Study area

This study was carried out at a high flat land (moor) of a calcareous geology in the Castilla y León region (Spain). Under semiarid climate, the native vegetation is holm-oak forest (Quercus ilex subsp ballota). However, during the nineteenth century most of these forests were converted into agricultural land, some of which remain until now. During the 1950s afforestation with Pinus halepensis was carried out on the less dense forest areas and in abandoned agricultural lands. Therefore, we selected these three types of land-use in these calcareous moors.

Soil sampling

Three plots were used. In each plot, three adjacent points of the three different land uses studied (agricultural, pine forest and natural oak forest) were selected. Four depth levels were considered (2-5 cm, 10-15 cm, 20-30 cm and 30-40 cm) and sampled at one sampling point randomly selected along the experimental area.

Soil analysis

Sieved soil samples (<2 mm) were analysed. Soil pH was measured in a 1:2.5 soil:water suspension using a glass electrode. Total organic C (TOC) and total N were determined using a LECO CHN 2000 Analyzer. Olsen P was also determined.

Phosphorus-31NMR was used to characterize the structural composition of alkali-soluble P (^{Turrión et al., 2010}). Phosphorus was determined in all the extracts by the ammonium molybdate-ascorbic acid method (^{Murphy & Riley, 1962}). The ³¹P-NMR spectra were recorded with a Bruker DPX-300 spectrometer operating at 121.49 MHz using a 90-degree pulse length, an acquisition time of 0.96 s and a total accumulation time of 2 h and 3800 scans.

Statistical analysis

A two fixed-factor factorial experimental design was considered. The first factor was the land use effect on soil properties (agriculture, pine forest and holm oak forest), while the second factor was the soil depth effect, with four samplings along the profile: 2-5 cm, 5-10 cm, 20-30 cm and 30-40 cm, including also the interaction between factors. General linear mixed models were fit with PROC MIXED procedure in SAS software.

Land use effect on soil properties

Significant effects of land use and depth were observed in all soil parameters considered, while land use by depth interaction was not significant for anyone (Table 1). Total organic carbon concentration (TOC) in soil decreased significantly with depth and was higher in the natural oak forest. Total nitrogen concentrations (N) were lower in the agricultural land and pine forest than in oak forest soils at all depths studied decreasing also with depth (data not shown). The pine forest soil showed significantly higher C/N ratio than soils from the other two land uses. Depth was also significant (Table 1), being this ratio lower on the surface than in deeper soil layers in all land uses.

Soil P in the studied land-uses

Total P concentrations in the NaOH-EDTA extract of the agricultural soil ranged from 16.8 to 101.1 mg*kg⁻¹, whereas they ranged from 22.9 to 37.6 mg*kg⁻¹ and from 9.42 to 34.03 mg*kg⁻¹ in oak and pine forest soils, respectively. In the agricultural soil, total inorganic P represented around 75% of total P in the first layers of the profile, decreasing in the oak and pine forest soils to around 20%. However, total organic P mainly represented the total P of this alkaline extract in oak and pine forest soils, representing around 83% of total P in the first layers of those profiles, but just 30% of total P in the first layers of the agricultural soil.

Our results indicate that only a small amount of P remains in the soil after this sequential extraction, confirming previous works suggesting that most of the total P is recovered by EDTA-NaOH extraction for soils (^{Turner et al., 2005}). Labile P forms concentrations (AEM-P and Olsen-P) were significantly higher in the agricultural soil (data not shown), with the greatest effects of land use in the surface soil layers (2-15 cm).

Inorganic orthophosphate represented around 62% (at 2-5 cm) of total P extracted by NaOH-EDTA in the agricultural land soil and around 20% (at 2-5 cm) in pine and oak forests soils. Phosphate monoesters was the main functional class of Po in soils from all land uses, representing 32%, 68%, and 64% of Po in the surface samples in the agricultural land, pine forest and oak forest, respectively. Moreover, monoesters concentrations decreased significantly with depth in all the land uses (P<0.001). Phosphate diesters include nucleic acids (DNA and RNA), phospholipids, and teichoic acids, and they represented between 5 to 12% of the total P extracted by NaOH-EDTA in the studied soils. Phosphate diesters usually constitute less than 10% of soil Po, but higher proportions have been detected in some forest soils (Cade-Menun et al., 2000). Diester P is more labile and accessible to microbial and enzymatic attack compared with monoester P (^{Zhu et al., 2013}) and also, diester dominates Po inputs to soil from plants and microbes (^{Turner et al., 2005}). Both reasons can explain the lower amount of diaester P observed in all the studied land use systems comparing to the monoester P forms. DNA-P concentrations were similar and low in all depths studied and in all the land uses. It is widely accepted that an increase or decrease in the proportion of diester P in soils is associated with corresponding changes in microbial P compounds (Cade-Menun et al., 2017). In contrast, easily mineralizable phosphate diesters in soils can accumulate when conditions for microbial decomposition become limited by climate (^{Makarov et al., 2002}), or by acid soil pH and high content of free Al (^{Turrión et al., 2000}), but this is not the case of the studied soils. Pyrophosphate-P was also very similar among land uses (not significant), but significant differences were observed with depth (P<0.05) (Table 2).