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Since the 1960s, the rate of reactive nitrogen (N) released to the environment by human activities has increased immensely (Galloway et al., 2003). This has led to increased productivity in forest ecosystems, but is also of major environmental concern in terms of the leaching of nitrate into ground and surface water reserves and negative impacts on biodiversity in natural habitats, including reduced production of fruit bodies of ectomycorrhizal (EcM) species (Arnolds, 1991).
EcM fungi form symbioses with tree roots and feed host trees with N taken up from the soil in exchange for photosynthetically derived carbon (C) (Smith & Read, 2008). Normally, > 90% of the nutrient-absorbing roots of EcM trees are colonized by their symbiont fungi (Taylor et al., 2000), and most nutrients, including N, therefore enter the plant via the fungal pathway. Nitrogen may be taken up from the same pool as accessed directly by plant roots, that is, ammonia, nitrate and, to some extent, amino acids (Persson & Nasholm, 2001), but, complementarily, many EcM fungi can also mobilize N from sources not directly available to plant roots, for example peptides, proteins or other organic N sources, such as chitin (Lindahl & Taylor, 2004). As N often is the major limiting nutrient in temperate and boreal forests (Tamm, 1991; Hyvönen et al., 2008), this contribution by the fungal symbionts may be crucial for tree growth under such conditions.
In the field, elevated N deposition or fertilization affects the biomass and community composition of EcM fungi. It has been recognized that the production of fruit bodies of EcM fungi is depressed by increased N deposition (Arnolds, 1991; Termorshuizen, 1993; Wallenda & Kottke, 1998). Below ground, species richness on root tips is also affected, although typically not as dramatically as above ground (Taylor et al., 2000; Lilleskov et al., 2002a,b; Avis et al., 2003; Toljander et al., 2006). Significant changes in fungal community composition with increased N availability seem to be the rule (Fransson et al., 2001; Lilleskov et al., 2002a,b; Avis et al., 2003; Toljander et al., 2006; Parrent & Vilgalys, 2007). Indeed, N availability has been shown to be a major determinant of EcM communities across north-western Europe (Cox et al., 2010). In a series of studies, Lilleskov et al. (2001, 2002a,b) demonstrated how the community changed along an N deposition gradient. In short, the results showed that, the higher the N deposition, the higher the dominance of species presumably using inorganic N sources successfully (nitrophilic species), and vice versa. Similar results were found by Taylor et al. (2000) with fungi isolated along a long-distance north–south European transect. The general trends among studies have been a decrease in Cortinarius spp. (Lilleskov et al., 2002a,b; Avis et al., 2003; Toljander et al., 2006), whereas specific Lactarius or Russula species increased with increased N loads (Lilleskov et al., 2002a,b; Avis et al., 2003).
Nitrogen availability influences the production of EcM root tips and external mycelia. Under extreme N-limited conditions, fungal biomass may increase as a response to N addition (Clemmensen et al., 2008), but, in general, EcM biomasses of both roots tips and, especially, external mycelia are reduced (Wallander & Nylund, 1992; Arnebrant, 1994; Wallenda & Kottke, 1998; Fransson et al., 2001; Nilsson & Wallander, 2003; Nilsson et al., 2005, 2007; Ostonen et al., 2011).
The objective of this study was to concurrently monitor the effects of N deposition on EcM fungi – such as changes in mycelial production, root tip abundance and community composition – along a stand-scale N deposition gradient. We took advantage of a forest edge-generated N gradient from high N deposition at the edge to two to three times lower N deposition in the interior forest. Deposition effects at forest edges are well described (e.g. Beier & Gundersen, 1989; Wuyts et al., 2008), and these serve as excellent experimental systems because the gradients are short (within 100 m) and steep, thereby limiting the variation in other site-related parameters. The study was performed as part of a larger research program monitoring N deposition effects on C and N storage and dynamics in forests. This enabled a unique possibility to co-analyze changes in mycorrhizal abundance and community structure with a wide range of environmental parameters. We expected that increased N availability driven by the N deposition gradient would decrease EcM mycelial production, EcM root tip abundance and EcM fungal diversity, as well as change the EcM fungal community structure and functioning.
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This study demonstrates, for the first time, concurrent changes in EcM community composition, mycelial production and root tip number along an N deposition gradient in a spruce forest edge. Previously, such effects have been reported in studies measuring only one or two of the above-mentioned mycorrhizal parameters (Wallenda & Kottke, 1998; Lilleskov et al., 2002a,b; Nilsson & Wallander, 2003; Nilsson et al., 2005; Cox et al., 2010). Nevertheless, the present study is in agreement with previous studies. Mycelial reductions were observed with increasing N availability from deposition or fertilization (Wallander & Nylund, 1992; Nilsson & Wallander, 2003; Nilsson et al., 2005, 2007, 2012), and this was also the case for root tip number (Wallenda & Kottke, 1998). Furthermore, community changes and lower species richness seem to be general with increasing N loads (Lilleskov et al., 2001, 2002a,b; Avis et al., 2003; Cox et al., 2010).
The mycelial response to the increased N deposition near the forest edge was far greater than the accompanying decrease in root tips (Fig. 1). Therefore, the decrease in mycelia produced must be explained, at least partially, by other factors. Changes in community composition with inherently different mycelial production may be one explanation for the low mycelial production up to 25 m from the edge. The observed increase in contact exploration types at the expense of short-distance exploration types towards the forest edge supports this hypothesis (Fig. 3). Alternatively, either the N status of the soil or the host trees regulate the mycelial production. Accumulating evidence indicates that the plant N status is a key controller of below-ground C allocation (Lilleskov et al., 2008; Högberg et al., 2010) and thus the availability of C for investment in the external mycelial network. In theory, proximal variables, such as available soil N for fungal uptake or below-ground C allocated to the fungi, should predict mycorrhizal responses better than more distal variables, such as N deposition (Lilleskov & Parrent, 2007). The production of mycelia seemed to decrease at 90 m in comparison with 50 and 75 m. Although nonsignificant, it is intriguing that a lower mycelial production was found when, at the same time, no leaching was present at 90 m. One explanation could be that the species dominating the community at 90 m had a more effective N uptake capacity than those dominating at the other distances. This emphasizes the need for more information on the performance of individual species in order to tie together changes in community composition and function. However, the mesh bags may not accurately record the true EcM mycelial abundance in the soil, as discussed further below.
Six of the nine measured N parameters correlated with one to seven of the mycorrhizal parameters (Table 4). Throughfall N and soil solution NO3-N correlated with both root tip number and the two richness parameters, and the latter correlated with most mycorrhizal parameters. These single correlation analyses are summarized in the ordination analysis in which the same six N parameters correlate with the community data (Fig. 4). Previously, N deposition, soil mineral N, foliar N and root N have been shown to be good predictors of mycorrhizal changes (Lilleskov et al., 2001, 2002a,b, 2008; Toljander et al., 2006; Cox et al., 2010). In our study, extractable mineral N did not correlate strongly with N deposition or soil solution NO3-N, and therefore not with changes in mycorrhizal parameters across the gradient. The peak at 25 m, in particular for extractable NH4-N, was created by one extreme value, which indicates the occurrence of a local hot spot, possibly related to some type of animal interference (data not shown). The reason for the N gradient not being pronounced for all N variables in the bulk soil may be a legacy of the period of lower N deposition, that is, before the farming practices were intensified and the local farm started to produce mink and poultry. In addition, as the stand is relatively young (25 yr), the edge effect on deposition may only have been pronounced for a short period after the trees reached a certain height.
The decrease in T. fibrillosa with increasing N deposition and the tendency of T. asterophora to behave oppositely are in agreement with Toljander et al. (2006), who studied the EcM community composition across a natural nutrient gradient. However, other studies found no such responses of Tylospora spp. to N availability (Jonsson et al., 2000; Lilleskov et al., 2002a,b). Similarly, contradictory results were reported for C. geophilum, which often dominates below-ground EcM communities, including those of spruce forests. Responses to N availability range from an increase (Fransson et al., 2001) to no response (Avis et al., 2003) to indications of preference for low-N sites (Lilleskov et al., 2002a,b; Toljander et al., 2006), the latter being in accordance with this study. An increase in specific Lactarius or Russula species with increasing N availability is probably the most common species response to N reported in the literature (Lilleskov et al., 2002a,b; Avis et al., 2003; Parrent et al., 2006; Cox et al., 2010). The negative correlation between L. quietus and distance to the forest edge therefore corroborates these earlier findings. As L. quietus may be associated with roots from occasional oak trees within the spruce stand, this conclusion must be taken with great caution. Unfortunately, it was not possible to amplify plant DNA from the root tips stored in ethanol in spite of repeated efforts using several different primer systems. The identity of the host in the samples identified as L. quietus therefore remains unknown. The oak regeneration was scattered throughout the gradient, and so we may have had mixed spruce and oak roots in our samples. As oak, like spruce, may host all exploration types, we regard it as probable that the oak-associated community would change in parallel with the spruce-associated community. The correlations between the changes in exploration types, mycelial production and N leaching should, however, still hold true even if we have both spruce and oak roots in our samples. The other dominating fungi are all well-known spruce associates and are often among the most frequent in spruce forests (Jonsson et al., 2000; Fransson et al., 2001; Peter et al., 2001; Toljander et al., 2006), but several species, including the two dominants T. fibrillosa and C. geophilum, are also known from oak.
As exemplified by C. geophilum, there are sometimes contradictory results between studies with regard to the response of single species to N availability. The problem may be caused by differences in the quantitative and qualitative N availability between forest and soil types. This underlines the importance of recording appropriate environmental data in ways that make these numbers available together with the species abundance data for cross-study meta-comparisons (Lilleskov & Parrent, 2007). In addition, the full dose–response to N availability of each species is rarely known and, hypothetically, a species may increase in one study and decrease in another. The use of multi-level experiments or replicated gradient designs is needed to address this, as discussed further by Lilleskov & Parrent (2007) and Cox et al. (2010). Furthermore, Toljander et al. (2006) discussed the problem in using short gradients for community studies, where samples within a certain distance may correlate with each other. We tried to reduce the impact of re-sampling individual fungal mycelia between cores by sampling with 2-m intervals (Lilleskov et al., 2004). Nevertheless, stand replication, as used by Cox et al. (2010), is needed to better unravel the environmental drivers for the EcM community composition observed.
The species recorded from the cloned mycelia were a subset of the root tip dataset. Only a few clones were analyzed and the mycelial production from 0 to 25 m from the forest edge was low. In addition, the mycelial community within the mesh bags may not accurately mirror the actual soil mycelial community. In particular, for example, Cortinarius spp. do not tend to grow into mesh bags even though these species produce abundant external mycelia (Kjøller, 2006). If the mycelial community data were to be analyzed more thoroughly, more clones should have been selected from each mesh bag or, ideally, 454 sequencing technology should have been applied (Wallander et al., 2010). However, this community is strikingly species poor compared with other EcM communities; therefore, less cloning or generation of 454 reads is actually needed to exhaustively sample the species richness. The inclusion of more clones or 454 reads would probably not have changed the percentages of EcM clones obtained along the gradient. An interesting result was that P. involutus, which was only found at low abundance (on root tips) in two soil cores (Table S2), was detected in 16 mesh bags from the entire sampled area (data not shown). Paxillus involutus is a long-distance exploration type within the Boletales, and a similar skewed relation between root tips and mycelia has been seen previously for other Boletales species (Kjøller, 2006). The higher frequency of non-EcM species from the clones of the present study, in comparison with a previous study (Kjøller, 2006), may be explained by the extraction method used. In the former study, mycelia were collected after wet sieving with forceps from the edge of the sieve, whereas, in the current study, mycelia were captured on 1.2-μm filter membranes. The latter method also captures some organic particles that probably will add to the subsequent detection of nonmycorrhizal clones. At the high N deposition of the forest edge, the high frequency of non-EcM species probably reflects the increase in saprotrophic fungi at the expense of EcM fungi.
In conclusion, this study has documented a large negative impact of N deposition on EcM biodiversity and biomass in soil and on roots. In particular, the almost entire loss of external mycelia under high-N conditions should be of concern. The supply of nutrients other than N to the tree may be impaired and, at the same time, N retention and uptake by mycelia in the topsoil are reduced. A lower abundance of N-retaining mycorrhizal mycelia under high N deposition may thus further exacerbate the risk of N losses from forest ecosystems by leaching (Nilsson et al., 2012). Moreover, the mycorrhizal and mycelial changes, via altered carbohydrate flux from above ground, must feed back on important processes, such as soil C sequestration and food web structure. The response of EcM fungi to N should therefore be incorporated into process-based soil C models that aim to incorporate N deposition effects on C dynamics in forest ecosystems.