Stable Water Use Efficiency under Climate Change of Three Sympatric Conifer Species at the Alpine Treeline.

The ability of treeline associated conifers in the Central Alps to cope with recent climate warming and increasing CO2 concentration is still poorly understood. We determined tree ring stable carbon and oxygen isotope ratios of Pinus cembra, Picea abies, and Larix decidua trees from 1975 to 2010. Stable isotope ratios were compared with leaf level gas exchange measurements carried out in situ between 1979 and 2007. Results indicate that tree ring derived intrinsic water-use efficiency (iWUE) of P. cembra, P. abies and L. decidua remained constant during the last 36 years despite climate warming and rising atmospheric CO2. Temporal patterns in Δ(13)C and Δ(18)O mirrored leaf level gas exchange assessments, suggesting parallel increases of CO2-fixation and stomatal conductance of treeline conifer species. As at the study site soil water availability was not a limiting factor iWUE remained largely stable throughout the study period. The stability in iWUE was accompanied by an increase in basal area increment (BAI) suggesting that treeline trees benefit from both recent climate warming and CO2 fertilization. Finally, our results suggest that iWUE may not change species composition at treeline in the Austrian Alps due to similar ecophysiological responses to climatic changes of the three sympatric study species.

Introduction

High-altitude forest ecosystems at the timberline-treeline transition have raised concern as they may undergo significant alterations due to climate warming and changes in ground-level air chemistry (Holtmeier and Broll, 2007; Wieser et al., 2009). Dendroclimatological studies conducted within the treeline ecotone of the Central European Alps have shown radial stem growth to be limited by low summer temperature (Carrer and Urbinati, 2004; Oberhuber, 2004; Büntgen et al., 2005; Frank and Esper, 2005; Oberhuber et al., 2008). During recent decades, several authors report treeline-associated conifers to reflect increased radial growth, putatively related to climate warming (Graumlich et al., 1989; Peterson et al., 1990; Jacoby and D’Arrigo, 1997; Rolland and Florence-Schueller, 1998; Bunn et al., 2005). Moreover, increasing atmospheric CO2 concentration may act in concert with climate warming to increase carbon accumulation within the treeline ecotone (cf. Graumlich, 1991; Saurer et al., 1997; Duquesnay et al., 1998; Sidorova et al., 2009). Notwithstanding, reduced radial growth has been attributed to late-summer drought under increasing treeline temperature in the European Alps (Büntgen et al., 2006; Carrer and Urbinati, 2006; Oberhuber et al., 2008; Wieser et al., 2009).

Stable isotope ratios of carbon and oxygen, i.e., 13C/12C and 18O/16O, respectively, may serve as dendrochronological proxies that facilitate mechanistic understanding of climate-related influences on physiological processes such as leaf gas exchange and stem wood formation (Loader et al., 2007; Weigt et al., 2015; and further references therein). In plant organic matter, δ expressing the 13C/12C ratio in relation to an international standard (Pee Dee Belimnite) depends on variables such as leaf conductance for water vapor (gw) that modify the net CO2 uptake rate (A; Farquhar et al., 1989). In addition, δ of plant organic matter (δ13C) is a function of atmospheric δ, which is accounted for by calculating discrimination of photosynthesis against 13C (Δ Farquhar and Richards, 1984) in relation to intrinsic water-use efficiency (iWUE), i.e., the ratio of A (rate of net CO2 fixation) versus gw (leaf conductance for water vapor). For a review see Brugnoli and Farquhar (2000).

When analyzing δ alone, the impacts of A (demand of CO2) and gw (supply of CO2) on iWUE are difficult to separate (Saurer et al., 2008). The oxygen isotope ratio (δ), however, may allow a distinction between biochemical and stomatal limitations of photosynthesis as it is not affected by the photosynthetic CO2 carboxylation but linked to gw (Barbour et al., 2000; Grams et al., 2007). It is, therefore, an ideal covariable to estimate to what degree photosynthesis and stomatal conductance modify δ (Scheidegger et al., 2000; Werner et al., 2012). Originally, this dual isotope approach was introduced for photosynthetic tissue and only recently tested conceptually for the interpretation of tree-ring data (Roden and Farquhar, 2012), although several critical points should be taken into account during interpretion. Among the most important issues that need to be considered are the facts that the δ of source and atmospheric water can vary spatially and temporarily and that post-photosynthetic and post-evaporative oxygen atom exchange processes could affect the initial leaf-level isotope signal (see below).

At the leaf level, δ of photoassimilates derive primarily from leaf water, typically being enriched in 18O compared to the source water (i.e., xylem water) through evaporative enrichment at the site of transpiration. This enrichment is counteracted by the so-called Péclet effect and transpiratory leaf cooling (for a review see Barbour, 2007), which may result in the above mentioned negative correlation between δ and g (e.g., Barbour et al., 2000; Grams et al., 2007). However, to an extent that may depend on species and site conditions, the signal is dampend by oxygen exchange with source water during biomass formation at the stem level (Gessler et al., 2013). This causes, at least partially, a decoupling between oxygen isotopic signatures of photoassimilates and the tree ring organic matter. However, in a recent report Weigt et al. (2015) confirmed that information may be exploited to relate ecophysiological responses of trees to environmental changes for both total sapwood organic matter and extracted cellulose. In any case, it appears advisable to confirm interpretation from the dual isotope approach by gas exchange assessments whenever possible.

Previous tree-ring carbon isotope studies carried out in tropical, arid, Mediterranean, temperate and boreal forest ecosystems have identified an increase in iWUE over the past 40 years in response to increasing atmospheric CO2 concentration (Penuelas et al., 2011; Saurer et al., 2014) and just recently supported by tree-ring δ13C dynamic global vegetation models comparisons (DGVMs; Frank et al., 2015). Tree growth on the contrary, remained stable or even declined, suggesting local site conditions to override a potential CO2-induced increase in growth (Penuelas et al., 2011; Silva and Anand, 2013; Levesque et al., 2014). Increasing iWUE accompanied by a reduced productivity has been attributed to the combined effect of elevated CO2 and climate change-induced soil drying (Penuelas et al., 2011; Saurer et al., 2014).

The impact of the steadily increasing CO2 level and concurrent climate change, however, still awaits clarification for treeline-associated conifers in the Central Austrian Alps where low temperatures limit tree growth (Oberhuber, 2007; Wieser et al., 2009). At treeline in the Central Austrian Alps ample precipitation during the growing season prevails every third to fourth day on average (Wieser, 2012), so that soil water limitation stays absent, allowing trees to meet their water demand (Tranquillini, 1979; Mayr, 2007; Matyssek et al., 2009). Hence, whole-tree conductance stays high and mainly depends on the evaporative demand in terms of irradiance and vapor pressure deficit (Wieser, 2012). Therefore, we hypothesize that treeline trees passively respond to the increasing atmospheric CO2 level (C), so that their leaf-intercellular CO2 concentration (C) rises in parallel, while iWUE remains unchanged. The hypothesis was evaluated by stable carbon and oxygen isotope sampling and radial growth analysis over the past 36 years (1975–2010) in stems of mature Pinus cembra, Picea abies and Larix decidua trees growing at the treeline of Mt. Patscherkofel in the Central Tyrolean Alps. Observed long-term trends in δ in tree rings and iWUE were at least for some years compared with in situ leaf-level gas exchange data, assessed at the same study site between 1979 and 2007 in adult P. cembra and L. decidua trees. Results are discussed in view of tree response to climate warming at the treeline ecotone.

Materials and Methods

Study Site, Climatic Data, and Tree Species

The study was conducted in a scattered stand at the lower edge of the treeline ecotone at 1950 m a.s.l. on Mt. Patscherkofel (47°12′37″ N, 11°27′07″ E), south of Innsbruck, Austria. The site is characterized by a cool subalpine climate, the possibility of frost during the entire year and a continuous snow cover from October through April. We used monthly mean temperatures and monthly total precipitation from 1975 to 2010 from a weather station nearby (Klimahaus Research Station and Alpengarten; 1950 m a.s.l.) for our analysis. Mean annual precipitation averaged 878 mm, with 58% falling during the growing season (May through September). Mean annual air temperature averaged 2.4°C, with summer maxima of up to 27°C and winter minima of -28°C.

The geology of Mt. Patscherkofel is dominated by gneisses and schist. The soil at the study site is a haplic podzol, being a typical soil type of the treeline ecotone in the Central Tyrolean Alps (Neuwinger, 1972). The water holding capacity of the soil (at 5–65 cm depth) at saturation (-0.001 MPa) averages 0.60 m3 m-3. Due to frequent precipitation during the growing season soil water potential rarely drops below -0.01 MPa, approximating soil water contents above 0.35 m3 m-3 (Wieser, 2012; including the dry summer of 2003, unpublished data).

The stand is composed of the dominant tree species Pinus cembra, accounting for 84% of the tree population, and accompanied by Larix decidua (9%) and Picea abies L. Karst (7%) at some locations. Trees grew either as isolated trees or in groups of four to five. The distance between single trees or tree groups was 20–30 m. From 20 P. cembra trees cored at the lower edge of the treeline ecotone Oberhuber et al. (2008) derived an expressed signal population (EPS) value of 0.94, reflecting a strong climate signal in the site chronology. From these trees we selected five trees which had the strongest correlation to the site specific mean tree-ring chronology, no missing rings, and regular ring boundaries. In addition we cored five dominant P. abies and L. decidua trees each to account for potential inter-specific differences of the three associated treeline species. In 2010 the trees were 69 ± 9 years old, with stem heights averaging 12 ± 1.3 m. The stem diameter at breast height (DBH) averaged 22 ± 3.2 cm.

Tree Ring and Basal Stem Area Increment

In fall 2010 we obtained two increment cores per trees at DBH using a 5-mm-diameter increment bore. For contrast enhancement of tree ring boundaries the cores were dried in the laboratory, non-permanently mounted on a holder, and the surface was prepared with a razor blade (Pilcher, 1990). Ring widths were measured to the nearest 1 μm using a reflecting microscope (Olympus SZ61) and the software package TSAP WIN Scientific. Ring widths of both cores from each sample tree were averaged and individual tree ring chronologies were then checked for dating accuracy using the COFECHA software (Holmes, 1994; Grissino-Mayer, 2001). As ring width may be biased by a negative correlation with the time course during tree maturation, ring width was converted to basal stem area increment (BAI) according to:

where R is stem radius inside tree bark and n is the year of tree ring formation (Fritts, 1976). Bark thickness was subtracted from stem radius. Finally BAI of each year were averaged over the five sample trees of each species.

Stable Isotope Analysis

δ and δ analyses for the years 1975–2010 were performed on the same cores as used for BAI assessment. Annual rings (early wood plus late wood) were cut exactly at ring boundaries by use of a scalpel and a reflecting microscope (Wild 308700). For each of the five study trees per species the two samples per tree ring were pooled and homogenized with a swing mill (Retsch MM301, Retsch Haan, Germany). In a subsample, we compared isotope signatures in bulk wood with those in cellulose for determining the necessity of cellulose extraction in our study trees. Cellulose extraction was performed using a modified version of the method of Brendel et al. (2000). The methodological comparison corroborated significant correlations in the cases of δ and δ (Figure ) as reported earlier from coniferous and other tree species (Jaggi et al., 2002; Sohn et al., 2013) and is in accordance with a recent report (Weigt et al., 2015). On average, δ in cellulose was 1.0–1.1 ‰ higher than in bulk wood (Table ), being smaller than 1.3–1.4 ‰ found in Picea abies by Borella et al. (1998) and Sohn et al. (2013). Mean δ in cellulose was 4.3–4.9 ‰ higher than in bulk wood (Table ) being somewhat lower than 5.9 ‰ in bulk wood of Picea abies (Sohn et al., 2013). Based on these findings and in accordance with a recent methodological study (Weigt et al., 2015), we used bulk wood samples rather than extracted cellulose for isotope analysis.

Comparison of Dashed lines reflect the one-to-one lines for comparison and the solid lines correspond to the linear regression analyses of the data points: δ: y = 1.03x + 1.86, r= 0.92, P < 0.001, n = 36; δ; y = 0.92x + 6.65, r= 0.73, P < 0.001, n = 18.

Regarding δ, 2.0 ± 0.02 mg of homogenized samples were weighed into tin capsules each (3.5 × 5 mm, IVA Analysentechnik e.K., Meerbush, Germany) and combusted to CO2 in an elemental analyzer (Eurovector EA3000) connected to an isotope ratio mass spectrometer (Isoprime, Elementar, Hanau, Germany). For δ analysis 0.7 ± 0.05 mg were weighed into silver capsules each (3.5 × 5 mm, IVA Analysentechnik e.K., Meerbush, Germany) to obtain CO at 1,430°C in a high-temperature pyrolysis system (HTO, HekaTech, Wegberg, Germany) which was connected via an open-split interface (Conflo III; Finnigan MAT, Bremen, Germany) to an isotope ratio mass spectrometer (Delta Plus; Finnigan MAT, Bremen, Germany). Isotope abundances were expressed using the δ-notation in ‰ relative to the international standards:

where Rsample is the molar fraction of the 13C/12C or 18O/16O ratio of the sample, and Rstandard that of the international IAEA standards V-PDB for carbon and V-SMOW for O. The analytical precision was <0.12 ‰ and <0.28 ‰ regarding δ and δ, respectively (expressed as standard deviation of the internal laboratory standard at the same sample mass).

Isotope Discrimination and iWUE

Tree ring specific δ were corrected for the progressive decline in atmospheric δ through calculating 13C discrimination (C):

To this end, δ with its nearly linear time course during 1980 through 2010 [1] was extrapolated for the years 1975 through 1979. In a simplified model, Farquhar et al. (1982) related Δ through plant physiological processes during CO2 fixation in C3 plants with the ratio of intercellular to ambient CO2 partial pressure (C/C):

where a (=4.4 ‰) refers to the slower diffusivity of 13CO2 relative to 12CO2 in air and b (=27 ‰) is the isotopic fractionation caused by enzymatic C fixation. C was obtained from published data[2]. It should be noted that Δ is determined by the ratio of chloroplast to the ambient CO2 mole fraction (C) rather than C, as used in equation 3, making the here calculated value sensitive to mesophyll conductance (g; Seibt et al., 2008). The latter varies in accordance to changes in environmental conditions such as temperature, irradiance, water and CO2 availability (Flexas et al., 2008). Consequently using C may be problematic if g to CO2 is not constant (Seibt et al., 2008). However, as information on mesophyll conductance of the three conifers under study is not available and published means of g would not improve results (Cernusak et al., 2013), we chose using the simplified linear model of Farquhar et al. (1982). Hence, iWUE, i.e., the ratio of the net carbon gain (A) versus leaf conductance for water vapor (gw), was calculated as follows:

where 1.6 is the ratio between the diffusivities of water vapor and CO2 in air.

Enrichment in 18O in tree rings over source water (Δ), resulting from incorporation of 18O-enriched photoassimilates into stem biomass, was calculated from d18O of tree ring organic matter (δ) and precipitation (δ) according to:

Precipitation is the only source of water on Mt. Patscherkofel and thus was assumed to reflect source water of trees. Furthermore, as there is evidence from a treeline in the Central Swiss Alps that mean values of δ and δ of soil water do not differ significantly, although soil water δ carries a distinct signal from snow melt water far into the growing season (Treydte et al., 2014), we used annual means of δO sampled on top of Mt. Patscherkofel (2246 m a.s.l.) (Umweltbundesamt Austria; personal communication) approximately 300 m south of the selected study trees for our ΔO calculation. We elevationally corrected δby a factor of 0.17 ‰ per 100 m of elevation (Umweltbundesamt Austria; personal communication) which is considerably lower than the global mean of 0.28 ‰ per 100 m of elevation (Poage and Chamberlain, 2001).

Leaf Level Gas Exchange Data

To illustrate long term trends in foliar CO2 and H2O gas exchange we compiled published gas exchange data of mature, field grown P. cembra and L. decidua trees carried out at our study site between 1979 and 2007 (Table ). Maximum net photosynthetic capacity at ambient CO2 (Amax; sensu Larcher, 2001) was assessed of sun exposed twigs from the upper canopy. Employed in situ were thermoelectrically climate-controlled cuvettes (Walz, Effeltrich, Germany) or a portable exchange system (CIRAS 1, PP Systems, Hitchin, Hertfordshire, UK). For methodological details see publications given in Table .

Statistical Analysis

Temperature, precipitation, vapor pressure deficit, Δ, BAI, C/C and iWUE trends were calculated for the time period 1975–2010 by least-squares linear regression analysis. For a given variable, differences among trends (slopes) between P. cembra, P. abies and L. decidua were assessed by the two-slope comparison test (Zar, 1999). We used repeated measures ANOVA to detect significant differences in the mean values (1975–2010) of Δ, BAI, C/C and iWUE of P. cembra, P. abies and L. decidua. Following Kunter et al. (2004) we used multiple least-squares linear regression models to assess the influence of atmospheric CO2 concentration (C) and mean growing season (May-Sep) air temperature (Tveg) and their interactions (explanatory variables) on tree-ring variables. For assessing the climatic impact on tree ring variables (BAI, Δ, and Δ) statistical analyses were based on mean monthly air temperature (°C) and total monthly precipitation (mm) throughout the study period (1975–2010). For each species Pearson’s correlation coefficients between BAI, isotope chronologies and both climate variables were calculated from August of the year prior to growth to September of the growth year. All the statistical analysis were conducted by use of the SPSS 16 software package (SPSS. Inc. Chicago, IL, USA), and a probability level of P < 0.05 was considered as statistically significant. As suggested by Sarris et al. (2013) we did not remove any age related trend from our tree-ring chronologies by conventional detrending procedures, thus avoiding the risk of removing any environmental signal or trend captured by our tree-ring series.

Results

Inter-annual Trends in Climate and Tree-Ring Indices

A warming trend is reflected at our treeline site during the growing seasons (0.50°C per decade P < 0.001) of 1975–2010, without concurrent trends in precipitation and vapor pressure deficit (Figure ). During the whole study period Δ and Δ chronologies were synchronized between the three studied species. In each species Δ13C increased over time (Figure ; Table ) whereas Δ decline (Figure ; Table ). The increase in Δ was accompanied by rising of Amax for both P. cembra (1979–2007) and L. decidua (1980–1993) by about 50%. Likewise, gw increase by about 75% in L. decidua (Table ).

Temporal variation in Data were fit by linear regression analysis: Tair: y = 0.053x-97.0, r= 0.30, P < 0.001; P: y = 0.075x-365.3, r= 0.00, P = 0.95; VPD: y = -0.055x + 13.3, r= 0.01, P = 0.49.

See Table for regression information.

The mean tree-ring Δ was highest in L. decicua, although the increase was significantly higher in P. cembra and P. abies (Table ). Temporal changes in Δ by contrast, did not differ significantly between the tree species (Table ). P. cembra showed the highest Δ while P. abies presented the lowest Δ and L. decidua displayed an intermediate mean (Table ). On average, growth of P. cembra was significantly higher than growth of P. abies and L. decidua (Figure ; Table ). During the whole study period all three species showed an increase in growth expressed as BAI, being significantly lower in P. cembra than P. abies, and L. decidua (Figure ; Table ).

Paralleling atmospheric CO2 enhancement (Figure ), tree ring derived C increased from 1975 through 2010 from 180 to 234 μmol mol-1 in P. cembra, from 185 to 227 μmol mol-1 in P. abies and from 213 to 256 μmol mol-1 in L. decidua (Figure ; Table ). Although species specific differences in the temporal change of C were not statistically significant different from each other, mean C was significantly lower in P. cembra and P. abies as compared to L. decidua (Table ). Averaged over the study period P. cembra showed the lowest and L. decidua, the highest C/C, while the C/C of P. abies was intermediate (Table ). The increase in C/C over time (Figure ; Table ) was significantly higher in P. cembra and P. abies than in L. decidua (Table ). In all the three species iWUE had remained stable during the study period (Figure ; Table ). However, we observed statistically significant between species, with P. cembra showing the highest and Larix decidua showing the lowest iWUE averaged over the study period (Table ).

(A) Ambient armospheric CO2 concentration (C, thin solid line) C, (B) C/C, and (C) iWUE chronologies of P. cembra (solid circle, solid line), P. abies (solid square, dashed line) and L. decidua (open circle, dotted line) between 1975 and 2010. See Table for regression information.

Effects C and Tveg on Δ, BAI, C/C and iWUE

Multiple linear regression analysis show that Δ and Δ of all species significantly increased with increasing ambient CO2 concentration (C) while growing season mean air temperature (Tveg) had no effect on Δ and on Δ (Table ). Growth (BAI) of P. cembra, and P. abies significantly increased with increasing C and Tveg. L. decidua presented a significant increase in BAI at increasing C without any response to Tveg (Table ). For all species we found a significant increase in C and C/C at higher C but not at higher Tveg (Table ). iWUE of P. cembra, P. abies, and L. decidua, however, did not significantly respond to increasing C and Tveg (Table ).

Δ, Δ, and BAI Response to Climate (Climate-Growth Relationships)

Climate-response relationships of Δ, and BAI differed both in time and in signal strength (Figures and ). In all three species Δ was significantly positive correlated with April throughout June temperatures (Figure ) and significantly negative correlated with January precipitation (Figure ). Previous-year August and October temperature also favored Δ in P. cembra and in P. abies, respectively (Figure ), whereas previous- and current-year August precipitation did so in P. abies (Figure ).

Pearson correlation coefficients between Horizontal gray lines indicates P < 0.05.

Pearson correlation coefficients between Horizontal gray lines indicates P < 0.05.

The effects of temperature and precipitation on Δ were clearly in opposite directions (Figures and ). In all the three species tree-ring Δ was negative correlated to air temperature from April to June of the current year and significantly positive correlated to previous-year December temperature (Figure ) as well as to January precipitation (Figure ). From Figure , previous- year August temperature had a negative correlation with L. decidua and current-year January temperature showed a negative correlation with L. decidua. Previous-year November and current-year March precipitation showed a negative correlation with Δ in L. decidua, as did June precipitation in P. cembra (Figure ).

We also found significant positive correlations between BAI and temperature during April, May, and June in P. cembra, P. abies, and L. decidua (Figure ). Current-year August temperature also favored BAI in L. decidua and P. abies, and also previous-year October temperature in P. abies (Figure ). The correlations between BAI and precipitation were weak, except for significant positive correlations in February and current-year August in P. cembra and a significant negative correlation in January in P. abies (Figure ).

Discussion

Similar growth and Δ, and Δδ responses were found over time in P. cembra, P. abies and L. decidua at the lower edge of the treeline ecotone in the central Austrian Alps. From 1975 throughout 2010 the three species increased Δ and BAI, while Δ showed a declining trend. Apparently, underlying response mechanisms were similar across the three studied species.

Our observed correlations for temperature and precipitation for Δ and Δ (Figures and ) are consistent with results reported for oak and pine trees at temperate sites in Switzerland (Saurer et al., 2008). Weather conditions prevailing during April through June predominantly were responsible for variations in tree-ring Δ, and BAI of P. cembra, P. abies, and L. decidua. We found positive correlations between April to June temperatures and Δ. Δ is strongly affected by net CO2 uptake rates, which at treeline are governed by both photon flux density and temperature (Treydte et al., 2001; McCarrol and Pawellek, 2004; Kress et al., 2011) as well as enhanced plant transpiration (Liu et al., 2015). Moreover, at our study site in situ net photosynthetic capacity of sun exposed twigs from the upper canopy of mature P. cembra and L. decidua trees measured under clear summer days also tended to increase between 1979 and 2007 (Table ), which might be attributed to both the observed increase in atmospheric Ca and Tair. Additionally, the temperature optimum of Amax for P. cembra increased from 12.5°C in 1956 (Pisek et al., 1969, 1973) to 15.0°C in 2002 (Wieser, 2004) and to 17.1°C in 2007 (Wieser et al., 2010), matching the observed increase in mean growing season air temperature of 0.9°C per decade (Figure ). An increase in net photosynthetic rates under elevated CO2 was also observed in P. mugo and L. decidua after nine years of free-air CO2 enrichment at the Swiss treeline (Dawes et al., 2013; Streit et al., 2014). Three years of ecosystem warming also increased carbon uptake of Pinus cembra at treeline in the Austrian Alps (Wieser et al., 2015).

Our precipitation signals suggest that trees do not suffer from moisture stress. Indeed the observed declining trend Δ and the strong negative correlations between Δ and growing season precipitation is consistent with the physiological isotopic responses (Barbour and Farquhar, 2000), suggesting that stomatal conductance is increased during the study period. Although a leaf physiological signal in δ will be dampened at the level of tree rings due to oxygen exchange with source water during cellulose biosynthesis, impact of gw on 18O in tree rings may be still detectable, even in whole wood analyses (Weigt et al., 2015). Ecosystem warming accompanied by unchanged VPD also increased g and hence also transpiration in boreal Picea abies (Bergh and Linder, 1999), Pinus sylvestris (Kellomäki and Wang, 1998), Picea mariana (Van Herk et al., 2011), Pinus cembra at treeline (Wieser et al., 2015), and Populus deltoides (Barron-Gafford et al., 2007). Thus, it seems that in cold environments under non-limiting water availability increasing temperatures counteract the diminishing effect of rising CO2 on leaf conductance (Saurer et al., 2014).

The observed positive correlations between BAI and April–June temperatures are also reflected in wood formation. At the study site wood formation of larch, spruce and pine generally starts in May, reaches its maximum in June, and terminates in August (Havranek, 1981; Loris, 1981; Gruber et al., 2009). Beside summer temperatures (Figure ) other climatic variable like winter and August precipitation (Figure ) are also known to influence radial growth at treeline as shown for P. cembra by Oberhuber (2004), reflecting minor soil water effects on tree growth at treeline (Tranquillini, 1979; Wieser, 2004). Although treeline trees are saturated with carbohydrates (Gruber et al., 2011), growth of trees at treeline is primarily affected by temperature dependent carbon sink activity during tissue formation (Hoch and Körner, 2003, 2012). The observed increase in BAI (Figure ) suggests that our treeline trees benefit from climate warming, although effects of CO2 fertilization on growth may not completely ruled out (Table ). Four years of experimental air warming with open-top-chambers also stimulated radial growth of Picea glauca seedlings at the subarctic treeline in southwest Yukon, Canada (Danby and Hik, 2007). Thus, when growth is stimulated and there is plenty of water g can increase as indicated by a decline in Δ (Figure ) along with increasing A, resulting in a constant iWUE (Figure ).

Elevated atmospheric CO2 is expected to affect plant carbon-water relationships, as a decline in stomatal conductance is often observed when plants are exposed to elevated CO2 (Battipaglia et al., 2013). If stomatal conductance declines under increasing CO2 in combination with an increased or unchanged carbon assimilation, this will decrease the C to C ratio and thus decrease Δ. Conversely, in all three study species Δ increased from 1975 throughout 2010, while tree-ring derived iWUE remained stable (Figure ) although ambient CO2 concentration increased by 60 μmol mol-1 (Figure ). The stability of iWUE resulted as C drifted upward paralleling the rise in C (Figure ). Likewise, in Picea schrenkiana at treeline in the western Tianshan Mountains in China iWUE remained also unchanged from 1985 to 2010 (Wu et al., 2015). No change in iWUE (i.e., homeostasis) over the last 100 years was also reported for three conifer species in the Selkirk Range (Rocky Mountains, Idaho, ID, USA) by Marshall and Monserud (1996). Other studies by contrast observed a 20% increase in iWUE from the 1960 throughout 2000 in mature trees in tropical, arid, Mediterranean, wet temperate and boreal forests distributed through Europe, Asia, Africa, America, and Oceania (Penuelas et al., 2011; Saurer et al., 2014; Frank et al., 2015). In these latter studies, increasing iWUE was attributed to the combined effect of increasing CO2 and climate change-induced soil drying that reduced stomatal aperture. Soil drought can be ruled out along the treeline ecotone of the Central Alps (Mayr, 2007; Wieser, 2012). Occurrence of soil drought strongly depends on site conditions such as precipitation patterns, water holding capacity of the soil, and evaporative demand. Ample precipitation and moderate evaporative demand in general cause soil water availability to be sufficiently high to meet the trees’ water demand at treeline in the Central Tyrolean Alps (Mayr, 2007; Wieser et al., 2009). As a consequence, treeline trees are rarely forced to restrict transpiration (Tranquillini, 1979; Benecke et al., 1981; Matyssek et al., 2009; Wieser and Leo, 2012; Wieser et al., 2014, 2015). Given the ample soil water availability whole-tree conductance of P. cembra, P. abies, and L. decidua remains high for CO2 uptake because leaf conductance for water vapor depends only on the evaporative demand driven by irradiance and vapor pressure deficit (Wieser, 2012).

Beside climate warming and increasing C, nitrogen deposition could also be important for explaining the observed increase in tree growth as increasing nitrogen deposition during the 1980ties (Smidt and Mutsch, 1993) has been suggested as a possible growth stimulator. However, there is evidence that nitrogen contents per needle dry mass are higher in trees at treeline as compared to trees growing at lower elevation sites (Körner, 1989; Birmann and Körner, 2009). Furthermore, since 1988, nitrogen deposition at treeline in the Tyrolean Alps is steadily declining (Amt der Tiroler Landesregierung, 2015), and a nitrogen fertilizer experiment at the alpine treeline in the Swiss Alps showed little or no growth stimulation (Keller, 1970). Thus, it seems that presently nitrogen deposition is insufficient to explain observed growth trends at treeline as reported previously by Tranquillini (1979) and Nicolussi et al. (1995).

Conclusion

Treeline trees respond to the increasing atmospheric CO2 level (C) in a way that their leaf-intercellular CO2 concentration (C) drifted upward paralleling the rise in atmospheric CO2 while iWUE remained stable over the last 36 years. The stability in iWUE was accompanied by an increase in BAI suggesting that treeline trees benefit from both recent climate warming) and CO2 fertilization. In addition, treeline trees are rarely forced to restrict transpiration due to ample soil water availability (Tranquillini, 1979; Matyssek et al., 2009; Wieser et al., 2015). A stable iWUE suggests an increase of both carbon gain and leaf conductance for water vapor as also indicated by stable C and O isotope analysis and direct gas exchange assessments. Furthermore, iWUE may not change species composition at treeline in the Austrian Alps due to similar ecophysiological responses to climatic changes of the three sympatric study species. Our finding that growth of treeline associated conifers increases with slowly rising ambient CO2 concentration and warming may be relevant for assessing complex growth models with empirical data, finally leading to model improvements and better estimations of forest-climate feedbacks.

Author Contributions

GW, RM, and TG conceived and designed the experiment. WO, AG, and ML performed the experiment. GW, WO, AG, and ML analyzed the data. GW, RM, and TG wrote the manuscript and WO and AG provided editorial advice.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Table 1

δ and δ difference between cellulose and bulk wood in annual growth rings of Pinus cembra, Picea abies and Larix decidua.

Species	δ13C [‰]	δ18O [‰]
P. cembra	1.1 ± 0.0	4.4 ± 0.4
P. abies	1.0 ± 0.1	5.0 ± 0.6
L. decidua	1.1 ± 0.1	4.9 ± 0.4

Table 2

A comparison of published maximum net photosynthetic capacity (Amax) and leaf conductance for water vapor of sun exposed shoots of mature Pinus cembra and Larix decidua trees at the lower end of the treeline ecotone on Mt. Patscherkofel.

Species	Year	Measured trees	Amax [μmol m-2 s-1]	gw [mmol m-2 s-1]	Reference
P. cembra	1979	1	3.4	n d	Havranek, 1981
P. cembra	2002	2	4.6 ± 0.2	n d	Wieser et al., 2005
P. cembra	2007	3	5.2 ± 0.7	n d	Wieser et al., 2010
L. decidua	1980	1	3.3	48	Benecke et al., 1981
L. decidua	1993	4	5.6 ± 0.9	85 ± 14	Volgger, 1995

Table 3

Regression information for Figures and .

Variable	Species	Equation	r2	P-value
Δ13C	P. cembra	y = 0.054x - 91.3	0.77	<0.001
	P. abies	y = 0.049x - 79.2	0.76	<0.001
	L. decidua	y = 0.030x - 40.1	0.49	<0.001
Δ18O	P. cembra	y = -0.062x + 159.9	0.50	<0.001
	P. abies	y = -0.075x + 184.7	0.60	<0.001
	L. decidua	y = -0.056x + 148.0	0.51	<0.001
BAI	P. cembra	y = 0.147x - 281.9	0.50	<0.001
	P. abies	y = 0.219x - 430.8	0.90	<0.001
	L. decidua	y = 0.273x - 538.4	0.77	<0.001
Ci	P. cembra	y = 1.81x - 3392.5	0.94	<0.001
	P. abies	y = 1.75x - 3296.0	0.94	<0.001
	L. decidua	y = 1.56x - 2873.3	0.94	<0.001
Ci/Ca	P. cembra	y = 0.002x - 4.33	0.77	<0.001
	P. abies	y = 0.002x - 3.88	0.76	<0.001
	L. decidua	y = 0.001x - 2.18	0.62	<0.001
iWUE	P. cembra	y = -0.085x + 249.4	0.09	0.067
	P. abies	y = -0.061x + 199.5	0.06	0.148
	L. decidua	y = 0.045x - 21.9	0.05	0.215

Table 4

Tree-ring carbon isotope characteristics (Δ18O, BAI, C/C, and iWUE) in P. cembra, P. abies, and L. decidua during the period 1975–2010.

	P. cembra		P. abies		L. decidua
	Change	Average (±SE)	Change	Average (±SE)	Change	Average (±SE)
Δ13C [‰]	1.9a	17.2 ± 0.7a	1.8a	17.3 ± 0.6a	1.1b	18.8 ± 0.484b
Δ18O [‰]	-2.2a	35.9 ± 0.9a	-2.7a	35.0 ± 1.052b	-2.0a	35.6 ± 0.883c
BAI [cm2]	5.3a	7.6 ± 3.8a	7.9b	5.7 ± 2.473b	9.8b	5.8 ± 3.3b
Ci [μmol mol-1]	65.2a	203.5 ± 19.6a	63.06a	208.5 ± 19a	56.2a	228.8 ± 16.9b
Ci/Ca	0.1a	0.6 ± 0.03a	0.1a	0.6 ± 0.03b	0.00b	0.6 ± 0.03c
iWUE [μmol mol-1]	-3.1a	81.0 ± 2.9a	-2.2a	78.4 ± 2.6b	-1.6a	67.8 ± 2.2c

Table 5

Summary of multiple linear regression models fitted to explain inter-annual changes (1975–2010) in Δ, BAI, CC, and iWUE of P. cembra, P. abies, and L. decidua in response to atmospheric CO2 concentration (C) and mean growing season (May-Sep) air temperature (Tveg).

Species	Variable	coefficient	SE	β	t-value	r-value partial	P-value
Δ13C
P. cembra	InterceptCaTveg	5.4830.034-0.058	1.2250.0040.067	0.903-0.091	4.477.584-0.865	0.8310.149	<0.001<0.0010.393
P. abies	InterceptCa Tveg	6.7970.031-0.064	1.0540.0030.058	0.925-0.111	6.4469.187-1.106	0.848-0.189	<0.001<0.0010.277
L. decidua	InterceptCa Tveg	11.8830.023-0.164	1.0270.0030.056	0.901-0.378	11.5726.917-2.900	0.769-0.451	<0.001<0.0010.007
Δ18O
P. cembra	InterceptCa Tveg	49.624-0.0390.054	2.3600.0080.130	-0.7420.060	21.030-5.1580.419	-0.6680.073	<0.001<0.0010.678
P. abies	InterceptCa Tveg	51.708-0.0500.143	2.3630.0080.130	-0.8420.143	21.886-6.5131.0103	-0.7500.189	<0.001<0.0010.278
L. decidua	InterceptCa Tveg	45.863-0.026-0.094	2.2190.0070.122	-0.574-0.120	20.671-3.671-0.768	-0.538-0.0132	<0.0010.0010.448
BAI
P. cembra	InterceptCaTveg	-19.9640.0790.357	5.4820.0180.301	0.6270.166	-3.6424.4721.184	0.6140.202	0.001<0.001<0.001
P. abies	InterceptCaTveg	-40.5830.1200.401	2.6430.0090.145	0.8550.169	-15.35413.9902.761	0.9250.433	<0.001<0.0010.009
L. decidua	InterceptCa Tveg	-53.5320.1590.272	5.3820.0170.296	0.8440.085	-9.9479.1280.919	0.8460.158	<0.001<0.0010.365
Ci
P. cembra	InterceptCa Tveg	-188.8301.119-1.033	19.0450.0621.047	0.991-0.054	-9.91518.130-0.987	0.953-0.169	<0.001<0.0010.331
P. abies	InterceptCa Tveg	-177.5001.100-1.014	17.3080.0560.952	0.996-0.054	-10.25519.623-1.065	0.960-0.182	<0.001<0.0010.294
L. decidua	InterceptCa Tveg	-113.9380.987-1.313	14.6920.0480.808	1.011-0.079	-7.75520.726-1.625	0.964-0.272	<0.001<0.0010.114
Ci/Ca
P. cembra	InterceptCa Tveg	0.0370.002-0.003	0.0540.0000.003	0.906-0.087	0.6808.752-0.843	0.836-0.145	0.501<0.0010.405
P. abies	InterceptCa Tveg	0.0870.001-0.003	0.0490.0000.003	0.918-0.096	1.7959.118-0.995	0.864-0.164	0.082<0.0010.347
L decidua	InterceptCa Tveg	0.3210.001-0.004	0.0410.0000.002	0.887-0.0193	7.8177.240-1.574	0.783-0.264	<0.001<0.0010.125
iWUE
P. cembra	InterceptCa Tveg	98.731-0.0620.534	9.9640.0320.548	-0.3720.189	9.909-1.9150.974	-0.3160.161	<0.0010.0640.337
P. abies	InterceptCaTveg	92.903-0.0530.534	9.0520.0290.534	-0.3510.210	10.263-1.7981.072	-0.2990.184	<0.0010.0810.291
L. decidua	InterceptCa Tveg	59.6440.0070.695	7.6610.0250.421	0.0520.317	7.7850.2701.650	0.0470.276	<0.0010.7890.108