www.sciencemag.org/content/352/6283/342/suppl/DC1
Supplementary Materials for Belowground carbon trade among tall trees in a temperate forest Tamir Klein,* Rolf T. W. Siegwolf, Christian Körner *Corresponding author. E-mail:
[email protected] Published 15 April 2016, Science 352, 342 (2016) DOI: 10.1126/science.aad6188
This PDF file includes: Materials and Methods Supplementary Text Figs. S1 to S14 Table S1 References
Materials and Methods Study site and experimental setup The study was conducted in a diverse mixed forest 12 km southwest of Basel, Switzerland (47°33’ N, 7°36’ E, 550 m a.s.l). The site is dominated by ~100 to 120-yearold coniferous (mostly Picea abies (L.) Karst., Larix decidua Mill., Pinus sylvestris L., and Abies alba Mill.) and deciduous trees (mostly Fagus sylvatica L., Quercus petraea (Matt.) Liebl., and Carpinus betulus L.; Fig. S8). Crown heights are 30-40 m forming a closed tree canopy with a leaf area index of around 5 2,3. The soil is a shallow, 30 cm deep, silty-loamy rendzina on calcareous bedrock. The climate is mild temperate, with mean January and July temperatures of 2.1 and 19.1 °C and mean temperature during the growing season (May-September) of 14.7 °C. Mean annual precipitation is ~900 mm. Between 30 July 2009 and 30 October 2014, five 37-40 m tall, 110-year-old Norway spruce (Picea abies) individuals were equipped with a free air CO2-enrichment (FACE) system based on a web of porous tubes1, which were installed with a 45 m height canopy crane (Figs. S1-S7). Flows of pure CO2, and thus, CO2 concentrations in the canopy, were controlled by a computer that also accounted for weather conditions: the FACE treatment was discontinued when temperatures were < 4 °C, photosynthetic photom flux density was < 100 μmol m-2 s-1, or wind speed was > 10 m s-1. Therefore, the operation of FACE was largely off during the coldest period from early November until early March (4 months). Throughout the rest of the year (March-October) median and mean CO2 concentrations in the tree crowns were 500-560 ppm and 530-590 ppm, respectively (60 sampling points in the canopy were monitored via infra-red gas analyser; LI-800, Li-Cor, Lincoln, NE, USA). The trees carrying a 13C label formed a group, facilitating CO2 enrichment and clear association of signals with the investigated trees. CO2 release was constrained to tree crowns, i.e. at 20-37 m aboveground, without downward CO2-flow and thus, preventing uncontrolled CO2 label transfer to the soil surface. Five similarly tall trees 5-30 m away from the labelled trees served as controls under unlabelled, ambient CO2 (A-trees). The CO2 gas employed for elevating the CO2 concentrations carried a constant 13C isotope signal (δ13C = -30‰, compared with -8‰ in the ambient air), permitting the tracing of the carbon flows in trees and into the soil, besides confirming the efficiency of the FACE system19,24. CO2-enrichment efficiency was also verified with 50 plant isometers distributed throughout the canopy1,2. These isometers were small pots planted with a grass (Echinochloa crus-galli) with the C4 photosynthesis pathway, i.e. without any apparent enzymatic 13C fractionation. The δ13C of isometers grown under labelled CO2 was on average -4.8‰ lower than that of isometers grown under unlabelled CO2, again reflecting an efficient FACE treatment. The CO2 gas employed for elevating the CO2 concentration carried a constant 13C isotope signal (δ13C = -30‰). Thus, δ13C measurements in organic matter facilitated the tracing of carbon flows in trees and soils, besides validating the efficiency of the FACE system. Fine roots from in-growth cores On 12 April 2013, we took 90 soil cores (12 cm in depth x 3.6 cm diameter): 9 cores per tree in the main rooting sphere (in 2 m distance around the tree trunks) were placed in triplets (10 cm distance within triplet) placed at angles of 120°, 240°, and 360° around each trunk (Figs. S9-S10). Root samples obtained from the three cores per triplet 2
were averaged to balance micro-scale heterogeneity. In these cored holes, we immediately installed similarly sized in-growth cores: cylinders made of a 2 mm stiff mesh (Sefar AG, Heiden, Switzerland), filled with sieved, root-free soil collected on-site and gently compacted. On 23 September 2014 (17 months later), the in-growth cores were recovered using a knife. Soil and in-growth cores were kept frozen at -20 °C and were defrosted prior to analysis in cold water (4°C) for 48h. Fine roots were extracted using a sieve (1 mm mesh) and tweezers and then classified into the following categories: P. abies, non-Picea, and dead fine roots. P. abies fine roots were selected by comparison to pure P. abies and pure Fagus sylvatica reference root collections from nearby sites. The distinct morphology of P. abies roots warranted the separation of P. abies roots from roots of other species. All fine root classes were dried at 80 °C for 48 h prior to biomass determination and isotopic analysis. Sampling of verified tree fine roots, stem cores, twigs, and herb rhizomes Lateral roots of labelled Picea trees and neighbouring unlabelled trees (1-4 m distance between stems) of three different species (Fagus sylvatica, Pinus sylvestris, Larix decidua) were extracted down to 5 cm soil depth on 10 and 27 March 2015 (Figs. S11-S13). Fine roots were sampled in the contact zone between roots of each labelled Picea and its non-Picea neighbour. Fine roots were also collected from the control, unlabelled Picea, and from nearby non-Picea trees (Fagus sylvatica, Pinus sylvestris). For this C-transfer study, we excavated roots from a total of 12 individual trees: three labelled Picea, three of their non-Picea neighbours, two unlabelled Picea, and four of their non-Picea neighbours (Fig. S8). On the same days, 3-5 top canopy twigs 1-2 years old, were sampled from trees of the various species neighbouring the labelled Picea trees for isotopic analysis (in addition to the samples of Picea). These samples were used to test the extent of potential, unintended label transfer across the canopy of these neighbouring species, i.e. from positions from crown parts close to and far away from the crowns of labelled Picea trees. Stem cores at breast height were sampled from the 12 individual trees used for C-transfer analysis (plus the 5 additional Picea trees that belonged to overall experiment) at 90° and 270° compass directions using a 200 mm long increment borer (core diameter 5.15 mm; Haglof, Sweden). The ten Picea trees (five labelled and five unlabelled) were cored on 23 and 26 September 2014; nine trees of the other three tree species (one neighbouring and two non-neighbouring from each species) were cored on 5 August 2015, with cores of neighbouring trees taken from stem sides proximal and distal to the labelled Picea trees. Stem cores were dried at 80 °C for 72 h and scraped using a scalpel for better reading of the tree-ring structure. Isotopic analysis was performed on the annual rings of 2010-2014, earlywood and latewood taken together. On 29 April 2015 1-7-years old rhizomes of three perennial plant species from the forest understorey (Paris quadrifolia, Mercurialis perennis, and Rubus fruticosus) were collected around each of the five unlabelled and five labelled Picea trees. Rhizomes were sectioned into annual segments, with their bark peeled off, and milled prior to isotopic analysis. Carbon isotope composition All tissue samples were dried at 80°C for 48h, weighed into tin capsules in aliquots of 0.3 to 0.8 mg and analyzed for carbon isotopes. The isotope analysis was 3
performed at the stable isotope facility at Paul Scherrer Institute, Villigen, Switzerland. Samples were analyzed using an isotope ratio mass spectrometer (IRMS) operating in continuous flow mode (Delta S, Thermo Finnigan MAT, Bremen, Germany) following combustion in an elemental analyzer (EA-1110 CHN, Carlo Erba Thermoquest, Milan, Italy), which was linked to the IRMS via a variable open-split interface (Conflo II, Thermo Finnigan MAT, Bremen, Germany). The precision of δ13C analyzes was < 0.1‰. The δ-notation expressed the isotopic deviation from the international reference standard (Vienna-Pee Dee Belemnite: V-PDB): δ13C = Rsample/Rstandard – 1 (‰) where R is the molar ratio of 13C to 12C for the sample and the standard, respectively. We applied an isotope mixing ratio calculation, following the basic equation a x n + (100-a) x m = p, where a is the contribution of one of two sources to a mixture (in %), n is its isotopic signature, m is the isotopic signature of the other source, and p that of the mixed product. Statistical analysis Differences in fine root δ13C among labelled and unlabelled Picea trees and among neighbouring and non-neighbouring non-Picea trees were tested in ANOVA and were significant at P
