Divergent patterns of foliar δ¹³C and δ¹⁵N in Quercus aquifolioides with an altitudinal transect on the Tibetan Plateau: an integrated study based on multiple key leaf functional traits

Abstract

Aims

With a close association with plant water availability, foliar δ¹³C had been investigated extensively in alpine regions; however, foliar δ¹⁵N has rarely been concurrently used as an indicator of plant nitrogen availability. Due to the positive correlations between leaf nitrogen content and foliar δ¹³C and δ¹⁵N found in previous studies, we expected that they should show consistent patterns along an altitudinal gradient.

Methods

To test our hypothesis, we measured foliar δ¹³C and δ¹⁵N in conjunction with multiple key leaf functional traits of Quercus aquifolioides, a dominant species of alpine forest on the eastern slopes of the Sygera Mountains, southeastern Tibetan Plateau from 2500 to 3800 m.

Important findings

(i) Contrary to our hypothesis, foliar δ¹³C exhibited a significant positive linear relationship with altitude; in contrast, foliar δ¹⁵N initially increased and subsequently decreased with altitude, the change in trend occurring around 3300 m. (ii) Our analyses indicated that leaf internal resistance and stomatal conductance, rather than photosynthetic capacity indicated by leaf N concentration, apparently explained the altitudinal variation in foliar δ¹³C, while differences in foliar δ¹⁵N were likely the result of soil N availability. (iii) Principal component analysis revealed a clear association between δ¹³C and a tradeoff between water loss and carbon gain, indicated by traits related to gas exchange such as leaf thickness, density, stomatal properties. In contrast, the second axis was associated with δ¹⁵N and nitrogen acquisition strategy in Q. aquifolioides across its altitudinal distribution, represented by traits related to nitrogen concentration and stomata per gram of leaf nitrogen.

INTRODUCTION

The relative proportions of stable isotopes of carbon and nitrogen can provide insights into nutrient and moisture dynamics in terrestrial ecosystems, because both isotopes are involved in several fundamental biogeochemical processes and are sensitive to environmental conditions (Handley et al. 1999). Therefore, variations in these isotopes have been used in a number of studies to explore plant responses to changing climates and environmental gradients (Adams and Grierson 2001; Dawson et al. 2002). In particular, it has been suggested that they vary along altitudinal gradients in alpine regions (Cordell et al. 1998; Cordell et al. 1999; Hultine and Marshall 2000; Körner et al. 1988).

As an important indicator of plant long-term water use efficiency, foliar δ¹³C at intra-specific level showed a strong and consistent increase with altitude (Hultine and Marshall 2000; Körner et al. 1988, 1991; Marshall and Zhang 1994; Sparks and Ehleringer 1997; Vitousek et al. 1990; Warren et al. 2001), although a nonlinear relationship has also been found in Asian alpine tree species (Li et al. 2009; Qiang et al. 2003; Zhao et al. 2008). Meanwhile, variation in key leaf functional traits, such as leaf mass per unit area (LMA), leaf nitrogen concentration per unit mass or area (N_mass, N_area), stomatal density (SD) that control to resource acquisition (i.e. carbon, nitrogen and water) and represent relatively independent aspects of plant ecological ‘strategy’ (Wright et al. 2004), has also been widely examined within alpine tree species, and these functional traits showed strong correlations with foliar δ¹³C. For instance, LMA and N_area generally increase with altitude, and positively correlate with foliar δ¹³C; in contrast, SD declines with altitude in many broad-leaved and coniferous woody species, and shows a negative relationship with foliar δ¹³C (Cordell et al. 1998; Hultine and Marshall 2000; Körner 1989; Read et al. 2014; Vitousek et al. 1990). It has been suggested that physiological mechanisms responsible for carbon isotopic variation are associated with shifts between supply and demand for carbon dioxide within the leaves (Hultine and Marshall 2000). Low SD and high LMA reduce leaf stomatal conductance and increase the distance of CO₂ transport to sites of carboxylation, respectively, and thus reduce CO₂ supply to the chloroplasts and subsequently increase δ¹³C of fixed carbon in leaves (Hultine and Marshall 2000; Vitousek et al. 1990). On the other hand, leaves with high leaf N_mass and/or N_area tend to have higher photosynthetic capacity because the protein in the photosynthetic apparatus represents up to 75% of total leaf organic nitrogen (Evans 1989; Poorter and Evans 1998), and thus have higher demand for CO₂ and subsequently have higher δ¹³C. To our knowledge, there have been no other investigations into the relative contribution of these different mechanisms to carbon isotope discrimination along altitudinal gradients in individual species.

In contrast to δ¹³C, δ¹⁵N is associated with terrestrial N cycling (Robinson 2001) and has been shown, in numerous studies, to vary along environmental gradients at regional to global scales (Aranibar et al. 2004; Cheng et al. 2009; Craine et al. 2009; Handley et al. 1999; Houlton et al. 2006; Murphy and Bowman 2009; Swap et al. 2004; Yang et al. 2013). For instance, because warm, dry ecosystems might have a relatively open nitrogen cycle and thus higher soil N availability (Robinson 2001), foliar δ¹⁵N increases with decreasing mean annual precipitation and with increasing mean annual temperature (MAT) across MAT ≥ −0.5°C (Craine et al. 2009; Handley et al. 1999). Surprisingly, there have been very few studies of plant δ¹⁵N at intraspecific level along altitudinal gradients. Sah et al. (2003) found that δ¹⁵N in the needles of Pinus roxburghii declined along an altitudinal gradient from 1200 to 2200 m in the Kathmandu valley of Nepal. In contrast, Vitousek et al. (1989) found no differences in δ¹⁵N in Metrosiderous collected from different altitudes along the Hawaiian coast. Although there is much published data about nitrogen isotopes in plants, at both regional and global scales, the effects of environmental variables on N isotope ratios are still far from complete. In general, it has been suggested that leaf δ¹⁵N is positively correlated with leaf N concentration and soil N availability at various spatial scales: across 15 temperate and tropical forest sites (Martinelli et al. 1999); within and among plant species at Glacier Bay, Alaska (Hobbie et al. 2000); across 67 grasslands in four regions of the world (Craine et al. 2005); within and among species and temperate forest stands (Pardo et al. 2006); among herbaceous species in temperate grassland (Craine et al. 2012). Specifically, in a research review, Craine et al. (2009) found that δ¹⁵N increased with increasing foliar N concentration at the global scale. However, so far, few studies have considered both leaf δ¹³C and δ¹⁵N simultaneously in alpine regions, and clearly, such an integrated study of both δ¹⁵N and δ¹³C, as well as other key leaf functional traits, would contribute to our understanding of plant ecological strategies, such as carbon, water and nitrogen use. Quercus aquifolioides Rehder & E.H. Wilson is a dominant evergreen sclerophyllous species of alpine forests in the Tibetan Plateau and adjacent Hengduan Mountains, growing across a wide range of altitudes, from around 2000 to 4500 m (Zhou et al. 1995). Therefore, Q. aquifolioides is an excellent species in the alpine region for us to examine the intraspecific variation in foliar δ¹³C, δ¹⁵N and other key leaf functional traits across altitudes. Due to the positive relationships between leaf N content and leaf δ¹⁵N and δ¹³C, as shown above, we hypothesized that they should increase consistently along such an altitudinal gradient.

In this study, in order to test our hypothesis that foliar δ¹³C and δ¹⁵N could exhibit consistent patterns across an altitudinal gradient, we therefore measured foliar δ¹³C, δ¹⁵N and other foliar traits in Q. aquifolioides at altitudes ranging from 2500 to 3800 m. Firstly, we examined whether δ¹³C, δ¹⁵N and other leaf functional traits exhibited similar trends in response to increasing altitude. Then, we addressed two issues: (i) identifying key functional traits that caused altitudinal variation in δ¹³C and δ¹⁵N; and (ii) determining the ecological consequences (e.g. water and nitrogen use strategies) of the differences in foliar δ¹³C and δ¹⁵N, along with other leaf functional traits, across the altitudinal gradient.

MATERIALS AND METHODS

Field sites

In September 2007, leaves of Q. aquifolioides were sampled from 13 sites between 2500 and 3800 m, covering a wider altitudinal range than in previous studies of oak species (Li et al. 2009; Zhang et al. 2005), on the eastern slopes of the Sygera Mountains, southeastern Tibetan Plateau. This represented the entire altitudinal distribution range of Q. aquifolioides in this region. In order to avoid topological effects, all our samples were collected from the sunny eastern slopes. Climate data at three elevations, representing the low (3000 m), middle (3500 m) and high (3800 m) altitudes, were collected from local meteorological stations located within the Sygera region. The MAT for the low, middle and high altitudes was 8.6, 1.8 and −0.73°C, respectively. The corresponding mean annual precipitation (MAP) values were 660, 800 and 1134mm. These values indicate that MAT declines linearly and MAP increases with increasing altitude in this region.

Mature leaves were collected from the south side of the crowns using a pruning pole. The slope of the land at the sites was 30–40%, with samples collected from 13 sites at nearly every 100 m from 2500 to 3800 m. At each site, 100 leaves from each of 5–10 individual mature trees were selected from the upper third of the canopy.

Leaf morphology

At each site, the leaf area (LA), mass (dry weight, LM) and leaf thickness (LT) of 10 leaves from each tree were determined. LA was measured using an LI-3000A portable area meter (Li-Cor, Lincoln, NE) and LT was measured with a digital caliper in the field. The measured samples were then placed in plastic bags and taken to the laboratory. Leaf samples were oven-dried for 48h at 80°C until they reached constant weight, and then LM was determined and LMA (the ratio of dry weight to LA) was calculated.

Stable carbon and nitrogen isotope compositions

For stable carbon and nitrogen isotope analysis, dried leaf samples were ground to a fine powder with a Tissuelyzer (Retsch, Haan, Germany) and each sample was split into two subsamples, one of which was sent to the Key Laboratory of Western China’s Environmental Systems (Ministry of Education), Lanzhou University, for the determination of δ¹³C and δ¹⁵N. All analyses were carried out with an elemental analyzer EA1108 (Carlo Erba, Milano, Italy) attached to a Delta Plus C isotope mass spectrometer and using a CONFELO Ⅱ interface (Thermo Finnigan MAT GmbH, Bremen, Germany). Carbon and nitrogen isotope ratios were reported as deviations from the standard Pee_Dee Belemnite (PDB) and atmospheric N₂ (defined as being 0‰), respectively, and expressed as per mil (‰). Therefore, the isotopic composition of a sample (δ¹³C or δ¹⁵N) was calculated as the ratio (‰):

where R_sample and R_standard are the ¹³C:¹²C (¹⁵N: ¹⁴N) ratios of the sample and the PDB (atmospheric N₂) standard, respectively.

Leaf carbon and nitrogen concentrations

Carbon and nitrogen concentrations of the leaves were determined, using the second set of leaf subsamples, by the Analytical Testing Center, Lanzhou University, China, with a CHN analyzer (Vario EL, Elementar, Germany). Leaf C and N concentrations per unit mass (C_mass, N_mass) were calculated, and then leaf C and N concentrations per unit area (C_area, N_area) were also calculated by multiplying C_mass and N_mass, respectively, by the LMA.

Stomatal measurements

Ten leaves preserved in FAA solution (38% Formaldehyde 5 ml: Glacial acetic acid 5 ml: 70% Alcohol 90ml) per individual were first bleached with a 4% sodium hypochlorite solution, and then immersed in tap water to remove the mesophyll and epicuticle, because leaves of Q. aquifolioides are hypostomatous. The remaining cuticle was then stained with safranin and mounted in glycerin on a microscope slide. Screening was then conducted using a BX51 light microscope (Olympus, Shinjuku-ku, Tokyo, Japan) equipped with a digital camera DP71, at magnifications of ×10 and ×40. The stomatal density (SD, number of stomata mm⁻²) and the length of stomata (SL, guard cell length, µm) were measured on micrographs using the Image-Pro Plus 5.0 software. We also calculated stomatal number per gram of nitrogen (St/N) and per unit leaf mass (St/LM) by dividing SD by N_area and LMA (Hultine and Marshall 2000), respectively.

Statistical analyses

We performed two sets of analyses. First, we characterized the relationships between elevation and the measured variables, and between foliar δ¹³C, δ¹⁵N and other traits using linear univariate regressions of the mean population values. We then used the principal component analysis (PCA) to assess the ecological strategy associated with the altitudinal variation in leaf traits within Q. aquifolioides. All statistical analyses were conducted in SPSS 16.0 (SPSS lnc., Chicago, IL).

RESULTS

Altitudinal variation in foliar δ¹³C and δ¹⁵N in association with other leaf traits

As altitude increased, foliar δ¹³C appeared to increase (Fig. 1A), while stomatal parameters including SD, SL and St/LM, all declined markedly (Fig. 1C–F). Leaf morphological traits such as LMA, LM and LT increased significantly with increasing altitude (Fig. 1B, 2B and C); whereas LA exhibited the opposite trend (Fig. 2A). In addition, C_area increased significantly with increasing altitude (Fig. 2D), whereas C_mass exhibited no such trend (R² = 0.177, P = 0.152).

By contrast, the relationships between foliar δ¹⁵N, N_mass, N_area, C:N, St/N and altitude were nonlinear, with a clear transitional zone at around 3300 m (Fig. 3, 1E; Table 1). Values of δ¹⁵N, N_mass and N_area increased linearly with altitude at low altitudes (up to 3300 m) and declined linearly, although not significantly, at high altitudes (above 3300 m). Conversely, C:N and St/N initially declined and then increased with altitude.

Relationships between foliar δ¹³C, δ¹⁵N and other traits

Foliar δ¹³C was significantly correlated with several leaf traits, including the morphological variables and stomatal parameters which affect the internal resistance and stomatal conductance of leaves (Table 2). For example, foliar δ¹³C exhibited significant positive correlations with LM, LT, LMA, and C_area and negative relationships with SD, SL and St/LM; in contrast, it exhibited relatively weak correlations with C_mass and LA (P = 0.053 and P = 0.094, respectively), but no correlations with N_mass, N_area, C:N and St/N (Data not shown). This suggests that altitudinal variations in δ¹³C could be caused by leaf internal resistance and stomatal conductance, rather than by photosynthetic capacity indicated by leaf N.

Foliar δ¹⁵N was also correlated with leaf traits, in particular N_mass, N_area, C:N and St/N (Table 3). It exhibited strikingly positive relationships with N_mass and N_area, and negative relationships with C:N and St/N, suggesting that altitudinal variations in δ¹⁵N may be closely linked to soil nutrient status, particularly soil N availability.

Ecological consequences of altitudinal differences in foliar δ¹³C, δ¹⁵N and other leaf traits

The 11 leaf traits were examined using PCA. The first PCA axis accounted for 47.00% of the total variation across the altitudinal gradient, compared with 27.24% for the second axis (Fig. 4). Out of all the traits, δ¹³C, LMA, C_mass, LT, SD, SL and St/LM contributed substantially to the first axis, while δ¹⁵N, N_mass, C:N and St/N contributed substantially to the second axis. Increasing scores on the first axis were associated with altitude; variables linked to water conservation, i.e. δ¹³C, LMA and LT also increased while SD, SL and St/LM declined (Fig. 4). On the second PCA axis increasing scores were also associated with increasing altitude; the variables associated with this axis reflected the plant’s nitrogen acquisition strategy, i.e. δ¹⁵N, N_mass, C:N, but exhibited nonlinear shifts, with a clear transitional zone at around 3300 m (Fig. 4). In addition, the two axes were orthogonal, suggesting that water and nitrogen use strategies in Q. aquifolioides operate independently along the altitudinal gradient.

DISCUSSION

Altitudinal variation in foliar δ¹³C

Our results suggest that foliar δ¹³C in Q. aquifolioides increases with altitude; this is consistent with many previous studies of altitude (Hultine and Marshall 2000; Körner et al. 1988, 1991; Marshall and Zhang 1994; Sparks and Ehleringer 1997; Vitousek et al. 1990; Warren et al. 2001). In general, most leaf N is bound up in photosynthetic enzymes, and

thus plants with higher N concentrations in their leaves are considered to have greater photosynthetic capacity (Evans 1989; Field and Mooney 1986). However, leaf N concentrations, particularly per unit area (N_area), exhibit an increasing trend with altitude in several species (Cordell et al. 1999; Körner 1989; Oleksyn et al. 1998; Read et al. 2014; Sparks and Ehleringer 1997; Suzuki 1998; Weih and Karlsson 1999). In this study, we found that N_mass and N_area increased with increasing altitude, but not in a linear fashion, and they were not correlated with foliar δ¹³C (Fig. 3). Furthermore, while both St/N and St/LM have previously been found to positively related to leaf CO₂ supply and thus negatively related to foliar δ¹³C (Hultine and Marshall 2000), only St/LM was significantly negatively correlated with foliar δ¹³C in our study (Table 3). This finding implies that CO₂ supply could cause the altitudinal variation in foliar δ¹³C in Q. aquifolioides. Similarly, a constant N_area value with increasing altitude has been found (Hultine and Marshall 2000), and a reduction in photosynthetic capacity has also been found in other species (Cabrera et al. 1998; Rada et al. 1998; Zhang et al. 2005). Consequently, the variation in the photosynthetic capacity of Q. aquifolioides may not be the main cause of the observed δ¹³C trend with altitude .

Another possible factor causing foliar δ¹³C to increase with altitude is the higher leaf internal resistance associated with thicker leaves (Morecroft et al. 1992) and increased LMA, which is an intermediate index of supply and demand for CO₂ (Hultine and Marshall 2000). This is because the longer diffusive path found in thicker leaves increases internal mesophyll resistance and thus reduces CO₂ concentrations at the site of carboxylation below P_int, the partial pressure in the substomatal cavities; such a decline in partial pressure would tend to increase foliar δ¹³C (Vitousek et al. 1990). Higher internal mesophyll resistance has been validated indirectly and directly in several species (Hultine and Marshall 2000; Kogami et al. 2001). Similarly, LA decreased while LM, LT and C_area increased significantly with increasing altitude (Fig. 3), thus contributing to the increasing LMA with altitude. Furthermore, strong positive relationships between foliar δ¹³C and LT or LMA were found in Q. aquifolioides (R² = 0.740, P < 0.001; R² = 0.655, P = 0.001, Table 2). Thus, leaf internal mesophyll resistance related to LMA could be one important factor causing the observed increases in foliar δ¹³C in Q. aquifolioides across the altitudinal range.

Besides internal mesophyll resistance, increases in foliar δ¹³C with altitude have also been attributed to a decrease in maximum foliar stomatal conductance (Meinzer et al. 1992; Morecroft et al. 1992), presumably determined by stomatal density and size (Franks and Beerling 2009; Woodward and Bazzaz 1988). Like many broad-leaved and coniferous woody species (Hultine and Marshall 2000; Körner et al. 1983; Schoettle and Rochelle 2000), SD in combination with stomatal size (as indicated by SL) declined linearly in Q. aquifolioides, indicating that stomatal conductance decreased with altitude (Fig. 2). Because plants in alpine regions often encounter low temperatures, high wind speeds and increased vapor pressure deficits between their leaves and the air (Tranquillini 1964), the reduced SD is the result of acclimation to reduce water loss, suggesting this could be more advantageous for water conservation than facilitating carbon gain at dry high-elevation sites (Schoettle and Rochelle 2000). At our study sites, the decline in MAT from 8.6, 1.8 to −0.73°C at altitudes of 3000, 3500, and 3800 m, respectively, also confirmed this view. Taken together, the main cause of the variation in foliar δ¹³C in Q. aquifolioides with altitude is likely leaf internal resistance and stomatal conductance affected by LMA, LT, SD and SL, rather than photosynthetic capacity as indicated by foliar nitrogen concentration.

Altitudinal variation in foliar δ¹⁵N

Foliar δ¹⁵N, as an integrator of terrestrial N cycling (Robinson 2001), has been found to have close relationships with various environmental factors (Amundson et al. 2003; Craine et al. 2009; Handley et al. 1999; Martinelli et al. 1999). However, there have been very few studies examining the relationship between foliar δ¹⁵N and altitude (Sah and Brume 2003; Virginia and Delwiche 1982; Vitousek et al. 1989). Even from these few studies, there are conflicting results. For example, foliar δ¹⁵N exhibited a decreasing trend with altitude in plants collected along gradients across California (Virginia and Delwiche 1982) and the Kathmandu valley in Nepal (Sah and Brume 2003), whereas in the tree Metrosiderous, studied near the Hawaiian coast, it exhibited no differences with altitude (Vitousek et al. 1989). In contrast to these results, we found that foliar δ¹⁵N in Q. aquifolioides showed a nonlinear trend with altitude: initially increasing significantly, and then declining (Fig. 3A). On the eastern slopes of the Sygera Mountains, MAP increases linearly while MAT declines from an altitude of 3000 m up to 3800 m. Foliar δ¹⁵N is positively correlated with MAT (when MAT ≥ −0.5°C) and negatively correlated with MAP at both regional and global scales (Craine et al. 2009; Handley et al. 1999), so the changes in MAP and MAT are unlikely to explain the nonlinear changes in foliar δ¹⁵N in Q. aquifolioides with altitude.

Since foliar N concentrations have been used broadly as an index of soil N availability (Asner and Vitousek 2005; Ollinger et al. 2002; Ordoñez et al. 2009; Reich et al. 2001; Xia and Wan 2008), and further δ¹⁵N is positively associated with terrestrial soil N availability on local to regional scales (Craine et al. 2009; Garten et al. 2011; Högberg 1997; McLauchlan et al. 2010), our data indicated that foliar δ¹⁵N had a strong positive relationship with leaf N concentrations (both N_mass and N_area), highlighting that the changing pattern of foliar δ¹⁵N with altitude may be closely associated with soil N availability. With increasing soil N availability, the production of ammonium, and subsequently nitrate, increases. Gaseous N loss during nitrification (Firestone and Davidson 1989) and the leaching of ¹⁵N-depleted nitrate (Högberg 1997; Koba et al. 2003) can lead to the remaining N pool (and subsequently the levels in plants) to be enriched in ¹⁵N. As N availability further increases, denitrificaiton begins to consume a larger proportion of the nitrate present (Hall and Matson 2003), further enriching inorganic N pools if denitrification does not completely consume the nitrate pool (Houlton et al. 2006). Although our data did not confirm these processes in detail due to lack of data on soil properties, i.e. soil δ¹⁵N, soil N content, and soil NH₄⁻-N and NO₃⁻-N content, they did indicate that foliar δ¹⁵N is closely related to soil N availability in Q. aquifolioides across the altitudinal range studied. Furthermore, there is much evidence that the natural abundance of ¹⁵N is greater in systems where nutrients are not limiting (Craine et al. 2009). Thus, higher foliar δ¹⁵N values appear to indicate a more open N cycle, with greater losses relative to turnover. If this is true, the varying pattern of foliar δ¹⁵N in our study suggests that the sites at around 3300 m might have a more open N cycle relative to the low- and high-altitudes occupied by Q. aquifolioides. In addition, compared to the decrease at high elevations (3400, 3500 and 3600 m), foliar δ¹⁵N slightly increased again at the highest elevations of 3700 and 3800 m, which have relatively higher precipitation. Since preferential leaching of isotopically light N into stream water (Amundson et al. 2003; Austin and Vitousek 1998; Högberg 1997; Vitousek 2002), we speculated that isotope discrimination of hydraulic leaching may be responsible for this slightly increasing change. To much better understanding the mechanisms underlying the pattern of foliar δ¹⁵N and nitrogen cycle across the altitudinal gradient in alpine regions, future studies are needed to quantify the effects of soil and climatic factors on foliar δ¹⁵N at multiple species and even community levels on alpine regions.

Intraspecific trade-offs in foliar δ¹³C and δ¹⁵N with other leaf traits at different altitudes

It is now widely accepted that, in vascular plants, there is a fundamental trade-off between rapid acquisition and conservation of resources (Grime et al. 1997; Reich et al. 1997; Wright et al. 2004), described as the ‘leaf economics spectrum’ (Wright et al. 2004); this is mostly based on comparisons between species. However, to date, only one study (Albert et al. 2010) has demonstrated that such relationships also occur within a species. Here we have shown that the same trade-off exists in Q. aquifolioides along a wide altitudinal gradient on the Tibetan Plateau. PCA identified one axis representing a water availability gradient from the low-altitude populations with lower δ¹³C, LMA and LT and higher SD, SL and St/LM, to the high-altitude populations with higher δ¹³C, LMA, and LT and lower SD, SL and St/LM (Fig. 4). Many environmental stimuli cause plants to regulate water loss and photosynthetic carbon gain, both dynamically by the opening and closing of stomata, and structurally by growing leaves with altered stomatal densities and pore sizes (Brodribb and McAdam 2011; de Boer et al. 2011). With the progressively declining water availability associated with low air and soil temperatures, high wind speeds and increased vapor pressure deficit between leaves and the ambient air in alpine regions, plants are able to enhance water use efficiency (δ¹³C) and LMA, and reduce stomatal density and size in order to optimize intraspecific water availability. Therefore, the first axis of our PCA reflected the intraspecific trade-off between water loss and photosynthetic carbon gain, and consequently the trade-off between persistence and growth in Q. aquifolioides across altitudes.

In contrast to the first axis, the second axis reflected a nitrogen use strategy in which the mid-altitude populations had higher foliar δ¹⁵N, N_mass and lower C:N, while the low- and high-altitude populations had lower δ¹⁵N, N_mass and higher C:N (Fig. 4). Although the N cycle is complex, the combination of foliar δ¹⁵N and N concentrations could strongly reflect terrestrial soil N availability from local to global scales in the above-mentioned many studies. The shifting patterns associated with foliar δ¹⁵N, N_mass and C:N in this study indicated that the mid-altitude populations experienced higher soil N availability, while the low- and high-altitude populations experienced lower soil N availability and in these populations the underlying metabolic processes are different. At altitudes above 3300 m, the reduced N availability could be caused by the very low MAT, which suppressed not only the decomposition and mineralization of organic matter, but also uptake of nutrients by roots. This is in line with the biogeochemical hypothesis (Reich et al. 2004). In contrast, at altitudes below 3300 m, there was low N availability; here, high leaf N relative to C at low MATs might arise as increasing N offsets the reduced efficiency of enzymes and physiological processes. This is in line with the temperature-physiological hypothesis (Reich and Oleksyn 2004). From the N availability perspective, therefore, the mid-altitudes around 3300 m may be the optimum zone for the growth of Q. aquifolioides.

CONCLUSIONS

Contrary to our hypothesis, our results indicated that foliar δ¹³C and δ¹⁵N in Q. aquifolioides exhibited different patterns along the altitudinal gradient on the Tibetan Plateau. Further, Variation in foliar δ¹³C, along with LMA, LM, LT, SD, SL and St/LM, may reflect trade-off between water loss and carbon gain. In contrast, variation in foliar δ¹⁵N, along with N_mass and C:N, may reflect nitrogen acquisition strategy in Q. aquifolioides across its altitudinal distribution.

FUNDING

This research was supported by the Program for New Century Excellent Talents in University (NCET-08-0257), the National Natural Science Foundation of China (30972337, 30930072 and 31170571) and Fundamental Research Funds for the Central Universities (lzujbky-2010-47 and lzujbky-2012-k20).

Conflict of interest statement. None declared.

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