Depth distribution of fungal activity
Because supplies of energy-rich C are largest in the surface litter, saprotrophic fungi are commonly found there and should obtain N from those sources (Lindahl et al., 2007). By contrast, ectomycorrhizal fungi acquire labile C from plant sugars, and therefore have a relative advantage over free-living heterotrophs in nutrient acquisition as the energy content of organic matter declines at deeper depths. Ectomycorrhizal fungi in the surface litter may rely on enzymatic breakdown by free-living saprotrophic microbes (Lindahl et al., 2005), whereas those active at greater depths presumably rely on their own enzymatic capabilities. Within ectomycorrhizal fungi, the enzymatic capabilities to acquire N from polymeric substances such as protein and chitin appear greater for hydrophobic than for hydrophilic taxa (Lilleskov et al., 2011). Accordingly, taxa with hydrophobic ectomycorrhizas should acquire N from greater depth and, because soil δ15N increases with depth, be higher in δ15N than taxa with hydrophilic ectomycorrhizas.
To assess whether different fungal groups acquired soil N from different depths, we compared natural abundance δ15N in fungal taxa in 2004 with soil in 2003 and compared fungi with soil from 15N-labeled plots sampled in 2003–2005 and in 2010 (Table 1, Fig. 5). In apparent support of our first hypothesis, fungal δ15N patterns at natural abundance were at their lowest in saprotrophic fungi, intermediate in hydrophilic ectomycorrhizal fungi, and at their highest in hydrophobic ectomycorrhizal fungi, presumably reflecting the use of N from shallow litter horizons or wood by saprotrophic fungi and two taxa of hydrophilic ectomycorrhizal fungi (Lactarius and Russula), and assimilation of deeper soil N by both hydrophilic (Laccaria and Amanita) and hydrophobic ectomycorrhizal taxa.
After plots were labeled with 15N in 2003, δ15N values were initially very high in the leaf litter (c. 350‰), but quickly declined (c. 50‰ by 2004) as new litter of low δ15N was added, 15N-labeled litter decomposed, and its N was incorporated into the Oea horizon (Table 1). In 2004, the year following application of the tracer, fungal δ15N followed expected patterns, with δ15N of saprotrophic fungi > hydrophilic ectomycorrhizal fungi > hydrophobic ectomycorrhizal fungi, again reflecting use of shallow soil N for saprotrophic and hydrophilic ectomycorrhizal fungi and use of deeper soil N for hydrophobic (and some hydrophilic) ectomycorrhizal fungi. However, Russula, Lactarius, and most of the saprotrophic fungi appeared to at least partially sample the 2003 litter cohort, as their δ15N values in 2004 in 15N-labeled plots were higher than any measured bulk pool for that year but lower than Oi values for 2003. Alternatively, DON and microbial N may have supplied N to these fungi, as they generally had similar δ15N values as the DON and microbial N in the Oea horizon in 2004 (Fig. 3); unfortunately, DON and microbial biomass were not measured in the Oi horizon, so precise comparisons are not possible. In contrast to these patterns, the hydrophilic taxa Inocybe, Amanita, and Laccaria had lower δ15N values than DON or microbial N from the Oea horizon, suggesting a possible contribution from mineral horizon N (Fig. 4, Table 1).
Finally, in 2010, 7 yr after 15N tracer was applied, soil and fungal δ15N patterns had changed, with the applied 15N migrating deeper down the soil profile. Between 2004 and 2010, fungal δ15N decreased more in the saprotrophic Rhodocollybia and in the hydrophilic Lactarius and Russula than in the hydrophobic Cortinarius and the hydrophilic Amanita and Laccaria. This indicates that these latter two taxa assimilated deeper N than Lactarius and Russula but did not increase in 15N relative to sources as much as other ectomycorrhizal taxa. These patterns were consistent with saprotrophic fungi and some hydrophilic ectomycorrhizal fungi using shallow soil N. In addition, the hydrophobic Tricholoma in 2010 was also high in δ15N, suggesting that taxa with hydrophobic ectomycorrhizas generally explore deeper horizons. High δ15N in Tricholoma have been reported before and attributed to high 15N enrichment between the mycelia and sporocarps (Zeller et al., 2007); here, our results indicate that acquisition of deeper, 15N-enriched N partially explains the high δ15N values of Tricholoma sporocarps.
To infer potential N sources, we paired δ15N measurements in 2004 at natural abundance with 2010 measurements from the FACE rings (Fig. 5). We assumed comparable 15N enrichment between sources and fungi for both sampling times. Lines of constant 15N enrichment for 2010 relative to 2004 indicate the probable δ15N at natural abundance of the source N, which could then be compared against the measured bulk soil pools. Based on high 15N labeling patterns in 2010 and the assumed similar 15N enrichment relative to sources in 2004 (natural abundance) and 2010 (15N tracer applications), Amanita, Cortinarius, and Laccaria acquired N from deeper in the soil profile (where 15N is higher) than Lactarius, Russula, and the litter-inhabiting Rhodocollybia. According to Eqn 4, the δ15N differences from 2004 to 2010 between sources and between sporocarps are not exactly equivalent, as they are related by the factor (1 + ε). However, if the estimated 15N enrichment value (ε) is 20‰, then they would only differ by 0.2‰ in 15N enrichment.
High 15N enrichments (between 25 and 32‰) of 15N-labeled 2010 values relative to natural abundance values in 2004 for Amanita and Cortinarius (as indicated by isolines on Fig. 5) are higher than any estimated soil pool enrichment. This implies that these two taxa access N pools that have retained more 15N than any measured bulk soil pool. Although additional pools other than bulk were not measured in 2010, the δ15N values in 2004 in the FACE plots for DON and microbial N were c. 30‰ higher than concurrently measured bulk values, indicating that pools such as these could potentially be similarly elevated relative to bulk pools in 2010. Such differences in δ15N between bulk pools and other soil fractions have previously been reported from 15N labeling experiments (Zeller & Dambrine, 2011), and presumably reflect variability in turnover times of different biochemical or biophysical fractions.
Resource partitioning in saprotrophic fungi
The saprotrophic taxa colonized different substrates, with Baeospora myosura commonly colonizing conifer cones (Rayner et al., 1985), Gymnopilus, Pholiota, Pluteus, and Oligoporus commonly colonizing wood (Pouska et al., 2010), and Mycena, Rhodocollybia, Marasmius, and Clitocybe commonly colonizing litter (Rayner et al., 1985). Baeospora and the wood decay fungi were similar in natural abundance δ15N (Fig. 2), suggesting that these taxa are using N of similar δ15N, with that N presumably relatively unprocessed and drawn from undecayed pine cones or wood. However, we point out that the litter decay fungi Clitocybe and Marasmius had similar values to Baeospora and the wood decay fungi in the regression model that included %N (Table 2). Some of the δ15N variability among taxa should accordingly be driven by variable proportions of 15N-enriched protein and 15N-depleted chitin, with higher %N and δ15N presumably correlating with higher relative protein content (Hobbie et al., 2012).
Pine cones closely matched Baeospora in 2004 (natural abundance), with a measured 15N enrichment in Baeospora relative to pine cones of 1.4‰. Higher δ15N values for the litter decay fungi Rhodocollybia and Mycena than for Baeospora, wood decay fungi, and the other two litter decay taxa suggest some degree of 15N enrichment during initial stages of decay of litter and subsequent acquisition of this N by Rhodocollybia and Mycena. The relatively high δ15N in Ramariopsis may indicate foraging for N in deeper soil horizons than other saprotrophic taxa. Whereas the other saprotrophic taxa feed on wood or fresh litter, Ramariopsis kunzei is typically reported as fruiting on ground, often buried below the surface litter (Arora, 1986), implying that it obtains N from deeper sources than fresh litter.
Depth distribution of ectomycorrhizal fungi and comparisons with other studies
Studies that have examined the depth distribution through soil profiles of ectomycorrhizal fungi can provide additional insights. In general agreement with the patterns reported here, some studies detected taxa of Lactarius and Russula in litter horizons or coarse woody debris, with Amanita, Cortinarius, Inocybe, and Laccaria found in deeper horizons (Dickie et al., 2002; Landeweert et al., 2003; Tedersoo et al., 2003). Although patterns were not as clear in other studies (summarized in Table S5), we note that fungal presence is unlikely to correlate precisely with fungal N source. For example, the density of the N resource will generally be greater in organic horizons than in mineral horizons, and therefore N uptake per unit of fungal biomass may be greater in organic horizons.
Some indication of the greater spatial extent of exploratory hyphae in taxa with hydrophobic vs hydrophilic ectomycorrhizas can be inferred from the study of Genney et al. (2006), in which fungal identity of both colonized root tips and of extraradical hyphae were assessed. In this study, 88% of the Cortinarius observations were of extraradical hyphae, compared with only 31% of the Lactarius rufus observations and 34% of the Cenococcum geophilum observations. Cortinarius has hydrophobic ectomycorrhizas, whereas Lactarius and Cenococcum have hydrophilic ectomycorrhizas. Similarly, a reanalysis of data in Peay et al. (2011) from Pinus-associated communities at forest edges and interior locations (10 m inside the edge) for the genera found at Duke FACE indicates that 77% of hydrophilic ectomycorrhizas were found at interior locations, compared with only 46% of hydrophobic ectomycorrhizas, with interior locations presumably associated with less extensive extraradical hyphal development than edge locations. As suggested in Hobbie & Agerer (2010), the apparently greater spatial exploration in hydrophobic than in hydrophilic taxa may lead to higher relative sequestration of 15N-depleted chitin in transport structures (extraradical hyphae) and the formation of 15N-enriched N within fungi that is metabolically available for sporocarp formation, therefore contributing to 15N enrichment of hydrophobic relative to hydrophilic taxa.
We conclude that saprotrophic fungi primarily assimilate N found in the Oi horizon (with the possible exception of Ramariopsis), and fungi with hydrophobic ectomycorrhizas primarily assimilate N from deeper horizons such as the Oea, supporting Hypothesis 1. However, taxa with hydrophilic ectomycorrhizas appeared to assimilate from the Oi horizon in some cases (e.g. Russula and Lactarius) and in other cases from deeper horizons (e.g. Amanita and Laccaria), which only partially supports Hypothesis 1. These differences in N acquisition largely corresponded to N isotope patterns at natural abundance.
Estimated 15N enrichment relative to bulk substrates was in the order: Lactarius > Russula > Rhodocollybia > Baeospora, and Cortinarius > Amanita. It appears that 15N enrichment relative to source is variable among taxa, and therefore contributes, along with source δ15N, to 15N differences among taxa in sporocarps, which supports Hypothesis 2. Higher δ15N values in some fungi than in any measured ecosystem pool suggest that fungi access N pools that are not captured in the relatively gross characterizations of litter and soil δ15N distributions presented here. In previous work, 15N enrichments have been analytically linked to partitioning of N between plants and fungi, within different fungal structures, such as extraradical hyphae vs sporocarps, or between protein and chitin (Hobbie et al., 2005, 2012). The results here suggest one approach to generate the data needed to test these analytical approaches.