microcosm establishment and treatment design
Fifteen terrestrial microcosms (1 m2 surface area) were established and maintained in the Ecotron controlled environment facility (Lawton 1996) for 312 days. The microcosms were constructed from grassland at Sourhope Experimental Farm (UK National Grid Reference NT855196) in Scotland. Soils from the site were collected, roughly homogenized by horizon, then treated with methyl bromide (CH3Br) to remove the seed bank, mesofauna and macrofauna. The soil profile was then reconstructed (to a depth of 303 mm) by horizon; the upper mineral horizon was split into two layers because the upper portion had greater organic matter content. Horizons and their depths were as follows: FH (35 mm), H (20 mm), Ah ‘upper’ (69 mm), Ah ‘lower’ (114 mm), AB (65 mm).
Seeds of the 10 most dominant plants at the Sourhope grassland were collected, glasshouse-germinated and introduced into the microcosms as seedlings in a randomly generated grid pattern that was repeated for all microcosm communities. In total, 384 seedlings were introduced into each community and were, in the same order of decreasing dominance as at the field site: Agrostis capillaris L., Festuca rubra L., Nardus stricta L., Anthoxanthum odoratum L., Poa pratensis L., Trifolium repens L., Holcus mollis L., Potentilla erecta L., Galium saxatile L. and Luzula multiflora (Ehrh.) Lej. All species established and persisted.
Each microcosm was housed within a 2 × 2 × 2-m Ecotron walk-in chamber. The Ecotron facility permits tight environmental control: simulated abiotic conditions in each chamber are effectively identical (Lawton 1996). Photoperiod was 18 h, including a gradual dawn and dusk of 2 h each. Temperature and relative humidity followed sine curves between 21·1 °C during the day and 9·5 °C at night, and 83% after rainfall (3·5 mm day−1) to a minimum of 63%, respectively.
Soil biota were introduced selectively to create communities that differed in organism body width. If the adult was not soil-dwelling, organisms were assigned to a size class based on their largest juvenile stage. We adhered to the general size classification for terrestrial decomposer food webs (Swift et al. 1979), establishing three treatments that formed a gradient of increasing functional complexity: (1) microbiota only (<100 µm diameter; primarily bacteria, fungi, Protozoa, Nematoda); (2) microbiota and mesofauna (100 µm to 2 mm diameter; primarily Collembola, Acari, Enchytraeidae); (3) microbiota, mesofauna and macrofauna (>2 mm diameter; primarily earthworms, slugs, insect larvae, staphylinid beetles). Hereafter, these treatments are referred to as microbiota, mesofauna and macrofauna treatments, respectively.
Soils were repeatedly inoculated with microbial solutions, derived using various physical extraction techniques (Bradford et al. 2002a) from Sourhope grassland turves. Microfauna, mesofauna and the majority of the macrofauna were also extracted from Sourhope turves using wet- (Nematoda and Enchytraeidae) or dry- (other mesofauna and macrofauna) funnel techniques. Two earthworm species (Allolobophora chlorotica Sav. and Lumbricus rubellus Hoffm.) and a mollusc (Arion ater L.) were obtained from biological suppliers (Blades Biological, Edenbridge, UK). These macrofauna species were present at the Sourhope grassland, but to obtain them in sufficient biomass to establish our experiment was deemed too destructive to the site.
Treatments were randomly assigned within five blocks defined by microcosm planting order. The block structure was used in all further set-up and in sampling, such that all procedures were conducted, in a random order, by block. Microcosms were constructed over 7 months: day 1 was assigned at the end of this period, the day after the last faunal inoculation was made. Microcosms were established gradually to permit adequate time for development of a ‘mature’ grassland plant community, as well as growth of the microflora and faunal communities. This timeline also permitted diminishing of the C and nutrient pulse, inherent to microcosm construction, prior to the experiment. Biomass and numbers of soil microflora and fauna once the microcosms were established (designated day 1) and on day 257 (the time of 13C-labelling and thus the start of the experimental work presented here) are provided by Bradford et al. (2002a), as are further details of experimental set-up.
We followed the movement of a 13C-label into plant foliar material, then through the soil community and back to the atmosphere as respired CO2 across a 52-day period. Communities were 13C-labelled using a mobile laboratory that administers a controlled flow of 13CO2 at ambient CO2 concentrations (≈ 370 µl l−1). This stable isotope-delivery system is described by Ostle et al. (2000). With this system, six microcosm communities could be labelled at one time. Two experimental blocks were pulse-labelled on day 255 and two blocks on day 257. Sampling of these blocks was staggered appropriately. The non-labelled block provided the natural abundance values for the measured C pools (see below).
To introduce the 13C-label, microcosm communities were capped with a chamber constructed from photosynthetically active radiation-transparent polyester sheeting (Mylar). Labelling continued for 6 h during the high-light (536 µmol m−2) period of the diurnal cycle. The rapid turnover time of air in the chambers meant that dilution of the 13CO2 by community respiration was minimal.
foliar, soil community and co2 analyses for 13c-label content
Clippings of plant foliar material were taken immediately prior to and immediately after 13C-labelling, and were used to estimate the amount of 13C-label assimilated by the plant community. To generate a representative community sample, the proportion of dry foliar biomass of each species to include was quantitatively based on plant species-abundance data, and within each microcosm at least five individuals of each plant species were sampled. Clipped material was dried at 40 °C and then milled under liquid N2.
Micro-arthropods (Collembola and Acari) were dry-extracted from 50 mm diameter organic horizon cores (55 mm depth) using Tullgren funnels. Enchytraeidae were wet-extracted from cores of the same size. At 2 days after the pulse, three such cores were sampled per microcosm for the micro-arthropods and three for the Enchytraeidae. Separate but single cores of the same size were used for extraction of Nematoda and determination of microbial biomass C. At 8 and 18 days after the pulse, the micro-arthropods and Enchytraeidae were extracted from three cores in total to minimize soil disturbance. Each core was halved longitudinally, with one half used for micro-arthropods and one for Enchytraeidae. This still provided a sampling area greater than recommended for extracting these fauna (Coleman et al. 2004).
Micro-arthropods were collected into salt water to kill the animals and prevent predation. Extractions continued across 48 h, but collected fauna were sorted and dried at 40 °C after each 24-h period to avoid biomass decay. Dry samples were weighed and milled under liquid N2. Enchytraeidae were freeze-dried prior to weighing. Nematoda were also freeze-dried, after extraction using a tray technique (Whitehead & Hemming 1965). Microbial biomass C was estimated using modified chloroform fumigation extraction (Vance et al. 1987), and provided for isotope analyses as potassium sulphate (K2SO4) extracts. All fauna were analysed whole (including gut contents). It was deemed too destructive to the communities to sample macrofauna and roots.
Community-respired CO2 samples were collected 2, 5, 8, 12, 18, 32 and 52 days post-labelling using a static cover-box technique (Holland et al. 1999). Gas samples from the cover-box headspaces were stored in 12 ml Exetainer tubes (Labco Ltd, High Wgcombe, UK) for <72 h prior to analysis.
All samples were analysed for C concentration and 13C/12C ratios by continuous-flow isotope-ratio mass spectrometry (IRMS) at the NERC 15N Stable Isotope Facility. Plant and soil biota samples were analysed using an elemental analyser (Carlo Erba/Fisons, Milan, Italy) linked to a modified IRMS (Dennis Leigh, Keele, UK) and CO2 samples using a trace gas preconcentration unit coupled to an Isoprime IRMS (Micromass, Manchester, UK). Analytical precision was ±0·1 δ13C‰. Internal gas standards and solid reference materials were calibrated to PDB (Pee Dee Belemnite) (Craig 1957).
To determine how the treatments affected assimilation and retention of the 13C-label, anova was used. Block was included as a non-interacting factor. To assess how the treatments affected the rate of change in 13C-label respiration, we used linear mixed-effects modelling. For this approach, fixed effects were time (continuous variable) and soil community treatment (categorical variable). To identify the unit of repeated measurement across time, the random effects were defined as chamber nested within block.
The linear mixed-effects model structure was used to determine how the treatments affected the utilization of the 13C-label by the microbial biomass C and Collembola pools. This model structure was unable to cope with missing values for the Nematoda, Enchytraeidae and Acari pools. Given that (as with the Collembola) there were no apparent time effects in these data, we dropped time from the model structure and analysed these pools using mean values across time for each replicate (either the mean value across all three time points, or only two time points when there was a missing value). All data were natural log-transformed prior to analysis. We follow the convention of reporting significance assuming α < 0·05. We report significance at one of three levels: P < 0·05, 0·01 or 0·001; for marginally significant (P < 0·1 but ≥ 0·05) results we report the actual P value. Following convention, we report non-significant effects as P > 0·05, but note that in all such instances the actual P value was ≥ 0·1.
Treatment effects on utilization of 13C-label by the biota pools can be assessed in three different ways, each defined by how the 13C-label values for a pool are expressed. The 13C-label mass values can be expressed (i) in terms of absolute mass of 13C-label; (ii) as their absolute mass relative to the 13C-label mass assimilated by the plant community; and (iii) as their absolute mass relative to the total mass of C in a biota pool. The second expression (mass relative to that assimilated by the plant community) gives information similar to the first expression (absolute mass), except that differences in 13C-label assimilation are corrected for. The first expression is relevant because, in contrast to experiments where a set amount of 13C-label is applied, the community treatments can influence the absolute uptake of the 13C-label. The third expression (relative to the total mass of C in a biota pool) permits assessment of whether the amount of C acquired by the pool that is derived from recent photosynthate, relative to that acquired from other substrates, is affected.