2.1 Study sites and sampling design
Study sites were the same as reported in Ibañez et al. (2021), distributed in two locations in the South‑West of the Iberian Peninsula: Doñana Natural Park (DN, 37° 15' 34" N, 6° 19' 55" W, 30 m a. s. l.) and Sierra Morena mountains (SM, 37° 39' 50" N, 5° 56' 20" W, 296 m a. s. l.). Both locations have Mediterranean climate regime with warm, dry summers, and mild winters (Peel et al. 2007). However, SM is slightly cooler and wetter than DN, with a mean annual temperature in SM of 16.8 ºC and in DN of 18.1 ºC, and a mean annual precipitation in SM of 648 mm and in DN of 543 mm. Grasslands in both locations are dominated by herbaceous annual species, including grasses, forbs, and legumes. Both locations are extensively grazed at similar stocking rates: DN grazed by goat and cattle (0.40 livestock units (LSU) ha−1), and SM by cattle and Iberian pigs (0.36 LSU ha−1).
Study plots were selected according to their tree composition, with representative canopy types of Iberian wood pastures (Costa Pérez et al. 2006). One pure Q. ilex stand, in the SM location (SM-ilex), and one pure Q. suber stand in the DN location (DN‑suber), both the most abundant stands in the Iberian Peninsula; one Q. ilex and Q. suber mixed stand (DN‑mixed), the next most abundant stand; and a pure Pinus pinea L. stand (DN‑pinea), a common tree plantation replacing traditional canopies (Costa Pérez et al. 2006).
Field work was carried out in spring (2016//04/05 – 2016/04/10), coinciding with the most productive moment of the system. Study treatments were therefore established according to plot (SM‑ilex, DN-mixed, DN‑suber and DN‑pinea), and canopy (open grassland, OG, and under the canopy, UC). At each treatment level we sampled 4 replicates, resulting in 40 sampling points, sampling aboveground biomass (Section 2.2), belowground biomass and soil (Section 2.3). In the DN‑mixed plot we discriminated between both Quercus species (Q. suber and Q. ilex) to establish sampling points. However, we performed preliminary comparative analysis in the DN‑mixed plot on environmental and vegetation characteristics under the canopy of both Quercus species and no relevant differences between Quercus species were found. DN-mixed results are then always presented combining both tree species.
2.2 Aboveground biomass sampling
At each sampling point we sampled same dominant species of each PFT including grasses (Bromus hordeaceus L. and Hordeum vulgare L.); forbs (Calendula arvensis L., Chamamelum mixtum L, Crepis capillaris L., Erodium moschatum L., and Geranium molle L.); and legumes (Ornithopus sativus Brot. and Trifolium subterraneum L.). Also, PFT composition from the same study plots (Ibañez et al. 2021b) was used to interpret and discuss our results.
2.3 Soil and belowground biomass sampling
Two soil cores of 9 cm2 surface and 0 – 10 cm depth were extracted at each sampling point. In the laboratory, one of the cores was processed for soil analysis, and the second core washed and filtered with a 0.2 mm pore size strainer to obtain belowground biomass (BGB).
2.4 Determination of carbon and nitrogen content and isotopic natural abundance
All collected samples, including soil, BGB, and the herbaceous layer sorted by PFT — grasses, forbs, and legumes —, were oven dried at 60ºC until constant weight, powdered with a ball mill (MM200, Retsch, Asturias, Spain) and tin capsuled (Courtage Analyse Service, Mont Saint-Aignan, France). For determining C content and δ13C in all our samples, except soils, we used glutamic acid and acetanilide laboratory standards, both calibrated with USGS40 international reference material. For determining the percentage of N and δ15N an additional standard was also used (N1), calibrated using IAEA-N1. Samples were prepared in the Institut de Biologie des Plantes (http://www.ips2.u‑psud.fr). Afterwards, samples were analysed in the Isolab of the Grassland Sciences group at ETH Zurich (http://www.gl.ethz.ch/), with a Flash EA 1112 Series elemental analyser (Finnigan MAT, Bremen, Germany), coupled to a DeltaplusXP isotope ratio mass spectrometer (Finnigan MAT, Bremen, Germany) via a 6-port valve (Brooks et al. 2003) and a ConFlo III interface (Werner et al. 1999).
For determining C content and δ13C in the soil, we used acetanilide and caffeine as standards, both calibrated using glutamic acid from IAEA, USGS40, and NBS-22. In determining the percentage of N and δ15N in the soil, tyrosine was also used as standard calibrated using IAEA-N1. Soil samples were prepared and analysed in the Isolab of the Grassland Sciences group at ETH Zurich, as described above.
Isotopic composition (δ13C and δ15N) were both calculated as deviation of the isotope ratio (R = 13C/12C or R = 15N/14N) of the samples from the ratio of the corresponding international standard (δ = (Rsample / Rstandard) − 1), VPDB for δ13C, and air-N2 for δ15N (Coplen 2011).
2.4 Data analysis
All data analyses were performed using R software (R Core Team 2019). We ran linear models on C and N content and isotopic composition (δ13C, δ15N) of each ecosystem compartment as function of plot (SM‑ilex, DN-mixed, DN-suber, DN-pinea), and canopy (OG, UC). Final models were selected by a stepwise procedure based on the Akaike information criterion (AIC), using the stepAIC function, MASS package (Venables and Ripley 2002). Linear models were also applied to explore the relationships between C and N dynamics within or between ecosystem compartments when applicable. Finally, we assessed differences among PFT on the C and N content and isotopic composition (δ13C, δ15N) by one way ANOVAs, and tukey post‑hoc tests, using the HSD.test function of the agricolae package (Mendiburu 2017). Only significant models (p < 0.05) are presented and discussed, selecting the most explanatory and parsimonious ones.