SEARCH

SEARCH BY CITATION

Keywords:

  • carbon allocation;
  • free-air CO2 enrichment (FACE);
  • fungi;
  • rhizosphere;
  • roots;
  • soil;
  • soil respiration;
  • stable isotopes

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  • • 
    How rapidly newly assimilated carbon (C) is invested into recalcitrant structures of forests, and how closely C pools and fluxes are tied to photosynthesis, is largely unknown.
  • • 
    A crane and a purpose-built free-air CO2 enrichment (FACE) system permitted us to label the canopy of a mature deciduous forest with 13C-depleted CO2 for 4 yr and continuously trace the flow of recent C through the forest without disturbance. Potted C4 grasses in the canopy (‘isometers’) served as a reference for the C-isotope input signal.
  • • 
    After four growing seasons, leaves were completely labelled, while newly formed wood (tree rings) still contained 9% old C. Distinct labels were found in fine roots (38%) and sporocarps of mycorrhizal fungi (62%). Soil particles attached to fine roots contained 9% new C, whereas no measurable signal was detected in bulk soil. Soil-air CO2 consisted of 35% new C, indicating that considerable amounts of assimilates were rapidly returned back to the atmosphere.
  • • 
    These data illustrate a relatively slow dilution of old mobile C pools in trees, but a pronounced allocation of very recent assimilates to C pools of short residence times.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Of all the carbon (C) assimilated by trees, about half is rapidly returned to the atmosphere by respiratory metabolism (Högberg et al., 2002), at least during the growing season. The other part enters various fast- and slow-turnover pools, the residence times of which are largely unknown (Körner, 2003). In particular, it is not known how quickly newly assimilated C compounds are invested into recalcitrant structures (e.g. stem wood), and how intimately (on what timescales) the various C pools (e.g. soil organic matter) and fluxes (e.g. root respiration) are tied to actual photosynthesis. For instance, C allocated to leaf respiration can be released within minutes, whereas C entering the root biomass pool can remain in the ecosystem for months or even several years. Carbon transferred to the recalcitrant soil organic matter pool, for example via root litter, may reside for thousands of years (Trumbore, 2000). We quantified the allocation of newly assimilated C to different forest compartments by taking advantage of the Swiss canopy-crane CO2-enrichment experiment (Pepin & Körner, 2002; Körner et al., 2005), in which naturally grown deciduous trees receive labelled CO2. The forest is not a plantation, so trees are of different size and age and live in interspecific competition for above-ground as well as below-ground resources.

Earlier direct quantifications of C allocation have used radiocarbon. However, these studies were either conducted on rather young trees (Hansen & Beck, 1990, 1994; Horwath et al., 1994), or were restricted to single trees (McLaughlin et al., 1979). The first forest-scale attempts used indirect evidence by interrupting phloem transport through girdling (removing or cutting of phloem). These experiments showed that allocation of photoassimilates to autotrophic respiration represents the largest flux of current assimilates (approx. 50%; Högberg et al., 2002). Autotrophic below-ground respiration is now more often defined by including not only roots, but also mycorrhizal fungi and microbes feeding on root exudates, altogether representing 50–65% of total soil respiration (Andrews et al., 1999; Högberg et al., 2001; Högberg et al., 2002; Bhupinderpal-Singh et al., 2003; Andersen et al., 2005). Stable C-isotope trials using pulse labelling in a grassland revealed that 4–6% of labelled C was respired by mycorrhizal mycelia within 21 h (Johnson et al., 2002). Slightly higher amounts (7–13%) of current assimilates have been found to be lost through exudation in potted tree seedlings (Phillips & Fahey, 2005). Such studies suggest that the largest amount of autotrophic respiration emerges directly from root respiration. Above-ground, assimilates are used mainly for structural growth (leaves, wood and fruits) and for cell metabolism.

The study of C allocation in mature forests is technically difficult without destroying the delicate plant–soil continuum, the widespread hyphal network of mycorrhizal fungi that forms the interface between roots and soil and allows the exchange of carbohydrates and nutrients. Stable isotopes serve as an ideal tracer to study C allocation, as only tiny amounts of tissue suffice for analysis. To apply isotopically labelled C, CO2-enrichment systems are a convenient tool as the supplemental CO2 is mostly of fossil fuel origin and therefore contains less 13C than ambient air. Given the many experimental systems in use, it is surprising that labelled C has rarely been used to trace the fate of C in the plant body and the ecosystem (Andrews et al., 1999; Matamala et al., 2003; Pataki et al., 2003; Steinmann et al., 2004). One reason may be that most tests did not last long enough, given that it takes several years until new C signals are detectable in large pools such as soil (Hungate et al., 1996). Furthermore, the assumption has to be made that CO2 enrichment does not exert major alterations of C allocation. CO2 effects cannot be determined as such, a long-term labelling of large control trees at ambient CO2 concentrations is all but impossible.

Here we present data for an array of assimilate pathways in an approx. 100-yr-old, diverse central European forest, studied over four growing seasons. We used 12 mature deciduous trees exposed to approx. 540 ppm CO2 using a specially designed free-air CO2-enrichment technology called web-FACE (Pepin & Körner, 2002). This system enriches tree crowns only, and the canopy is at a height that prevents downward draughts and direct CO2 diffusion from the crowns to the forest floor, as a lack of 13C signals in understorey herbs confirmed (Steinmann et al., 2004). This offers the unique opportunity to trace the fate of C in trees through stems into roots, soil and soil air, without confounding CO2 fluxes via understorey vegetation or direct diffusion. Therefore there is a clearly defined ‘port of entry’ for C, with all other parts of the system not directly affected by the label.

To calculate the potential 13C signal strength, we used C4 grasses grown in small pots, exposed in the tree crowns, as references for the isotope signals (‘isometers’). Repeated sampling of different forest compartments over four growing seasons allowed an estimation of the timing and mixing of new C in various C pools. We hypothesize that most of the carbohydrates formed by photosynthesis are invested in labile C pools, and we expect a rapid return of most of this new C to the atmosphere.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Site description and CO2-enrichment system

The experiment was performed in a diverse mixed forest located near Basel, Switzerland (47°28′ N, 7°30′ E; elevation 550 m asl) with tree heights of 30–35 m. The forest is situated on a silty-loamy rendzina and is characterized by a 15-cm-deep rock-free topsoil and a 15–30-cm-deep rocky subsoil (approx. 40% of the subsoil volume is made up of stones), underlain by fragmented limestone bedrock. In the upper 10 cm the soil has a pH of 5.8 (measured in distilled water extracts).

A 45-m free-standing tower crane equipped with a 30-m jib (crane arm) and a working gondola provided access to 62 dominant trees in an area of approx. 3000 m2. A group of 14 canopy-size broad-leaved trees [three Fagus sylvatica L., four Quercus petraea (Matt.) Liebl., four Carpinus betulus L., one Tilia platyphyllos Scop., one Acer campestre L. and one Prunus avium L.], covering a canopy area of 550 m2, were selected for CO2 enrichment. Of these, one slim individual of Quercus died, and CO2 enrichment on the one Prunus at the eastern edge of the plot was not sufficient, so these two trees were excluded from the study, leaving 12 individuals for the analysis. Eleven control trees (three Fagus, two Quercus, two Carpinus, two Tilia, two Acer) were located in the remaining crane area at sufficient distance from the CO2-release zone. In late September 2000, trees were exposed to a ‘warm-up’ CO2 treatment of a few weeks to mitigate the inevitably step-nature of the treatment (Luo & Reynolds, 1999). From spring 2001 onwards, trees were exposed to elevated, labelled CO2 from around mid-April to roughly the end of October, depending on bud break and leaf shedding. During the night, CO2 release was interrupted. In total, approx. 300 t pure CO2 was used per year. A more detailed description of the CO2-enrichment system is given by Pepin & Körner (2002).

The isotopic composition of the pure CO2 was monitored every week in year 1 and was found to be identical for all but one week. In year 2, a contract was made with the gas deliverer to guarantee the same source of CO2, so CO2 was monitored only at 2–3-wk intervals from year 2 onwards. Because of its fossil fuel origin, it was depleted in 13C relative to ambient atmospheric CO2 by −29.7 ± 0.3 vs approx. −8 (Fig. 1a), thus the fate of labelled photoassimilates could be traced. In spring 2004 we analysed honeydew that had been excreted by aphids as a reference for fresh photoassimilates (Pate & Arthur, 1998; Barbour et al., 2005). On average, we found honeydew δ13C values of −25.7 in control and −30.8 in labelled trees, which correlated very well with leaf δ13C (r2 = 0.93). The isotope values are expressed in the δ-notation: δ13C = (Rsample/Rstandard − 1) × 1000 () where R is the molar ratio of 13C to 12C for the sample and the standard, respectively.

image

Figure 1. (a) Mean annual δ13C ± 1 SE of the pure supplemental CO2 (n = 6–12 sampling dates). Top right, overall mean ± SE over 4 yr. (b) Mean annual δ13C ± 1 SE of C4 grass isometers (2001, Cynodon dactylon; 2002–04, Echinochloa crus-galli; n = 12–35 pots). Numbers represent differences in δ13C between grasses grown in control trees and trees exposed to labelled CO2 for single years; top right, average difference over 4 yr ± SE.

Download figure to PowerPoint

C4 isometers

The abundance of 13C in the CO2 was monitored with so-called isometers, C4 grasses [Cynodon dactylon (L.) Pers. and Echinochloa crus-galli (L.) P. Beauv.] grown in 50-ml containers (in a sand–clay mixture), placed in the tree crowns. In year 1, the grasses were also used to monitor the spatial distribution of the added CO2 in neighbouring trees. We therefore had more pots in the area surrounding the CO2-release zone (n = 35) than in the labelled area itself (n = 12). As the CO2 was concentrated mainly around the labelled trees (Pepin & Körner, 2002), the number of pots in the control area was reduced to 12 and, in turn, the number of pots in the labelled area was increased to 35. We assumed the δ13C difference between grasses exposed to labelled air compared with grasses grown in ambient air (5.9) to reflect the actual isotopic signal the canopy is exposed to, because the grasses consist exclusively of C that originates from the CO2 they assimilated, with no influence from old C reserves. To calculate the fractions of new (= labelled) C in other compartments, we used a rule of proportion where the isometer signal of 5.9 refers to 100% new C. We assumed that 13C fractionation is not influenced by elevated CO2 (Saurer et al., 2004).

The sensitivity towards 13C discrimination in response to changes in climatic factors is low under well watered and light-saturated conditions in C. dactylon (used in 2001) and even lower in E. crus-galli, which was used from 2002 onwards (Buchmann et al., 1996). Therefore δ13C values of these grasses exposed to labelled CO2 could be used to calculate time-integrated CO2 concentrations of the labelled CO2 using the following mixing ratio model with the CO2 concentration and isotope ratio of its two CO2 constituents (atmospheric and pure CO2 gas):

  • celev × δ13Celev = cpure × δ13Cpure + camb × δ13Camb(Eqn 1)

where celev is the CO2 concentration of the CO2-enriched air, and δ13Celev is the δ13C isotope ratio of the CO2-enriched air derived by C4 grasses (δ13C of leaves minus a discrimination factor of 5.5 for C. dactylon and 4.4 for E. crus-galli; Buchmann et al., 1996). cpure is the CO2 concentration by which the air was increased and was substituted by celev − camb, and δ13Cpure is the value of the CO2 in the tank (Fig. 1a). Camb is the atmospheric CO2 concentration (assumed to be 375 ppm), and δ13Camb is the δ13C of ambient air (assumed to be −8). CO2 concentrations were calculated by rearranging the equation and solving for celev. The seasonal means of these CO2 concentrations were compared with the seasonal mean CO2 concentrations measured with a nondispersive infrared gas analyser (LI-800, Li-Cor, Lincoln, NE, USA).

Tissue sampling

Leaves   We collected 20 leaf discs of upper canopy foliage of the five deciduous tree species in August of each year (in 2002 in June/July and September) using a metal puncher. To minimize microclimatic effects, only samples of sun-exposed leaves were harvested. Overall means were calculated by averaging over all trees, thus giving the more abundant species a stronger weight.

Litter  Fifty-six litter traps of 0.5 m2 were placed in a 6-m grid in the crane area. In autumn, the traps were emptied every second week, and litter was sorted by species and weighed. For δ13C determination, litter of one pretreatment and only one treatment year were chosen for analysis, for reasons of analytical costs (1999 vs 2003). The overall δ13C for each litter trap was calculated by pooling δ13C values of all species weighted by their biomass contribution. For comparison with fresh crown litter, five leaves per tree were sampled in autumn 2003, shortly before leaf abscission.

Wood  We used wood cores punched in 2004 with a custom-made 4-mm-diameter stainless steel core puncher, creating minimal tree wounding (Asshoff et al., 2006). Yearly growth rings were separated using a scalpel under a microscope.

Fine roots  In August 2004, fine roots (<1 mm diameter) were collected at c. 10 cm depth for each tree by digging near the stem close to the main roots, to make sure that only roots of a specific tree were included. Fine roots were picked by hand; roots of understorey species (mainly Hedera helix L.) and dead roots could be distinguished visually based on their colour, and were excluded. In the laboratory, loose substrate attached to the roots was removed mechanically by gentle shaking and kept for analysis (so-called rhizospheric soil, see below). The roots were enclosed in plastic bags filled with water to remove the remaining substrate in an ultrasonic cleaner (Bransonic 92), then rinsed with deionized water and oven-dried at 80°C.

Fungi  All fungal sporocarps on the site, and in the surrounding area within c. 100 m from the labelled zone, were harvested. Sporocarps from the unlabelled area were collected with >12 m distance from the edge of the CO2-enriched zone, which was identified as the demarcation zone based on stable δ13C values of mycorrhizal fungi. Sporocarps were specified by taxonomic experts and classified as either mycorrhizal or saprophytic, based on the taxonomic literature. Only the caps of sporocarps were used for isotope analysis.

Rhizospheric and bulk soil  The sedimented root-attached soil fraction (partly including dissolved organic C) was placed in glass cups and oven-dried at 60°C. Of this, 20 mg was weighed into tin capsules and 80 µl 2 m HCl was added to remove carbonates. Before isotope analysis, the acid-treated samples were air-dried for 24 h. In April 2005, soil cores from 0 to 6 cm depth were collected to analyse the δ13C in bulk soil (n = 5). The samples were washed through a 400-µm sieve, rinsed with deionized water, oven-dried at 60°C and ground. The carbonates were removed from the powder as described above.

Carbon-isotope analysis of organic samples

All organic material was oven-dried at 80°C for 48 h and ground with a steel ball mill (Mixer Mill, Retsch MM 2000, Haan, Germany), and 0.6–0.8 mg dried powder was packaged in tin capsules for δ13C analysis. Samples were then combusted in an elemental analyser (EA-1110, Carlo Erba Thermoquest, Milan, Italy). Via a variable open-split interface (Conflo II, Finnigan Mat, Bremen, Germany), gas samples were transferred to the mass spectrometer (Delta S, Thermo Finnigan Mat), which was operated in continuous flow mode. The precision for δ13C analysis was <0.1.

Soil air

Soil air was sampled from 170 ‘gas wells’ (permanently installed PVC tubes in the upper soil layer, 12 cm long, 2 cm in diameter). The top was sealed with a silicon septum. The bottom of the tube was open, and two vertical slits on each side were cut from the bottom up to 3 cm below soil surface to integrate the CO2 released from soil between 3 and 11 cm depth. The gas wells covered a test area of 60 × 70 m, and were placed in a grid of 3 m within the approx. 550-m2 CO2-enriched area and in a grid of 6 m in the larger control area. For details on the sampling and measurement procedure, see Steinmann et al. (2004).

To determine the δ13C of soil CO2, the Keeling plot approach (Keeling, 1958) was applied for each day and CO2 treatment separately. All data were corrected for isotope fractionation caused by slower gas diffusion of the heavier 13CO2 by subtracting 4.4 (Hesterberg & Siegenthaler, 1991). To estimate the effect of understorey vegetation on δ13C of soil air, total above-ground biomass of herbs and small shrubs was cut to the base on four circular plots (1 m radius) centred around the gas wells in July 2004. Measurements of soil-air δ13C were carried out 2 d before and 1–16 d after understorey removal (daily in the first week, every second day thereafter).

The isotope ratio of the soil air was determined with a gas bench II linked to a mass spectrometer (Delta Plus XL, Thermo Finnigan, Bremen, Germany). The CO2 concentration of every gas sample analysed was calculated from the calibration line with standard gas samples of known CO2 concentrations (340 and 5015 ppm). The area of the voltage signal peak of the mass spectrometer for CO2 (masses 44, 45 and 46) was integrated over time and was proportional to the CO2 concentration of the air sample. Reference gas samples were included with each series of measurements. Up to 20000 ppm, the CO2 concentrations agreed well (y = 1.04x, r2 = 0.99) with infrared gas analyser readings (Innova 1312, Innova, Ballerup, Denmark).

Statistics

The need for a canopy crane did not permit randomization of the treatment units (it would require several cranes), therefore a detailed investigation of a priori differences in physiology and morphology between control trees and those later exposed to CO2 was performed by (Cech et al., 2003). As no systematic differences between the two groups of trees were found, we could use single trees as treatment units for the statistical analysis.

Our main goal was to identify tree signals irrespective of species (n = 12 trees in labelled CO2; n = 11 control trees). In addition, tests were carried out using species as a factor, despite the low replication. Because Acer and Tilia were represented by only one tree in the labelled zone, they were pooled for the analysis and referred to as ‘others’. A repeated-measures anova was applied whenever data were collected in several years, with tree species, CO2 treatment and their interaction as fixed factors, and year as the repeated factor.

In the case of roots and soil, where data were collected only once, a two-way anova was performed with species and CO2-treatment as fixed factors. For the analysis of leaf litter data, traps were defined as replicates, and single pots were defined as replicates for canopy isometer analysis. Species were regarded as replicates in the case of fungi, including the fungal type (mycorrhizal or saprophytic) as a fixed factor.

Applying a Student's t-test, soil-air δ13C between treatments was compared using Keeling plot intercepts calculated for each treatment. For the soil-air CO2 analysis, gas wells were assigned to trees as described by Steinmann et al. (2004), resulting in 12 circles in the CO2-enriched and 35 circles in the control area, the diameter of which varied with tree diameter. These circles were regarded as replicates for the two-way anova, with tree species and CO2 treatment as fixed factors.

All errors refer to standard errors. Statistical analysis was carried out using r ver. 2.0.1 (R Development Core Team, 2004).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Isotopic composition of supplemental CO2

A constant isotope ratio of the added CO2 is a prerequisite for tracing the assimilated C. The 10th and 90th percentiles were −30.4 and −28.9, respectively, and reflect the temporal variation. Yearly δ13C means remained relatively constant (Fig. 1a), resulting in an average of −29.7 ± 0.3 over 4 yr.

C4 isometers

Seasonal mean δ13C of C4 grasses grown on control trees showed little variation between the four study years (Fig. 1b). More variation was observed in grasses exposed to labelled CO2, with significantly lower δ13C values (−19.6 ± 0.26, P < 0.0001). The new C signals, represented by the δ13C difference between grasses in ambient minus δ13C of grasses exposed to labelled CO2, did not change significantly between years (CO2 treatment × year, P = 0.32) and reached 5.9 ± 0.6 averaged over the 4-yr means.

The isometer-derived CO2 concentrations for 2001–04 were 514, 519, 596 and 566 ppm. In the first 2 yr, these concentrations corresponded well with independent readings of gas-sampling lines using an infrared gas analyser, and were somewhat higher than infrared gas analyser readings in the last 2 yr (mean CO2 concentrations for 2001–04: 520, 520, 580 and 550 ppm).

Leaves

In the pretreatment year (1999), trees later assigned to the CO2 treatment tended to have slightly less negative leaf δ13C (−26.7) than trees later used as controls (−27.5; Fig. 2a). A similar difference was found for leaf litter. These pretreatment differences were accounted for when calculating the tissue-specific contribution of new, labelled C. For the overall signal we used a pretreatment correction over all trees, whereas for signals in single species we applied a species-specific pretreatment correction. We have no obvious explanation for this a priori difference, because there are no measurable differences in soil parameters, including moisture. Leaves from CO2-enriched trees were significantly labelled starting from the first full year of treatment, and signals were four times higher than pretreatment differences (Fig. 2a). In August 2001, new C signals were 39% in Quercus, 63% in Fagus, 66% in Acer, 77% in Carpinus, and reached 100% in Tilia, possibly reflecting differences in branchlet C autonomy. The species-weighted average signal over all trees increased from year to year, reaching 97% new C by year 4.

image

Figure 2. (a) Leaf; (b) leaf litter δ13C of five deciduous tree species exposed to ambient (open bars) and 13C-depleted CO2 (closed bars), including a pretreatment year (1999, shaded area). Means ± 1 SE for each year and treatment are shown (n = 11–12 trees). Litter data are shown for year 3 only (2003), when both fresh litter picked in the canopy and trapped ground litter (0.5 m2 mesh traps, 30 cm above-ground; 15 traps under control trees and five under labelled trees) were analysed. Leaves were collected in mid-summer; litter was collected in October–November. P values for labelling effects (anova): (*), P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Download figure to PowerPoint

Leaf litter

Leaf litter collected with litter traps in pretreatment year 1999 was −29.6 in the area later used as a control, and −29.0 in the area later exposed to labelled CO2 (Fig. 2b). In 2003, pretreatment-corrected new C signals in litter reached only 28%, averaged over all traps, whereas in freshly fallen litter collected in the canopy, a 90% signal was measured in accordance with fresh leaf signals (Fig. 2a). Litter collected with traps near the ground (25–35 m below canopy) had probably been mixed with litter from the surrounding area during autumn storms, which reduced the signal in ground litter compared with litter from the canopy. Ground litter from control trees was therefore collected at sufficient distance from the labelled zone to minimize mixing with labelled material. In 2003, the isotopic signal strength of ground litter in the labelled zone was strongly species-specific and signals were significant in all species except Fagus. This, together with large variations in biomass contributions ranging from <1% (Acer) up to 90% (Fagus), explained most of the variation in δ13C between different traps.

Wood

Wood δ13C in trees later exposed to labelled CO2 was −27.3, whereas trees later assigned to the control treatment exhibited slightly less negative values (−27.1) in pretreatment year 1999 (data not shown). Over all trees, pretreatment-corrected signals of newly formed wood weighted by species were 71% in year 1, and reached 91% in year 4.

Fine roots

Fine roots consisted of 38% new C over all trees in August 2004, 3.5 seasons from the start (Fig. 3a). Quercus exhibited the strongest signals, followed by Carpinus and Fagus, whereas the weakest signals were measured in the Acer tree and, surprisingly, in the Tilia tree, which always produced the strongest label in leaves and wood.

image

Figure 3. (a) Mean δ13C ± 1 SE of fine roots (<1 mm) for five tree species exposed to ambient (open bars) and 13C-labelled CO2 (closed bars) in year 4 of carbon isotope labelling (2004). Numbers above graph indicate number of trees sampled. (b) Left panel, mean soil δ13C ± 1 SE, which was attached to fine roots (rhizospheric soil) shown in (a); right panel, bulk soil δ13C ± 1 SE at 0–6 cm depth in April 2005. Number of samples shown below graph. In the lower part of all panels, mean δ13C differences ± SE between samples collected in the control and labelled areas are shown with results for the labelling effects of the one-way anovas (ns, not significant).

Download figure to PowerPoint

Fungi

Over all years, sporocarps of 85 different fungal species were found (33 presumably from mycorrhizal and 52 from saprophytic fungi, of which 11 mycorrhizal and 21 saprophytic fungi were found in the labelled zone). All mycorrhizal species belong to the ectomycorrhizal type. The δ13C analysis of fungal sporocarps clearly confirmed the taxonomic classification of species into saprophytic and mycorrhizal (P < 0.0001), the latter always exhibiting more negative δ13C values (Fig. 4). In the labelled forest zone, no 13C labels were found in saprophytic fungi even after 4 yr (Fig. 4a). By contrast, labels in sporocarps of mycorrhizal fungi growing under labelled trees had already reached 62% in year 1. This signal did not increase with time, and was identical in 2003 (Fig. 4b). For no obvious reason, the 13C signals in mycorrhizal fungi were reduced to 41% in year 4, the year following an exceptional drought in 2003. In the reference area, large variations in δ13C values were found between species of the same type of fungus (−26.6 to −20.7 for saprophytic species; −28.2 to −22.4 for mycorrhizal species). Also, within the same species and year substantial variation occurred, reaching an extreme range of −27.7 to −21.1 in Mycena crocata.

image

Figure 4. Mean δ13C ± 1 SE of fungal sporocarps classified as (a) saprophytic; (b) mycorrhizal species. n = Number of species found. Mean δ13C differences between sporocarps collected under control (open bars) and 13C-labelled trees (closed bars) are shown by numbers in graph. (*), P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Download figure to PowerPoint

Soil

Acidified soil particles that had been attached to the fine-root surface contained 9% new C by year 4 (Fig. 3b, left), whereas no signal was found in acidified bulk soil of the same rooting zone in April 2005, shortly before the CO2-enrichment system was set in operation for the fifth season (Fig. 3b, right).

Soil air

Already in May 2001, 3 wk after the first full growing season of CO2 enrichment began, soil air tended to be labelled (Fig. 5a). From June 2001 onwards, new C signals remained statistically significant throughout the study period, including winter data. The contribution of new C increased almost steadily during the first growing season, reaching 29% in October 2001, and was around 35% between June and October during normal years (2002, 2004). In October 2003, at the end of an exceptional drought, new C signals in soil air reached 51%. At the beginning of the growing season (April–May), new C signals were always less pronounced than later in the season. As soil CO2 labels in 2002–04 remained in the same range as in October 2001, a steady state had already been reached one season after continuous labelling of the canopy commenced. Cutting the understorey vegetation around our gas wells (3.14 m2) did not alter soil-air signals, suggesting that signals were not affected by the light ground cover and mainly reflected the respiration of tree roots and root-associated microbes/fungi.

image

Figure 5. (a) Seasonal variation of δ13C in soil air at 3–11 cm depth over 4 growing seasons under trees exposed to ambient (open symbols; n= 59 gas wells) and 13C-labelled CO2 (closed symbols; n= 25). Values derived from Keeling plot. Except for the first measurement date, all isotope signals were statistically significant as assessed by t-test. Error bars are SE of Keeling plot intercepts. Months between the growing seasons are shaded. (b) Mean soil CO2 concentrations ± 1 SE of the same samples used for isotope analysis. *, Significantly higher CO2 concentrations in soil air under CO2-enriched trees. (*), Lower CO2 concentrations in the CO2 enriched area (reverse CO2 effect). For statistical analysis samples were assigned to circles around trees (n = 35 circles around control trees; 12 around CO2-enriched trees). P-values for the CO2-effects of two-way ANOVAs with species and CO2-treatment as factors are shown. (*), P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Download figure to PowerPoint

During summers with normal weather conditions, CO2 concentrations of the same gas samples collected for isotope analysis were higher in the area where crowns received CO2 enrichment. For half the sampling dates, the difference was significant (Fig. 5b). The largest increase in CO2 concentration (+123%) was measured in October 2002 after a wet summer. During a centennial drought in summer 2003, the canopy CO2-enrichment effects on soil-air CO2 concentrations diminished, and were even reversed in December 2003. At the same time the contribution of new C, as assessed by 13C signals, reached a maximum (Fig. 5a).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

After labelling photoassimilates in tree canopies with 13C-depleted CO2 for 4 yr, new C signals were found in all forest compartments investigated except bulk soil and sporocarps of saprophytic fungi (Fig. 6). Our data illustrate a very intense and rapid C flow from canopy to soil biota, a slow penetration of fine roots (suggesting an approx. 10-yr turnover), and an almost complete replacement of old C in new growth rings of trees by year 4. Below we discuss these results separately for each forest compartment.

image

Figure 6. New carbon (C) signatures as assessed by 13C-tracer signals of forest compartments classified as fast-turnover C pools (left panel) and slow-turnover C pools (right panel). The maximum steady-state C-isotope difference between C4 grasses (isometers) grown in crowns of control and CO2-enriched trees is shown as a dashed line and refers to 100% new (=13C-labelled) carbon. Myco, mycorrhizal fungi; Sapro, saprophytic fungi; Rhizo soil, rhizospheric soil.

Download figure to PowerPoint

Canopy CO2 environment

The vigorous apical growth of top-canopy branches made it necessary to slightly elongate and move the CO2-release tubing system every year, to maintain the desired CO2 concentration around the upper canopy (Asshoff et al., 2006). Isometers had to be newly installed every year. This explains the variation in yearly average CO2 concentrations measured by infrared gas analysis and isometers across this rough forest canopy. Frequent watering and exposure to the high irradiance in the upper canopy minimized the biochemical 13C discrimination in the C4 grasses, as evidenced by the rather stable δ13C readings of control grasses. The agreement between CO2 concentrations derived from isometer signals and infrared gas analysis of air samples confirms the steady exposure of the forest canopy to the 13C-tracer signal.

Delayed leaf signals

Surprisingly, leaf tissue had fully adjusted to the novel stable isotope signal only after 4 yr. During leafing, no decrease of mobile C reserves takes place (Hoch et al., 2003), suggesting that our data should not be seen as evidence for a dependence of leaves on old C to build a new canopy. More probably, the data indicate that new C rapidly enters a given pool of mobile C (presumably in wood tissue) and mixes with this pool before entering leaf construction. In other words, the data suggest substantial turnover of mobile C pools along the assimilate transport path. The resulting dilution process of old by new C can theoretically go on for years, but the degree of dilution (and hence C turnover) is still remarkable, and also points to a large reserve pool compared with annual leaf C needs, which had already been confirmed for this site (Hoch et al., 2003). Based on our first year's results, 30% of C found in new foliage is from previous years. This does not preclude that leaves may still produce three to four times their own C cost per year (Poorter et al., 2006). As the lower part of the canopy is not exposed to elevated CO2, unlabelled lower-canopy foliage could, alternatively, have dampened the new C label. The largest amount of old C in new foliage was found in Quercus leaves (39%), a late-flushing species reaching maximum photosynthetic rates only later in the season (Morecroft & Roberts, 1999).

New carbon signals in leaf litter

Nearly identical new C signals in canopy litter compared with fresh leaves in 2003 suggest that structural biomass contains the same mixture of unlabelled and labelled C as mobile carbohydrates or amino acids, which are recycled during leaf senescence. Therefore the weak signals in leaf litter collected with traps near the ground must have resulted from dilution with litter from surrounding trees during autumn storms.

New carbon signals in tree rings

There may be two reasons why stem wood did not attain a 100% new C signal after 4 yr. First, early wood formation, just like foliage, may draw C from a slowly diluting mobile C pool in stem-storage tissue (see above); second, the lower canopy (<15 m) is not exposed to labelled CO2, perhaps causing a dilution of the isotope signal. If we attribute the lack of a 100% tree-ring signal at the base of the stem exclusively to the contribution of C from lower-canopy foliage, the data suggest this canopy layer contributes, at most, 9% of total C (because 91% was labelled). This is an interesting result, illustrating the dominant role of upper-canopy foliage for tree growth. Most probably this is true only for upper-canopy trees, as they are exposed to a large light gradient, whereas in the lower canopy light is distributed more evenly. If we also account for unknown mobile C-pool dilution (particularly through ray tissue), the contribution of the subcanopy becomes even smaller.

Fine root signals

Fine roots are often assumed to turn over rapidly, but the bulk fine-root fraction in forests has been shown to last several years (Gaudinski et al., 2001; Matamala et al., 2003). The 38% new C signal in the <1-mm fine-root fraction found here in the fourth season suggests that our samples represent a mixture of new and older (>4-yr) fine roots. However, similarly to leaves, fine roots could also be built from a slowly diluting C pool (see above). Assuming a linear increase in new C, fine roots would reach a 100% signal in approx. 10 yr (10-yr root C turnover), which is substantially longer than suggested by data for a Pinus taeda forest (4.2 yr) and a temperate deciduous Liquidambar styraciflua forest (1.25 yr; Matamala et al., 2003).

Contrasting labels in sporocarps of mycorrhizal and saprophytic fungi

Based on the high (a few days) turnover rate of arbuscular mycorrhizal hyphae (Staddon et al., 2003), we assume that ectomycorrhizal hyphae are also rapidly recycled. Therefore the pronounced allocation of new C to ectomycorrhizal fungi might indicate that large amounts of C are rapidly released back to the atmosphere. As hyphae of single genets can cover areas up to 300 m2 (Bonello et al., 1998), sporocarps collected in the CO2-enriched area could be linked to trees exposed to elevated as well as ambient CO2. This might explain why sporocarps consisted of at least 40% old C during the whole study period. A labelling gradient with increasing radial distance from the treated area suggested a signal influence of approx. 20% at 6–12 m outside the CO2-enriched area. So the fungal signal in the labelled area should reflect the reciprocal influence of nonlabelled trees surrounding the 550-m2 test area. It is very unlikely that mycorrhizal fungi had acquired C from sources other than their host plant, such as soil or leaf litter (Högberg et al., 2001; Treseder et al., 2006). The variability in δ13C of mycorrhizal fungi we observed between years might partly reflect C obtained from either overstorey or understorey trees, depending on years. Understorey trees are well known to exhibit more negative δ13C (for Fagus, −34.4 in the understorey compared with −28.0 in the overstorey), and this signal could translate to their fungal partner (Högberg et al., 1999). Alternatively, fungal species composition might have been altered in response to elevated CO2 as shown earlier by Fransson et al. (2001), resulting in a shift in δ13C caused by species-specific values.

As no label was detected in saprophytic fungi after four treatment years (Fig. 4a), these fungi decomposed C compounds that were >4 yr old, in accordance with the results of Hobbie et al. (2002). This was somewhat surprising, as at least a few of the species found are known to decompose leaf litter (e.g. Mycena galopus; Ghosh et al., 2003).

Soil carbon signals

The fact that new C signals in soils were found exclusively in the rhizospheric fraction, but not in bulk soil, suggests that soil C input had taken place mainly via fine roots (exudates, rhizosphere microbes). As these are relatively short-lived compounds, we assume that our signal reflects contributions to the labile C pools, as has been shown in previous studies (Hagedorn et al., 2003; Lichter et al., 2005), and is likely to be accompanied by a stimulation in soil respiration, as shown earlier (Körner & Arnone, 1992; Heath et al., 2005). In contrast to our experiment, an increase in soil C was found in a L. styraciflua forest exposed to elevated CO2 (Jastrow et al., 2005), which is probably the result of strongly enhanced root production and root turnover (Matamala et al., 2003; Norby et al., 2004). In general, bulk soil signals are usually very small (5%; Jastrow et al., 2005) and are therefore difficult to detect (Hungate et al., 1996).

Soil air

Our data suggest that, after reaching a quasi-steady state within a year, new C contributes 35% to respired CO2 emerging from soil under normal weather conditions during three seasons, which is lower than described earlier (55–65%; Andrews et al., 1999; Högberg et al., 2001; Bhupinderpal-Singh et al., 2003; Andersen et al., 2005). This may reflect real differences between forests to some extent, but may also have other explanations. For example, during a severe drought in summer 2003, when no plant-available water was present down to 1 m depth (Leuzinger et al., 2005), contributions of current assimilates to total soil CO2 rose to 51%, similar to the studies mentioned above. We assume that during the drought microbes feeding on older, unlabelled C were less active and contributed less to respired CO2 (Fig. 5b), whereas root respiration continued, perhaps supported by hydraulically lifted water (Caldwell et al., 1998) or by water from greater depths, and exceeded heterotrophic respiration, thus causing the strong new C signals in this year (Fig. 5a). The more pronounced soil-air signal in this year might also have resulted from the interaction of drought and elevated CO2 on stomatal conductance. Drought leads to less negative δ13C in assimilates, but elevated CO2 relieved drought stress during that extraordinary dry summer. Actually, drought led to higher stomatal conductance in trees exposed to elevated CO2 (S. G. Keel, unpublished data), causing δ13C in concurrent assimilates to become even more negative in CO2-enriched trees, thus adding to the strength of the tracer signal imposed by artificial labelling. The generally smaller signals at the beginning of the season (during leafing) indicate that soil-air signals are driven mainly by current assimilates, which are less abundant under a flushing canopy in April and May than after full canopy development.

Steadily increasing soil-air isotope signals during the first treatment season highlight the velocity by which new C is allocated below-ground, and the importance of recently assimilated C for below-ground metabolism. This is supported by previous studies, which have shown a close temporal linkage between climatic conditions and the isotopic composition of respired CO2, suggesting that these photoassimilates are respired within <10 d after assimilation (Ekblad & Högberg, 2001; Bowling et al., 2002; Scartazza et al., 2004; Steinmann et al., 2004; Tang et al., 2005).

As demonstrated by increased soil-air CO2 concentrations under the CO2-enriched canopy area, roots, microbes feeding on exudates, and/or mycorrhizal fungi respired more CO2 in response to elevated CO2. This confirms earlier findings of increased soil CO2 efflux in response to CO2 enrichment under more artificial test conditions (Zak et al., 2000; King et al., 2004; Niinistöet al., 2004; Heath et al., 2005). Hence the new C fluxes measured here are likely to have been affected by CO2 enrichment as they are higher than ‘normal’.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

This in situ 13C-labelling study yielded direct evidence on the path and speed of C flows in mature deciduous forest trees. The data indicate a high degree of mixing between newly assimilated C and old mobile C stores before investment into structural growth. While new tissue such as leaves and fine roots may correspond quantitatively to 100% new C, their actual isotopic composition proves a high degree of dilution with old C; it takes several years to replace old by new C, even in zones of most active growth. On the other hand, new C signals appear strongly and rapidly (within days) in soil CO2, suggesting a massive flow of new C to the rhizosphere, and fungal symbionts in particular. We conclude that C loaded to the phloem (as indicated, e.g. honeydew δ13C of aphids) enters the rhizosphere largely undiluted. However, before C is invested in tree tissues, it is rapidly mixed (and diluted) with old C. The size of the C-reserve pool and its mobility thus determine the new C-signal strength in tree tissue. Four years suffice to arrive at 90–100% C replacement in leaves and new tree rings, but fine roots still retain 60% old C, which we attribute to their greater-than-expected longevity.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

We thank Erwin Amstutz and Olivier Bignucolo for crane operations and on-site support, Markus Wilhelm for the taxonomic classification of the fungi, Katharina Steinmann for sharing her experience and her data collected in 2001, and Roman Asshoff for providing the wood data. Maya Jäggi greatly supported the stable isotope analysis. The CO2-enrichment experiment was funded by the Swiss National Science Foundation projects 3100-059769.99, 3100-067775.02 and 5005-65755 (NCCR Climate), and the Swiss Canopy Crane by the Swiss Agency for the Environment, Forest and Landscape. We thank three anonymous reviewers for helpful suggestions on the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  • Andersen CP, Nikolov I, Nikolova P, Matyssek R, Haberle KH. 2005. Estimating ‘autotrophic’ belowground respiration in spruce and beech forests: decreases following girdling. European Journal of Forest Research 124: 155163.
  • Andrews JA, Harrison KG, Matamala R, Schlesinger WH. 1999. Separation of root respiration from total soil respiration using carbon-13 labeling during Free-Air Carbon Dioxide Enrichment (FACE). Soil Science Society of America Journal 63: 14291435.
  • Asshoff R, Zotz G, Körner C. 2006. Growth and phenology of mature temperate forest trees in elevated CO2. Global Change Biology 12: 848861.
  • Barbour MM, Hunt JE, Dungan RJ, Turnbull MH, Brailsford GW, Farquhar GD, Whitehead D. 2005. Variation in the degree of coupling between δ13C of phloem sap and ecosystem respiration in two mature Nothofagus forests. New Phytologist 166: 497512.
  • Bhupinderpal-Singh, Nordgren A, Löfvenius MO, Högberg MN, Mellander PE, Högberg P. 2003. Tree root and soil heterotrophic respiration as revealed by girdling of boreal Scots pine forest: extending observations beyond the first year. Plant, Cell & Environment 26: 12871296.
  • Bonello P, Bruns TD, Gardes M. 1998. Genetic structure of a natural population of the ectomycorrhizal fungus Suillus pungens. New Phytologist 138: 533542.
  • Bowling DR, McDowell NG, Bond BJ, Law BE, Ehleringer JR. 2002. C-13 content of ecosystem respiration is linked to precipitation and vapor pressure deficit. Oecologia 131: 113124.
  • Buchmann N, Brooks JR, Rapp KD, Ehleringer JR. 1996. Carbon isotope composition of C4 grasses is influenced by light and water supply. Plant, Cell & Environment 19: 392402.
  • Caldwell MM, Dawson TE, Richards JH. 1998. Hydraulic lift: consequences of water efflux from the roots of plants. Oecologia 113: 151161.
  • Cech PG, Pepin S, Körner C. 2003. Elevated CO2 reduces sap flux in mature deciduous forest trees. Oecologia 137: 258268.
  • Ekblad A, Högberg P. 2001. Natural abundance of C-13 in CO2 respired from forest soils reveals speed of link between tree photosynthesis and root respiration. Oecologia 127: 305308.
  • Fransson PMA, Taylor AFS, Finlay RD. 2001. Elevated atmospheric CO2 alters root symbiont community structure in forest trees. New Phytologist 152: 431442.
  • Gaudinski JB, Trumbore SE, Davidson EA, Cook AC, Markewitz D, Richter DD. 2001. The age of fine-root carbon in three forests of the eastern United States measured by radiocarbon. Oecologia 129: 420429.
  • Ghosh A, Frankland JC, Thurston CF, Robinson CH. 2003. Enzyme production by Mycena galopus mycelium in artificial media and in Picea sitchensis F1 horizon needle litter. Mycological Research 107: 9961008.
  • Hagedorn F, Spinnler D, Bundt M, Blaser P, Siegwolf R. 2003. The input and fate of new C in two forest soils under elevated CO2. Global Change Biology 9: 862872.
  • Hansen J, Beck E. 1990. The fate and path of assimilation products in the stem of 8-year-old Scots pine (Pinus sylvestris L.) trees. Trees – Structure and Function 4: 1621.
  • Hansen J, Beck E. 1994. Seasonal-changes in the utilization and turnover of assimilation products in 8-year-old Scots Pine (Pinus sylvestris L.) trees. Trees – Structure and Function 8: 172182.
  • Heath J, Ayres E, Possell M, Bardgett RD, Black HIJ, Grant H, Ineson P, Kerstiens G. 2005. Rising atmospheric CO2 reduces sequestration of root-derived soil carbon. Science 309: 17111713.
  • Hesterberg R, Siegenthaler U. 1991. Production and stable isotopic composition of CO2 in a soil near Bern, Switzerland. Tellus Series B – Chemical and Physical Meteorology 43: 197205.
  • Hobbie EA, Weber NS, Trappe JM, Van Klinken GJ. 2002. Using radiocarbon to determine the mycorrhizal status of fungi. New Phytologist 156: 129136.
  • Hoch G, Richter A, Körner C. 2003. Non-structural carbon compounds in temperate forest trees. Plant, Cell & Environment. 26: 10671081.
  • Högberg P, Plamboeck AH, Taylor AFS, Fransson PMA. 1999. Natural C-13 abundance reveals trophic status of fungi and host-origin of carbon in mycorrhizal fungi in mixed forests. Proceedings of the National Academy of Sciences, USA 96: 85348539.
  • Högberg P, Nordgren A, Buchmann N, Taylor AFS, Ekblad A, Högberg MN, Nyberg G, Ottosson-Löfvenius M, Read DJ. 2001. Large-scale forest girdling shows that current photosynthesis drives soil respiration. Nature 411: 789792.
  • Högberg P, Nordgren A, Ågren GI. 2002. Carbon allocation between tree root growth and root respiration in boreal pine forest. Oecologia 132: 579581.
  • Horwath WR, Pregitzer KS, Paul EA. 1994. C-14 allocation in tree soil systems. Tree Physiology 14: 11631176.
  • Hungate BA, Jackson RB, Field CB, Chapin FS. 1996. Detecting changes in soil carbon in CO2 enrichment experiments. Plant and Soil 187: 135145.
  • Jastrow JD, Miller RM, Matamala R, Norby RJ, Boutton TW, Rice CW, Owensby CE. 2005. Elevated atmospheric carbon dioxide increases soil carbon. Global Change Biology 11: 20572064.
  • Johnson D, Leake JR, Ostle N, Ineson P, Read DJ. 2002. In situ (CO2) C-13 pulse-labelling of upland grassland demonstrates a rapid pathway of carbon flux from arbuscular mycorrhizal mycelia to the soil. New Phytologist 153: 327334.
  • Keeling CD. 1958. The concentration and isotopic abundances of atmospheric carbon dioxide in rural areas. Geochimica et Cosmochimica Acta 13: 322334.
  • King JS, Hanson PJ, Bernhardt E, DeAngelis P, Norby RJ, Pregitzer KS. 2004. A multiyear synthesis of soil respiration responses to elevated atmospheric CO2 from four forest FACE experiments. Global Change Biology 10: 10271042.
  • Körner C. 2003. Carbon limitation in trees. Journal of Ecology 91: 417.
  • Körner C, Arnone JA. 1992. Responses to elevated carbon dioxide in artificial tropical ecosystems. Science 257: 16721675.
  • Körner C, Asshoff R, Bignucolo O, Hättenschwiler S, Keel SG, Pelaez-Riedl S, Pepin S, Siegwolf RTW, Zotz G. 2005. Carbon flux and growth in mature deciduous forest trees exposed to elevated CO2. Science 309: 13601362.
  • Leuzinger S, Zotz G, Asshoff R, Körner C. 2005. Responses of deciduous forest trees to severe drought in Central Europe. Tree Physiology 25: 641650.
  • Lichter J, Barron SH, Bevacqua CE, Finzli AC, Irving KE, Stemmler EA, Schlesinger WH. 2005. Soil carbon sequestration and turnover in a pine forest after six years of atmospheric CO2 enrichment. Ecology 86: 18351847.
  • Luo YQ, Reynolds JF. 1999. Validity of extrapolating field CO2 experiments to predict carbon sequestration in natural ecosystems. Ecology 80: 15681583.
  • Matamala R, Gonzalez-Meler MA, Jastrow JD, Norby RJ, Schlesinger WH. 2003. Impacts of fine root turnover on forest NPP and soil C sequestration potential. Science 302: 13851387.
  • McLaughlin SB, McConathy RK, Beste B. 1979. Seasonal-changes in within-canopy allocation of photosynthate-14C by White Oak. Forest Science 25: 361370.
  • Morecroft MD, Roberts JM. 1999. Photosynthesis and stomatal conductance of mature canopy Oak (Quercus robur) and Sycamore (Acer pseudoplatanus) trees throughout the growing season. Functional Ecology 13: 332342.
  • Niinistö SM, Silvola J, Kellomäki S. 2004. Soil CO2 efflux in a boreal pine forest under atmospheric CO2 enrichment and air warming. Global Change Biology 10: 13631376.
  • Norby RJ, Ledford J, Reilly CD, Miller NE, O’Neill EG. 2004. Fine-root production dominates response of a deciduous forest to atmospheric CO2 enrichment. Proceedings of the National Academy of Sciences, USA 101: 96899693.
  • Pataki DE, Ellsworth DS, Evans RD, Gonzalez-Meler M, King J, Leavitt SW, Lin GH, Matamala R, Pendall E, Siegwolf R, Van Kessel C, Ehleringer JR. 2003. Tracing changes in ecosystem function under elevated carbon dioxide conditions. Bioscience 53: 805818.
  • Pate J, Arthur D. 1998. δ13C analysis of phloem sap carbon: novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globulus. Oecologia 117: 301311.
  • Pepin S, Körner C. 2002. Web-FACE: a new canopy free-air CO2 enrichment system for tall trees in mature forests. Oecologia 133: 19.
  • Phillips RP, Fahey TJ. 2005. Patterns of rhizosphere carbon flux in sugar maple (Acer saccharum) and yellow birch (Betula allegheniensis) saplings. Global Change Biology 11: 983995.
  • Poorter H, Pepin S, Rijkers T, De Jong Y, Evans JR, Körner C. 2006. Construction costs, chemical composition and payback time of high- and low-irradiance leaves. Journal of Experimental Botany 57: 355371.
  • R Development Core Team. 2004. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org
  • Saurer M, Siegwolf RTW, Schweingruber FH. 2004. Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years. Global Change Biology 10: 21092120.
  • Scartazza A, Mata C, Matteucci G, Yakir D, Moscatello S, Brugnoli E. 2004. Comparisons of δ13C of photosynthetic products and ecosystem respiratory CO2 and their response to seasonal climate variability. Oecologia 140: 340351.
  • Staddon PL, Ramsey CB, Ostle N, Ineson P, Fitter AH. 2003. Rapid turnover of hyphae of mycorrhizal fungi determined by AMS microanalysis of 14C. Science 300: 11381140.
  • Steinmann KTW, Siegwolf R, Saurer M, Körner C. 2004. Carbon fluxes to the soil in a mature temperate forest assessed by C-13 isotope tracing. Oecologia 141: 489501.
  • Tang JW, Baldocchi DD, Xu L. 2005. Tree photosynthesis modulates soil respiration on a diurnal time scale. Global Change Biology 11: 12981304.
  • Treseder KK, Torn MS, Masiello CA. 2006. An ecosystem-scale radiocarbon tracer to test use of litter carbon by ectomycorrhizal fungi. Soil Biology and Biochemistry 38: 10771082.
  • Trumbore S. 2000. Age of soil organic matter and soil respiration: radiocarbon constraints on belowground C dynamics. Ecological Applications 10: 399411.
  • Zak DR, Pregitzer KS, King JS, Holmes WE. 2000. Elevated atmospheric CO2, fine roots and the response of soil microorganisms: a review and hypothesis. New Phytologist 147: 201222.