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Owing to their effects on internal concentrations of carbon dioxide (CO2) (Ci), intercepted radiation and atmospheric and soil water deficits modify the ratio of 13C : 12C in plant carbon (δ13C, e.g. Leavitt & Long, 1986; Livingston & Spittlehouse, 1996; Korol et al., 1999). For example, rates of photosynthesis are generally greater in sunlit foliage than in shaded foliage, leading to a greater reduction in Ci and a greater depletion in 13C (Leavitt & Long, 1986). Even more well described is the reduction in Ci caused by stomatal closure in response to water deficits (e.g. Guehl et al., 1995; Stewart et al., 1995; Lauteri et al., 1997; Panek & Waring, 1997).
The relatively large body of literature dealing with δ13C in conifers shows that a number of other factors (e.g. altitude, nutrition, hydraulic architecture) also play roles in determining δ13C (e.g. Walcroft et al., 1996; Korol et al., 1999; Hultine & Marshall, 2000; Warren & Adams, 2000) in wood and foliage. For example, δ13C varied with shoot water potential at a rate of −0.18‰ MPa−1 in Quercus spp. (Damesin et al., 1998) and with altitude at a rate of approx. 2‰ km−1 in numerous species and studies (e.g. Körner et al., 1991). Most of the δ13C studies have been ‘observational’ and there are few field-based, controlled tests of the effects of environmental conditions (e.g. availability of radiation, water, nutrients) on δ13C in plant tissues (e.g. Morecroft & Woodward, 1990). From one controlled field experiment, Högberg et al. (1993) provided evidence for the effects of nitrogen fertilization on δ13C signatures in foliage of Picea abies and Pinussylvestris.
As for conifers, both simple assessments of water availability (e.g. Macfarlane & Adams, 1998) and modelled water balances (Dupouey et al., 1993) show moderately strong relationships with δ13C signatures of the wood in recent (e.g. < 10 yr) annual growth rings of hardwoods. Over the longer term, increases in atmospheric concentrations of CO2, and variations in the leaf-to-air humidity gradient and in leaf temperature will all influence δ13C (Comstock & Ehleringer, 1993). For both conifers and hardwoods, modelled water balances for the growing-season, and sometimes for periods as short as 1 month, provide the strongest relationships (up to 93% of variance in δ13C explained) between δ13C of wood and indices of environmental conditions (Dupouey et al., 1993; Livingston & Spittlehouse, 1996). Like most, these models were developed using data from more or less ‘ideal’ sites with homogeneous vegetation and soil.
Recently, Yoneyama et al. (1997) provided a first report of the isotope ratios in phloem sap for wheat that was followed by Pate & Arthur (1998) and Pate et al. (1998) for Eucalyptus globulus. Part of the attention on phloem sap originates from a desire to obtain a better estimate of the δ13C signatures of leaf sugars – some of the first products of carbon fixation – and thus an estimate of the effects on δ13C of current environmental conditions. Hence the determination of δ13C in phloem sap, containing carbon assimilated in a period of hours to days before sampling, is useful to assess short-term variations in Ci/Ca (Brugnoli et al., 1998; Pate & Arthur, 1998). Such data are required to help explain why the relationship between foliar δ13C signatures and current environmental conditions varies so greatly among species, genera and climates. In the case of beech, new season’s foliage, like that of many other species, is formed largely from stored carbon and nutrients (e.g. Millard & Proe, 1993; Kozlowski & Pallardy, 1997) and thus more likely to carry a δ13C signature that reflects the environmental conditions for carbon fixation in previous seasons than the current one. Clearly, the determination of δ13C in phloem sap provides a strong guide to Ci/Ca during the present growing-season. Yoneyama et al. (1997) used aphids to sample phloem sap, whereas Pate et al. (1998) relied on phloem sap that bled from shallow incisions in the bark. Irrespective of the method, the ability to obtain phloem sap, will ‘enable more rigorous tests of prediction and reality for δ13C and δ15N, and determination of the uses of natural abundance measurements in improving our understanding of C and N metabolism and transport’ (Yoneyama et al., 1997).
Water availability is a limiting factor for growth of beech (Fagus sylvativa) in central Europe (Ellenberg, 1995). Climate models for central Europe predict an increase in temperature and longer periods of drought during the growing-season (Enquete Kommission, 1994). Reductions in soil water availability will, arguably, strongly influence patterns of competition between drought-sensitive beech and less sensitive species. Both mature and regenerating stands of beech grow on the shallow, rendzina soils derived from limestone that are widespread in southern Germany (e.g. Schwäbische Alb). These soils often have poor water-holding capacity. Equally importantly, there is strong political and social support for the transformation of spruce monocultures into site-adapted deciduous forests with beech as the dominant species. At the present time, we lack the necessary physiological basis for reliable prediction of either the likely success of re-establishment of beech on shallow, rendzina soils or effective management strategies, especially the management of stand density.
Advantage was taken of the opportunity presented by a replicated field trial in southern Germany to characterize the interacting effects of radiation and water availability on δ13C signatures in phloem sap, foliage and wood, in order to obtain information about temporal variation in Ci. By combining these data with the determination of water potential of twigs and soil, tree water use and stand transpiration, it was hoped to validate δ13C signatures of different tissues as indices of water availability and incident radiation in beech forests that were sensitive to typical management treatments. The trial included paired sites that differed mainly in aspect on either side of a narrow valley and, within each site, replicated plots of differing stand density. The broad hypotheses, based on past records of growth and vegetation analysis, were that the more south-facing site would have less available water and greater radiation than the more north-facing site, and that thinning would increase the availability of water. Additionally, an aim was to characterize ‘phloem bleeding’ as a means of sampling phloem sap from beech. This sampling technique is a prerequisite to characterizing δ13C signatures in the phloem sap of adult beech trees, since other techniques are difficult to use in the field (aphid technique) or introduce C compounds into the sampling solution (Schneider et al., 1996), thereby confounding interpretation of δ13C signatures.
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The first hypothesis (i.e. that aspect would determine water availability) was broadly supported by the data. On the basis of previous studies of beech (Saurer & Siegenthaler, 1989; Schleser, 1990, 1992; Dupouey et al., 1993), the 20-yr record in tree rings shows that water availability was less at the SW site since wood from this site was significantly less depleted in 13C than wood from the NE site. Equally, August was the driest month during the 1999 growing-season, and in this month phloem sap from the SW site was also much less depleted in 13C than sap from the NE site. The relation between soil water availability and δ13C signature is illustrated in Fig. 2, and soil water potential was distinctly more negative at the SW site than at the NE site at the time of the August sampling. Only slight differences in δ13C signatures of the phloem were observed between sites (SW and NE) in September when soil water potential had increased because of heavy rainfall. Pate & Arthur (1998) and Pate et al. (1998) showed recently that the δ13C signature of phloem sap from E. globulus was an excellent predictor of current water availability. Here, phloem δ13C was closely (albeit not robustly) related to water potential, although the slope of the regression line (approx. −3.9‰ MPa−1) was much greater than that recorded for Quercus spp. (−0.18‰ MPa−1, Damesin et al., 1998) and still greater than that recorded for Pinus spp. in much more seasonally arid environments in Australia (Warren et al., 2001). The analysis of the relationship between δ13C and water potential remains speculative, and further work is needed to confirm the linear range for beech.
As with previous studies where growth (measured as either BA increment or radial growth) has been compared with δ13C, there was reasonable agreement between the two parameters (see Fig. 4) in the present study. However, both growth and δ13C exhibited a variable ‘time-lag’ with environmental conditions (rainfall, radiation) that precluded a significant correlation. For growth, the lag effect has been at least partly explained by Sass & Eckstein (1995). The mean area of the first xylem vessels formed in each growth ring of beech is strongly influenced by water availability in the previous summer. Sass & Eckstein (1995) concluded that vessel formation at the annual beginning of cambial activity was mainly controlled by ‘internal factors’ with little influence of rainfall, while towards the end of cambial activity vessel formation was strongly influenced by recent rainfall.
Based on Pate & Arthur (1998), it could be proposed that water availability would be the major influence (or even the sole determinant) of the δ13C signature of carbon in beech tissues and phloem sap contents. If this were the case then thinning, which almost universally increases the amount of water available to remaining trees (e.g. Breda et al., 1995), should produce tissues that are more depleted in 13C than controls (the second hypothesis). However, in August when water supply on both sites was strongly limited by climatic conditions as indicated by the increase in soil water potential (Fig. 2), plots with the least tree density and BA (10 m2 ha−1) produced phloem that was the least depleted in 13C. An alternative explanation is that thinning – which caused a drastic decrease in LAI – has substantially increased the radiation intercepted by remaining trees, thereby increasing rates of photosynthesis and reducing Ci (e.g. Ehleringer et al., 1986; Leavitt & Long, 1986; Pearcy & Pfitsch, 1994; Israeli et al., 1996; Berry et al., 1997). A significant though weak relationship was also observed between hours of sunlight (a measure for the plant-available radiation) in the growing-season and δ13C in wood for the SW site. In part, the expression of an irradiance effect in δ13C only when water was in limited supply may stem from the rather low light compensation point of beech (approx. 350 µmol m−2 s−1, Kreuzwieser et al., 1997). Only when Ci is already constrained by partly closed stomata will the response to light (reduction owing to increased rates of carbon fixation) be realized.
Apart from direct tests of the hypotheses, the data allow useful comment on several other aspects of the interactions of climate and soils/landforms. It can be argued that, for any given climatic conditions, soil/landform characteristics hold soil water more ‘tightly’ at the NE site, against both evapotranspiration and evaporation, than at the SW site. This is mostly due to the poor water storage capacity (see soil descriptions and soil water potential, Fig. 2) of the soil at the SW site relative to that on the NE site. When water was in plentiful supply (e.g. in July), trees on the SW site transpired freely (Fig. 3a), at higher rates than trees on the NE site, and phloem carbon was significantly less depleted in 13C at the SW site (Fig. 6). A similar pattern of δ13C was seen in September. According to the literature, the higher rate of depletion of available soil water at the SW site (Fig. 2) should cause at least partial closure of stomata and a lower δ13C. Indeed, this was confirmed in August when water flux densities in the xylem of trees at the SW site fell by more than half. In addition SWP doubled and phloem δ13C was increased by at least 5‰. This pattern was partially reversed for the NE site – water fluxes in the xylem of trees at the NE site were greater than at the SW site during the dry month of August (Fig. 3b), which is supposed to be a consequence of the open stomata. In addition, SWP actually increased between July and August (Table 2), and δ13C signatures of phloem sap were little changed from those in July (Fig. 6). These results are strongly suggestive that during the dry month of August, the supply of available water at the NE site had been moderately well maintained, whereas it had been substantially depleted at the SW site (see Fig. 2).
In particularly dry years and the year immediately thereafter, there were few differences in δ13C of wood between the SW and NE sites. For example, the mean δ13C of wood from the years 1985, 1986 and 1989–92 was −25.5‰ at the NE site and −25.3‰ at the SW site. It is concluded that the soil/landform characteristics set ‘lower limits’ to water availability that are not particularly different between the two sites but that, in any given period, availability is largely determined by exposure to radiation and thence by vapour pressure deficit. The results are consistent with the findings of Walcroft et al. (1997) that the observed close relationships between soil water availability (e.g. Stewart et al., 1995) or cumulative transpiration (e.g. Livingston & Spittlehouse, 1996) and δ13C owe much to the integration of a number of environmental variables (e.g. precipitation, irradiance, temperature and air saturation deficit) by Ci, and to the direct relationship of transpiration and δ13C through stomatal conductance.
It was not intended to model δ13C and water availability at the available sites and, in any event, the required long-term climatic data (radiation and evaporation) were lacking. Moreover, these sites pose considerable difficulties for water balance modelling (in contrast to most sites for which models have been generated) owing to their shallow surface soils, rocky subsurface soils that quickly grade to ‘blocky’ parent material, and steep slopes. It must also be noted that water balance–δ13C relationships have seldom been tested beyond sites at which they were generated. It remains to be seen if on-going research can develop a successful water availability model for these sites and, then, if it has any predictive capacity for δ13C. Given the observed strong influence of radiation on phloem δ13C (and on wood δ13C), inclusion of radiation in the latter will probably improve sensitivity (Walcroft et al., 1997).
The sugar and carbon isotope analysis of the phloem sap of beech proved instructive, first for the range of δ13C values observed, and secondly for the relative concentrations of carbon isotopes and sugars.
Over the growing-season in 1999, the mean δ13C of sap from the 12 experimental plots (six per site) varied by more than 13‰: from approx. –20‰, values close to those observed for foliage from desert plants (e.g. Ehleringer & Cooper, 1986), to approx. –33.5‰, values typical of plants in high-rainfall, tropical forests (e.g. Jackson et al., 1993). This range is perhaps best interpreted in light of the soil profile at the SW site which, when coupled with the steep slope, clearly holds little water for plants for any length of time. Growth of beech on this site is probably a fine balance between evapotranspiration and rainfall during the growing-season, and is not buffered by a huge soil water storage pool. Changes to this balance via global warming may influence growth and vitality of beech, and may cause changes in the vegetation composition owing to a shift in competition patterns. On the other hand, the extreme values of δ13C for phloem sap are integrated within the tree to produce wood and foliage with δ13C signatures close to those found in other studies of beech (e.g. Schleser, 1990, 1992; Dupouey et al., 1993). The ability to sample phloem sap from beech, as demonstrated here, should enable future studies to elucidate relationships among carbon transport and metabolism and account for the differences in δ13C signatures among recently fixed carbon, transported sugars, and developing wood and foliage.
Phloem sap from beech has a sugar ‘profile’ comparable to that of other hardwoods for which there are data (e.g. Zimmerman & Ziegler, 1975; Pate & Arthur, 1998; Pate et al., 1998). While the sap was notably rich in sucrose (> 200 mM), it also contained glucose and fructose at concentrations in the range 0–10 mM. Sugar concentrations varied greatly between trees within treatments and sites, and never as predictably with δ13C as shown by Pate et al. (1998) for E. globulus in late summer. There, sugar concentrations in phloem sap increased as water availability (as indicated by δ13C) decreased. While there is some suggestion (Fig. 7) that sugar concentrations in beech phloem sap may be related to δ13C, the relationship is tenuous and largely obscured by tree-to-tree variation. Even when only those data from trees under the greatest water stress are considered (SW site in August), sugar concentrations were independent of δ13C. Pate & Arthur (1998) attributed their result to ‘independent responses of the sugar-loading systems of the mesophyll : minor vein system to current water status of the leaf, xylem and parent plant’. The differences between the two studies can be attributed to plot-level climate or (given the high tree-to-tree variability) even individual tree microclimate. Severe shortages of water for beech (as indicated by δ13C of −20‰) are restricted to days, or at most weeks, whereas in western Australia the ‘summer drought’ may last for 3 months or more.
This study has shed some light on past accounts of the abundance of carbon isotopes in beech, and should stimulate further attempts to model the relationships among environmental variables and δ13C. δ13C signatures in the phloem of beech are sensitive to seasonal changes in water availability, whereas δ13C signatures in the foliage are far less sensitive. In addition, the data suggest an interactive effect of low water availability and increased radiation on δ13C in the phloem sap. The demonstrated ability to sample phloem sap of mature beech in the field, both directly and easily, opens the door to many future field studies – most obviously of carbon and nutrient transport but also of hormone transport between shoots and roots.