soil was collected for 13C analyses on three post-­labelling days (i.e., 5, 12 and 49 days after labelling), but when compared with plant material, its 13C atom% excess values showed only a slight enrichment (enrichment values ranged from 0.001 to 0.01 atom excess% and the highest enrichment occurred on the fifth post-labelling day). On the other hand, the overall chase period averages showed that in ambient O3 treatments the enrichment in soil was 0.004 atom% excess, whereas in elevated O3 treatments average enrichment was 0.006 atom% excess (O3 main effect, P = 0.050).

When 13C excess values were analysed, temperature-treated trees had a significantly higher amount of excess 13C in their foliage (temperature main effect, P = 0.020) and in fine roots (temperature main effect, P = 0.018). Temperature-induced increases in foliage and root excess 13C amounts were especially clear on peak enrichment days, in foliage this occurred on the first post-labelling day (0 day, Figure 3a) and in fine roots on the 5th post-labelling day, but thereafter this temperature effect on 13C amount was less clear (time × temperature interaction for foliage P  < 0.005, and for fine roots P = 0.023). Overall label return via soil respiration was increased by 32% in elevated temperature treatments (temperature main effect, P = 0.059) when compared with trees grown under ambient temperature. The temperature effect on soil respiration was again clearest on peak enrichment day, i.e., 2 days after labelling (Figure 3b, time × temperature interaction effect). Principal component analysis (Figure 4) also revealed temperature treatment effects on tree growth, overall soil respiration rates and amounts of excess 13C in foliage and soil respiration. Hence, temperature-treated trees fixed more 13C since they also had larger foliage biomass and in general, since trees were bigger, their soil respiration and thereby also 13C return via soil respiration were larger (Figure 4). Soil 13C excess amounts showed a marginally statistically significant O3 response (O3 main effect, P = 0.069), as in elevated O3 exposures (average 13C excess amount 304 mg m2) soil contained 44% more excess 13C than in ambient O3 treatments (average 13C excess amount 211 mg m2). Principal component analysis also showed that O3 treatments were mainly loaded on PC2 and this component explained ∼20% of the total data variation (Figure 4). Based on PCA, O3 effects were mainly seen in fungal components, as mycorrhizal infection and sporocarp production seemed to be stimulated under mild O3 stress, and also soil excess 13C amounts were more related to O3 treatments.

Figure 3.

(a) 13C excess amount in leaves and (b) 13C label return via soil CO2 efflux in gt15 trees during the pulse-chase period in 2008. Values are means ± SE. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Figure 3.

(a) 13C excess amount in leaves and (b) 13C label return via soil CO2 efflux in gt15 trees during the pulse-chase period in 2008. Values are means ± SE. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Figure 4.

The PCA biplot diagram showing the loading plot of tree and fungal growth, soil CO2 efflux, 13C excess atom% and 13C excess amount variables superimposed on the score plot of O3 and temperature treatments in gt15 trees. The total variation of data explained by principal components PC1 and PC2 was 41 and 20%, respectively. The PC1 separates temperature treatments and PC2 O3 treatments. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Figure 4.

The PCA biplot diagram showing the loading plot of tree and fungal growth, soil CO2 efflux, 13C excess atom% and 13C excess amount variables superimposed on the score plot of O3 and temperature treatments in gt15 trees. The total variation of data explained by principal components PC1 and PC2 was 41 and 20%, respectively. The PC1 separates temperature treatments and PC2 O3 treatments. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Partial 13C budgets for the 12th and 49th post-labelling days are shown in Figure 5a and b. In general, 13C budgets revealed that O3 had a tendency to reduce the proportion of excess 13C fixed in the foliage and simultaneously increase the proportion of 13C fixed in the soil (O3 main effects, P = 0.069 for foliage and P = 0.081 for soil, data pooled over measurement days). On the other hand, increased temperature reduced the amount of excess 13C in soil (temperature main effect, P = 0.041, data pooled over measurement days), probably indicating that 13C was lost faster from soil via increased soil respiration.

Figure 5.

Partial 13C budgets showing 13C excess amount allocated to leaves, roots and soil (a) 12 days and (b) 49 days after 13C labelling. Values are relative proportions of excess 13C fixed in different solid compartments on these two post-labelling days. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Figure 5.

Partial 13C budgets showing 13C excess amount allocated to leaves, roots and soil (a) 12 days and (b) 49 days after 13C labelling. Values are relative proportions of excess 13C fixed in different solid compartments on these two post-labelling days. C, control; O3, elevated O3 alone; T, elevated temperature alone; O3 + T, elevated O3 and elevated temperature in combination (n = 4 per treatment).

Discussion

Our model system was used to study the interactive effects of moderate warming and chronic O3 exposure on silver birch C allocation. We were able to demonstrate that both moderate warming and chronic ozone exposure affected silver birch C allocation, though sometimes treatment effects were genotype dependent, and also some counteractive effects of O3 on temperature responses were observed.

Silver birch growth under changing climate

As hypothesized, moderate temperature elevation was able to enhance silver birch above- and below-ground biomass accumulation and stem growth, although there were some exceptions from this generalization. The above results are in accordance with the previous leaf area measurements from the same experiment (Mäenpää et al. 2011) as well as with Carter (1996) and Peltola and Kellomäki (2005) who suggested that the deciduous trees benefit from climate warming. The temperature effect on stem elongation growth was more pronounced than that on diameter growth. According to Way and Oren (2010), larger enhancement of stem elongation compared with stem diameter growth could finally lead to reduced stem taper and make trees more vulnerable to, e.g., wind damage. Temperature treatments also usually increased root biomass, except in gt14 trees at the final harvest. In contrast to previous warming studies with ­different tree and grass species (Hartley et al. 2007, Way and Sage 2008), R : S ratios were not thus decreased due to warming, but in general larger trees had larger root systems also.

Before the leaf abscission period, all genotypes had the largest leaf biomass in elevated temperature treatments, but at the end of season O3 counteracted this temperature effect. Hence, temperature treatment alone caused delayed leaf senescence, but like in Mäenpää et al. (2011), temperature increase could not fully counteract these negative effects of O3. Similar partial compensation for the negative O3 effect was also reported for elevated CO2 in paper birch trees (Riikonen et al. 2008). Photosynthetic : non-photosynthetic tissue ratios, instead, revealed more genotype-dependent treatment responses. Especially, gt14 invested in foliage under enhanced temperature and also showed delayed leaf abscission, whereas gt15 invested more in root biomass especially before the leaf abscission period.

Based on P : nP ratios in the final harvest, elevated O3 accelerated leaf senescence and abscission, this result being in agreement with previous O3 studies (Pell et al. 1997, Riikonen et al. 2004, Ribas et al. 2005). However, this O3 effect was seen only in gt15 under ambient temperature, while in gt25 an O3 effect became evident under ambient and elevated temperature indicating again a large variation in O3 sensitivity within the Finnish birch populations in accordance with Pääkkönen et al. (1997). Negative O3 effects were also seen in stem height growth and stem biomass accumulation, consistently with Oksanen et al. (2007, 2009). Both variables indicated that the fastest-growing genotypes (gt14 and gt25) showed the largest O3-caused stem growth reductions and hence were the most O3-sensitive genotypes, whereas O3 effects on other two genotypes were negligible (gt12) or even slightly stimulative (gt15). Similarly, Häikiö et al. (2007) pointed out one O3-thriving genotype among a hybrid aspen population. In contrast to many previous studies made with fast-growing birches (Oksanen et al. 2009, Matyssek et al. 2010a), we did not observe any O3-caused R : S ratio reductions. Root : shoot ratio responses in general are highly variable because of intra- and interspecies variation, growing conditions and ontogenetic drift (Reich 2002).

Below-ground responses are complex and variable

In contrast to previous O3 studies (Andersen 2003, Kasurinen et al. 2004), there was no clear soil respiration stimulation or decrease due to O3 stress. Lack of a clear O3 effect on soil respiration was probably linked to negligible changes in coarse + fine root biomass in the present study. In addition, summer 2008 was very rainy in eastern Finland, and the rainfall between June and August was 43% higher than the long-term average (1971–2000; Finnish Metereological Institute 2008). Since O3 fumigation was automatically stopped during the rain, the O3 exposure in O3 treatments was low and might have also affected the magnitude of observed O3 responses. However, recently Nikolova et al. (2010) showed that fine root turnover was increased in O3-exposed beech trees, whereas root biomass was not changed and this fine root turnover change probably caused an increase in soil respiration rates too. Furthermore, in contrast to our study, O3 exposure clearly stimulated soil respiration rates beneath beech and Norway spruce trees during a humid year, whereas in dry years tree species-specific drought tolerance determined the magnitude of O3 effects on soil respiration responses (Nikolova et al. 2010). Interestingly, in our study, elevated O3 modified the temperature response of soil respiration in most of the studied genotypes. Hence, the most consistent temperature-induced increase in overall soil respiration rates was observed in gt14, whereas in gt12 O3 partly reduced the temperature-caused stimulation in soil respiration in O3 + T trees, and in other genotypes temperature effects were only seen in ambient O3 (gt25) or elevated O3 (gt15). A stepwise linear regression model also confirmed that, in addition to soil moisture conditions, overall tree biomass explains a significant amount of variation in soil respiration rates. Our results and other experiments (Niinistö et al. 2004, Bronson et al. 2008) with increased air + soil temperatures suggest that also tree-mediated temperature effects (i.e., indirect warming effects) can be significant modifiers of soil respiration responses under changing climate.

There was an O3-induced stimulation in mycorrhizal root growth measured as total infection level in this study, this finding being in accordance with previous O3 field studies (Kasurinen et al. 1999, 2005, Haberer et al. 2007). Previously, Kasurinen et al. (2005) showed that the clearest O3 effects were seen at the mycorrhizal root level, and recently Grebenc and Kraigher (2007) also observed that in mature beech trees the clearest O3 effects on root systems were seen in the mycorrhizal component. However, in the Kranzberg forest experiment, O3 stress effects on root biomass and C flux to roots were minor (Nikolova et al. 2010, Andersen et al. 2010), and observed changes in mycorrhizal root growth and diversity were suggested to be linked to O3-induced changes in phytohormone signalling between the shoot and roots (Matyssek et al. 2010b).

Interestingly, also sporocarp production was enhanced at the final harvest due to O3 stress. This result is in contradiction with the previous field study, where O3 reduced total sporocarp production beneath the silver birch trees (Kasurinen et al. 2005). However, sporocarp production responses are also fungal-species specific, and in the current study the main species forming sporocarps belonged to the genus Laccaria, whereas in a previous field study (Kasurinen et al. 2005) there were several different fungal species (e.g., Peziza badia, Amanita muscaria, Thelephora terrestris, Lactarius mammosus and Cortinarius spp).

13C allocation in gt15 trees was changed due to O3

Although trees were exposed to high air CO2 levels during the labelling (even close to 3000 ppm levels), the duration of this CO2 increase period was very short. In addition, all labelled trees were treated with 13C gas similarly and therefore we do not expect that the labelling method itself would change the outcome of this study. The pulse-labelling procedure led to clear 13C enrichment in tree foliage and roots, whereas the extent of soil 13C enrichment was lower. 13C signal in soil respiration was also clear. In leaves, the highest enrichment was observed immediately after the labelling (0 day), whereas in soil respiration the highest peak of label was seen 2 days after the labelling though there was some label return already on Day 0. In 13CO2 labelling studies, where the labelling gas has been in contact with soil surface, diffusion of 13CO2 in the soil pores can cause a large abiotic 13C return to the atmosphere (Subke et al. 2009). Since we enclosed trees inside labelling chambers of which the soil compartment was fully excluded, we can assume that most of the label return was biotic. Our data thus support previous studies in which 13C label return beneath deciduous trees started less than 0.5–1.5 days (Epron et al. 2011) and maximum label return via soil respiration also occurred 2–4 days after labelling (Plain et al. 2009, Epron et al. 2011).

According to PCA, temperature treatments were related to tree growth, soil respiration and also to the amount of 13C excess fixed in leaves and roots that returned via soil respiration. In temperature treatments trees were bigger and could fix more 13C during labelling and since their soil respiration rates were also higher, more 13C label was returned beneath temperature trees during the chase period. Principal component analysis also revealed that there were some O3 effects on below-ground variables, as the amount of 13C excess in soil, sporocarp production and total mycorrhizal infection levels were related to each other in O3 treatments. Thus, in gt15 temperature effects were clearly seen in above tree-level processes, whereas O3 effects were mainly manifested in the fungal compartments. In addition, partial 13C budgets (13C allocation patterns) also show that elevated O3 caused a shift in 13C allocation. Clearly, less 13C label was fixed into the foliage, while at the same time more 13C label was found in soil beneath the trees. Ozone stress has been suggested to impair phloem loading and cause accumulation of sugars in the foliage, but this should have been reflected as increasing 13C excess proportions in leaves. However, it is also possible that the quality of exudates released via rhizodeposition may have changed due to O3 stress (Andersen 2003), and this was reflected in soil 13C excess amounts. Since root samples were harvested less often than leaf samples, it is also possible that some treatment-induced changes in the relative proportions of root 13C excess amounts were masked due to less frequent sampling. We did not analyse sporocarp 13C excess amounts.

Conclusions

Moderate temperature elevation during the growing period has the potential to enhance silver birch growth and soil respiration in boreal forests, but also tree genotype and prevailing O3 levels can modify these responses significantly. Based on stem biomass and height growth data, the fastest-growing silver birches are also the most sensitive to chronic O3 stress. On the other hand, fungal growth responses and 13C-labelling experiment both showed that mild O3 stress can stimulate C flux from tree to root symbionts and soil. Changed P : nP ratios suggested that genotypes have different growth strategies, at least during their sapling period, as some of the genotypes clearly invested more in foliage relative to woody mass under temperature treatments. In addition, O3 had a tendency to enhance leaf senescence and abscission processes in some genotypes and thereby its effects on subsequent growth of these genotypes are likely to be cumulative.

Funding

This study was funded by the Academy of Finland (projects 109933 and 122297) and the University of Eastern Finland (spearhead project ‘Changing climate and biological interactions related to forests’).

Conflict of interest

None declared.

Acknowledgments

We thank Marjo Alavillamo and staff of UEF Research Garden for their help with carrying out this experiment. We also thank staff of the Finnish Forest Research Institute for their help with the micropropagation and planting of the experimental trees. Special thanks are due to Timo Oksanen for the technical assistance. Laboratory technicians Virpi Tiihonen and Jaana Rissanen are acknowledged for their help with the field work and laboratory analyses.

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