Summary

1. Research has indicated that increases in nitrogen (N) deposition can greatly affect ecosystem processes and functions. There is limited information about the effects of long-term N addition on soil nematodes and their functional composition, although nematodes are the most abundant multicellular animals on Earth.
2. We conducted a field experiment in 2004 with four levels of N addition (0, 60, 120, and 240 kg N ha^-1 yr^-1) in a subtropical Cunninghamia lanceolata forest. Soil samples with three depths (0–20, 20–40 and 40–60 cm) were collected and the community structure, diversity and trophic groups of soil nematodes were determined in 2014.
3. N addition significantly increased the abundance of bacterial- and fungal-feeding nematodes, but decreased the abundance of plant-feeding nematodes at the 0-20 cm soil layer. Accordingly, the plant parasite index and enrichment index decreased but the basal index and channel index increased, which weaken the importance of the plant-based energy channel, but enhance the importance of the fungal-based energy channel. N addition had no effects on the diversity of soil nematodes in three soil depths. Structural equation modeling analysis indicated that N loading directly changed plant-feeding (total r²=0.42) nematodes, or indirectly affected bacterial- (r²=0.43), fungal- (r²=0.31) and plant-feeding nematodes via change soil nutrients, soil water content and pH.
4. These findings suggest that N addition can change the community structure and energy channels of soil nematodes, which would affect soil processes and food web functions in forest soils under future environmental change scenarios.

Experimental design

In December 2003, 12 experimental plots were established in a 6-ha section of the Chinese fir plantation. Each plot was 20 m × 20 m and had similar site conditions. In each plot, a central area of 15 m × 15 m was designated for treatments, arranging one of four levels of N. The treatment codes N0, N1, N2, and N3 indicated the loading of 0, 60, 120, and 240 kg N ha^-1 yr^-1, respectively. Each treatment was represented by three randomly assigned replicate plots. Urea [CO(NH₂)₂] was used as the N source, and the required quantity was dissolved in 20 L of water per plot. From January 2004 to January 2014 (when plots were sampled), 20 L of N solution was sprayed onto the soil surface in the central area (15 m × 15 m) of each plot once per month. Control plots (N0) were sprayed with the same volume of water without CO(NH₂)₂.

Soil sampling and the extraction and identification of nematodes

In January 2014 (after 10 years of N application), soil cores were collected from each plot (the treated central area) at 0–20 cm, 20–40 cm, and 40–60 cm soil depths. In each plot, five soil cores (3 cm in diameter) at each depth were randomly taken and combined to form one composite sample per depth per plot. The litter layer at the sample locations was removed before cores were collected. There were 36 soil samples (3 depths × 12 plots) in total.

After visible roots and stones were removed, the soil samples were passed through a 2-mm-mesh sieve and then stored at 4 °C for chemical analysis and nematode extraction. Soil water content (SWC, %, g of water per 100 g dry soil) was measured by comparing weights before and after oven-drying at 105 °C for 24 h, and soil pH was determined in a 1:2.5 (w/v) soil suspension. Concentrations of total N (TN, g/kg dry soil) and total phosphorus (TP, g/kg dry soil) were measured. Total C (TC, g/kg dry soil) was determined using an elemental analyzer (Perkin Elmer Instruments series II, USA). Contents of soil nitrate (NO₃⁻-N, mg/kg fresh soil) and ammonium nitrogen (NH₄⁺-N, mg/kg fresh soil) were determined. Available P (AP, mg/kg dry soil) was extracted with a sodium bicarbonate solution (0.5 M, 30 min extraction), and the extracted phosphate was then quantified by the molybdenum blue procedure. The response variables for soil total C, total N and total P; for NO₃⁻-N, NH₄⁺-N and available P were indicated by principal component scores (both PC1 scores were employed), which responded to the PCT and PCA in the data figure, respectively.

Nematodes were extracted from a 100-g subsample of fresh soil from each composite soil sample with Baermann funnels. The extracted nematodes were collected in a 4% formalin solution, and then counted and identified with a DIC microscope (ECLIPSE 80i, Nikon). The first 100 individuals encountered were identified to the genus or family level and classified into four trophic groups, including bacterial-feeding nematodes (BF), plant-feeding nematodes (PF), fungal-feeding nematodes (FF) and omnivorous-predatory nematodes (OP).