In most forests, fungi are key players in the below-ground cycling of nitrogen (N) and carbon (C), with saprotrophic fungi mainly decomposing surface litter and woody debris and ectomycorrhizal fungi more active in organic and mineral horizons below the litter layer (Lindahl et al., 2007). Yet information on how ectomycorrhizal fungi differ in exploration for N is scarce. In one approach that may provide some insight into how ectomycorrhizal taxa differ in their N acquisition strategies, Agerer (2001) proposed that ectomycorrhizal fungi could be classified based on the extent and method of hyphal exploration of the soil, with fungi possessing hydrophilic ectomycorrhizas usually classified into contact, short-distance, or medium-distance smooth exploration types, and fungi with hydrophobic ectomycorrhizas generally classified into medium-distance fringe and long-distance exploration types. Peay et al. (2011) proposed that these exploration types may correlate with strategies for acquiring C from roots, with medium- and long-distance exploration types more likely to colonize roots at greater depth or further from trees than short-distance exploration types. Exploration types may be adapted for specific N forms, with taxa with hydrophobic ectomycorrhizas focused on insoluble forms of N such as protein or chitin, and taxa with hydrophilic ectomycorrhizas focused on soluble forms of N (Trudell et al., 2004; Hobbie & Agerer, 2010; Lilleskov et al., 2011).
Nitrogen isotopes (15N : 14N, expressed as δ15N) have been a useful tool in exploring fungal functioning. For example, several studies have demonstrated that saprotrophic fungi are lower in δ15N than ectomycorrhizal fungi, presumably for two reasons: saprotrophic fungi assimilate primarily wood-derived or litter-derived N (15N-depleted) and ectomycorrhizal fungi assimilate deeper soil N (15N-enriched) (Kohzu et al., 1999; Hobbie, 2005); ectomycorrhizal fungi transfer 15N-depleted N to their host plants, leading to 15N-enriched fungal biomass (Hobbie & Colpaert, 2003). Hobbie & Agerer (2010) observed that δ15N patterns in sporocarps correlated with hydrophobicity of ectomycorrhizas, with hydrophobic exploration types c. 3‰ higher in 15N than hydrophilic exploration types. Soil δ15N increases with depth (Billings & Richter, 2006; Hobbie & Ouimette, 2009), and Agerer et al. (2012) correlated hyphal exploration depth in Ramaria taxa with fungal δ15N. The higher δ15N in hydrophobic than in hydrophilic ectomycorrhizal fungi may accordingly indicate that hydrophobic taxa are active at deeper depths than hydrophilic taxa.
One way to assess whether source N or transfer of 15N-depleted N controls fungal δ15N is to use 15N labeling to generate data that can be compared against natural abundance patterns. 15N labeling should change the δ15N of N sources but not change 15N effects of internal partitioning and N transfer to host plants. Therefore, by comparing natural abundance and tracer results, differences in the source δ15N of different fungal taxa can be estimated, because we can assume that the 15N enrichment (ε) between sporocarps and source N is the same in both cases. This information can then be used to estimate the 15N enrichment between sources and sporocarps and the probable sources for sporocarp N. Although tracer 15N labeling studies have also been useful to study N dynamics in many terrestrial ecosystems (Currie et al., 1996; Hofmockel et al., 2011; Wang & Macko, 2011), 15N labeling has yet to be examined systematically in sporocarps of different fungal taxa, despite the potential insights into both fungal functioning and the interpretation of natural abundance δ15N.
Here, we used information on natural abundance and tracer 15N patterns in sporocarps and other ecosystem pools at the Duke free air CO2 enrichment (FACE) study in a Pinus taeda forest. Although we report differences in some cases between elevated and ambient CO2 treatments in δ15N of different pools, we do not focus on treatment effects of elevated CO2. Instead, we use the 15N-applied label to provide general insights into fungal functioning that are unavailable from studies solely at natural abundance.
At the Duke FACE site, the forest floor was labeled with tracer concentrations of 15N in 2003. Ecosystem samples were collected in 2003, 2004, 2005, and 2010, allowing the 15N label to be tracked over time as it was assimilated by biota and migrated from surface to deeper horizons. In soil collected from 2003 to 2005, 15N labeling patterns were initially high in surface layers and then decreased over time, while increasing over time at greater depths (Hofmockel et al., 2011). We suggest that fungal 15N labeling patterns will reflect two factors: the δ15N of the soil and litter pools from which fungi obtain N; and 15N enrichment between sources and sporocarps arising from 15N and 14N partitioning within fungi. Therefore, measuring fungal δ15N at two different time points under changing background concentrations of 15N labeling allows the second factor to be accounted for and can improve estimates of the soil horizons from which fungi acquire N (Fig. 1). This can be expressed mathematically in the following equations, where δ15Nft1 and δ15Nft2 are the fungal signatures at time t1 and t2, δ15Nst1 and δ15Nst2 are the source signatures at time t1 and t2, and ε is the 15N enrichment between sources and sporocarps.
Multiplying both sides by (1 + δ15Nst1) and solving for δ15Nft1 we get:
A similar equation can be written for the fungal signature at time t2:
By measuring isotopic patterns in ecosystem pools and fungi at the Duke FACE site, we tested the following hypotheses:
- Saprotrophic fungi will generally use N sources found close to the surface and ectomycorrhizal fungi generally use deeper N. Ectomycorrhizal fungi with hydrophobic ectomycorrhizas will use N from deeper depths than taxa with hydrophilic ectomycorrhizas.
- Both source differences and 15N enrichment relative to sources contribute to 15N differences among different fungal taxa, with 15N enrichment relative to sources greater for ectomycorrhizal fungi than for saprotrophic fungi.