Author for correspondence: Dr Martina Peter Tel: +41 1 739 21 11 Fax: +41 1 739 22 15 Email:email@example.com
• Changes in above- and below-ground ectomycorrhizal species composition are reported following nitrogen addition for 2 yr to a subalpine spruce (Picea abies) stand.
• The macrofungal sporocarp production was recorded before and during N addition. Belowground ectomycorrhizal diversity was measured by PCR-RFLP analysis of the internal transcribed spacer (ITS) region of rDNA extracted from single mycorrhizal root tips before, and after 1 and 2 yr of fertilization.
• Sporocarp surveys showed that diversity of the ectomycorrhizal community was drastically reduced following 1 yr of N addition, whereas the saprobic fungal community was not affected. The impact on belowground ectomycorrhizal diversity was less pronounced with no change either in the number of ectomycorrhizal taxa or in Simpson’s index of diversity. However, a change in belowground species composition 2 years after N addition was observed with significant changes in abundances of single species.
• Species which produced large sporocarps accounted for 25% of all sampled root tips. At least 44% of all ectomycorrhizas were formed by species belonging to the Thelephoraceae and Corticiaceae, taxa which produce inconspicuous sporocarps.
• Addition of N caused a shift in ectomycorrhizal abundance from species forming large sporocarps to species with no or resupinate sporocarps.
In temperate climates, almost all species of forest trees are ectomycorrhizal. Fungal partners of these symbioses acquire fixed carbon from their photoautotrophic host, but in turn provide the host with nutrients, protect its root system from microbial pathogens, and enhance its drought tolerance (Smith & Read, 1997). Therefore, the ectomycorrhizal symbiosis is a crucial factor in forest tree health. Because this symbiosis is generally regarded as an adaptation to nutrient limited conditions (Read, 1991), the impact of air pollution in industrialized zones, particularly the enhanced deposition of atmospheric nitrogen (N), on ectomycorrhizal fungi is currently a matter of debate. A decline in species richness and abundance of macromycetous sporocarps in Europe in the 20th century has been reported, and ectomycorrhizal species seem to be particularly affected (Arnolds, 1991). Similar trends were observed in long-term inventories of macromycetes in a Swiss forest ecosystem, in which the proportion of ectomycorrhizal species decreased compared to that of the saprotrophs since the 1980s (Egli & Ayer, 1997). Enhanced N deposition was hypothesized to be the main reason for such changes (Arnolds, 1991; Rühling & Tyler, 1991). Several field experiments (overview in Wallenda & Kottke, 1998) investigating the response of the fungal community to N fertilization support this hypothesis, in that most ectomycorrhizal species had reduced sporocarp formation, whereas only minor effects were observed on saprobic species.
Using sporocarp presence as an indicator of species diversity of ectomycorrhizal fungi entails several problems (Watling, 1995): Sporocarp formation varies both in space and time and depends on a range of external factors. Detailed long-term investigations are, therefore, required to record the existing fungal community. But even if this is provided, sporocarp inventories only partly reflect the ectomycorrhizal species composition on root-systems. The lack of sporocarps does not necessarily indicate the absence of a particular fungal species at the root level (Arnolds, 1991). Furthermore, many species which do not form large, conspicuous sporocarps have been found to be important in forming ectomycorrhizas (e.g. thelephoroid, corticoid, and ascomycete fungi; Gardes & Bruns, 1996a; Kårén & Nylund, 1997; Erland et al., 1999, Jonsson et al., 1999a; Jonsson et al., 1999b; Mahmood et al., 1999).
The question of whether the observed decrease of ectomycorrhizal sporocarps at higher N levels reflects a qualitative and quantitative reduction of these fungal taxa in the soil, that is a reduction of mycelial biomass and of the number of ectomycorrhizas formed by these species, can only be answered by investigating species diversity and abundance at the root level. There are a few studies which include belowground aspects in N-deposition investigations by monitoring morphotypes, number of mycorrhizal root tips, or ergosterol content in fine roots (Menge & Grand, 1978; Alexander & Fairley, 1983; Brandrud, 1995; Kårén & Nylund, 1997; Nilsen et al., 1998). Generally, no or only minor changes were observed belowground in response to N addition, whereas the aboveground sporocarp formation was negatively affected. In some studies, the percentage of colonized root tips was found to be reduced shortly after fertilizer application (Menge & Grand, 1978; Tétreault et al., 1978; Haug et al., 1992). In these experiments, the N fertilizer was applied at once, which aggravates the comparison to the situation of constant low inputs, for example from atmospheric deposition. To our knowledge, in only two studies has the ITS-PCR-RFLP method been applied to investigate the impact of N deposition on ectomycorrhizal fungi. Kårén & Nylund (1997) classified mycorrhizas into morphotypes, which in turn were studied using the ITS-PCR-RFLP method in a simulated N deposition experiment. Morphotype analyses indicated that a shift in species composition might occur. However, the number of RFLP-types in the examined plots did not change due to fertilization, and since they did not analyse a sufficient number of samples with the ITS-PCR-RFLP method, they were not able to draw conclusions at the level of single species. Lilleskov et al. (1998) studied the effect of enhanced N availability on above- and belowground diversity of ectomycorrhizal species over a short, steep N deposition gradient near a NH3 production facility. By ITS-typing of the fungal symbiont on root tips, they found a reduced number of species forming ectomycorrhizas at higher N levels.
In the present study, a N-addition experiment was performed using a long-term fertilizer, which should simulate a continuing, high N deposition on the forest soil. The aims of the present study were: (1) to monitor the responses of ectomycorrhizal and saprobic sporocarp production to N addition over time; (2) to determine whether the shift in the fungal species composition noted by morphotype analyses in previous N deposition studies is confirmed by ITS-PCR-RFLP-analyses and how this shift is expressed at the species level; (3) to compare the impact of N addition on above- and belowground ectomycorrhizal diversity, and in particular (iv) to see if single ectomycorrhizal species whose sporocarp production is affected by N fertilization show the same changes at the root level. The experiment was performed in a Norway spruce stand where detailed sporocarp surveys had been carried out for 3 yr before starting the fertilization.
Materials and methods
Study site and experimental design
The experiment was performed in a reforested 35-year-old spruce forest in an native subalpine Picea abies stand. The stand was located in the Flysch zone at 1350 m above sea level (asl). The average atmospheric N input in this region is estimated to be 15–20 kg N ha−1 yr−1 (Eugster et al., 1998). Sporocarp surveys had been carried out in an area of 1100 m2 (20 m × 55 m, divided into 1 m2 subplots) since 1994, 3 yr before starting the current experiment. To assess the effects of N addition, we used a clumped segregation design with four replicates. The size of each replicate was 8 m × 8 m, divided in subplots of 1 m2. The four replicates were situated next to each other resulting in two 16 m × 16 m quadrats, one for the control and one for the treatment. A buffering zone of 10 m separated the two quadrats. We chose a segregation design to avoid the risk of the control replicates being affected by N leaching. An amount of 150 kg N ha−1 yr−1 was added as ammonium nitrate. The addition was done on 20 m × 20 m to obtain a fertilized zone of 2 m surrounding the treatment plot. We used a long-term fertilizer in solid, globular form (Osmocote 23 + 0 + 0; Hauert Dünger, Switzerland) in which the nutrient is wrapped in an organic mantle and continuously released over 5 to 6 months. The globules were added twice per year in May and October, from spring 1997 onwards.
We included three additional plots of 8 m × 8 m – unsampled replicates (UR) – for sporocarp survey to control for effects of root and soil sampling on sporocarp production.
The term relevé will, hereafter, be used for the recordings in the replicate plots of 8 m × 8 m in the subsequent years. Plot will be used for the experimental quadrats of 16 m × 16 m, which contain the four replicate plots.
Sporocarps of all macromycetes were counted and mapped weekly with a resolution of 1 m2 from spring 1994 onwards in the 1100 m2 area. The inventories started in spring after snow melt, and ended by the onset of winter. To avoid double counting, mapped caps were marked using methylene blue.
PCR-RFLP analysis of the ectomycorrhizas
Fine roots were sampled in spring 1997 before the onset of the fertilization experiment, in spring 1998, and in spring 1999. For each relevé, 64 samples were collected as follows: in the centre of each square meter, a short piece of fine roots (approx. 3 cm) was extracted with tweezers from the organic horizon (approx. upper 5 cm), put into 1.5 ml tubes, transported to the laboratory in cooling boxes, and stored at 4°C no longer than 1 wk until processing. The roots were placed into water and carefully rinsed under a dissecting microscope. From each sample, one single mycorrhizal root tip was randomly selected for PCR-RFLP analysis. A short root was considered mycorrhizal if a mantel was present. The mycorrhizas were placed into 1.5 ml tubes and stored at −20°C before DNA extraction. The rest of the sample was stored at the same temperature as spare material. In total, 1536 samples were analysed.
DNA was extracted following the protocol described by Kårén et al. (1997) and amplified according to the PCR protocol in (Gardes and Bruns, 1996b). Before amplification, dye-labelled deoxicytidine triphosphates ([F]dCTPs, PE Applied Biosystems) were added to the PCR reaction mixture in a ratio dNTP to [F]dCTP of 400 : 1. We used the universal primers ITS1 and ITS4 developed by White et al. (1990). Running PCR products on 1.5% agarose gels and visualizing them with ethidium bromide checked success of amplification. For removing fluorescent by-products and unincorporated [F]dNTPs, PCR products were purified following the Simplified Ethanol Precipitation Procedure described in the Protocol book of ABI PRISM BigDye Terminator Cycle Sequencing (PE Applied Biosystems). To obtain RFLP patterns, 8 µl aliquots of the PCR product were digested with the restriction enzymes MboI, HinfI, and TaqI according to the manufacturer’s recommendations (MBI Fermentas). RFLP products were purified again as described earlier in this section, except that the ethanol concentration was not reduced. Fragment sizes were determined on an ABI Prism 310 Genetic Analyser using GeneScan Analysis and Genotyper Software (PE Applied Biosystems). Taxotron software (Pasteur Institute, Paris, France) was used to analyse and interpret the RFLP patterns using single-linkage cluster analysis following the description of Kårén et al. (1997), with minor modifications: because we determined the fragment sizes on a Genetic Analyser, we directly imported the values in the fragment size (MW) file. The error in determination of the fragment sizes between 50 bp and 500 bp was set at 5 bp: as we used restriction enzymes that cut the two DNA-strands unequally (‘sticky-end’ enzymes), we obtained double peaks with a shift of 2–4 bp, plus a maximum shift of 1 bp was observed in repetitions.
RFLP patterns were compared to a reference database consisting of approx. 180 DNA patterns of different species from most major basidiomycetous, ectomycorrhizal genera (32 different genera, 19 families; M. Peter, unpublished). This database comprised all the species which were found in the experimental area of 1100 m2 and close to it (distances up to approx. 20 m). It was not possible to delimit all species with this set of restriction enzymes, especially in the family of Cortinariaceae. There are four groups where the fungal species show the same RFLP pattern and which were in root samples:
Cortinarius group 1: C. colus, C. angelesianus, C. damascenus, C. decipiens, C. jubilarius, C. paleaceus, C. paleiferus, C. parvannulatus, C. subsertipes, C. evernius
Cortinarius group 2: C. uraceus, C. bataillei, C. gentilis
Cortinarius group 3: C. spilomeus, C. limonius, Rozites caperata
Cortinarius group 4: C. anomalus, C. latobalteatus
The term RFLP-type refers to a single species or a group of species showing a unique RFLP pattern.
Calculations and statistical analyses
For univariate analyses, ANOVA in Statview (SAS, 1998) and Genstat (Payne et al., 1993) was used. Time series were treated as repeated measures to test interactions of treatment-by-time. This provides evidence of whether the progression of a parameter over time is different among the treatments. In Genstat, contrasts were calculated on the data from 1999, 2 yr after the start of the fertilization treatment. To test the effect of N addition on single RFLP-types, we applied paired t-tests (within treatments between years) and unpaired t-tests (between treatments within years). Unless otherwise noted, significance level was P < 0.05. Diversity was calculated using Simpson’s diversity index: 1 − D = 1 − Σ(Pi)2. Pi for sporocarp species was measured as the number of 1 m2 subplots in each relevé in which the species was observed, devided by the number of subplots totalled over all species. For mycorrhizas, Pi is the frequency of each RFLP-type in each relevé. To determine the influence of N on the number of RFLP-types, we used rarefaction (Hurlbert, 1971) because the number of successfully analysed mycorrhizas by ITS-RFLP differed between the relevés. The estimated number of RFLP-types was calculated for a sample size of 26 mycorrhizas, which was the lowest number of RFLP patterns within a relevé.
Similarities of the relevés regarding sporocarp or mycorrhizal communities were measured using van der Maarel’s index of similarity (Wildi & Orlóci, 1996). This index is based on the contingency table, the most original way to compare community recordings. Species frequencies are taken into account, and zero observations were not considered as indicators of similarity. The index provides values between 0 for total dissimilarity and 1 for total similarity. Sporocarp abundances were measured in terms of number of fruitbodies. To reduce the influence of species with very high sporocarp numbers, all values were log transformed before calculating similarity. For mycorrhizas, RFLP-type frequencies were determined as the proportions of mycorrhizal root tips colonized by each of the RFLP-types in each relevé. The percentage values were square-root transformed to decrease the influence of abundant types. RFLP-types and sporocarps which were found in only one relevé (unique species) were omitted from the analysis to reduce the influence of unusual species. Similarity of the communities was analysed by ordination using principal coordinates analysis (PCOA) in MULVA-5 (Wildi & Orlóci, 1996).
Effects of N addition on aboveground diversity (sporocarp production)
In total, sporocarps of 85 fungal species were observed from 1994 to 1999 in the 1100 m2 area. Twenty-nine of these species were ectomycorrhizal. Mean species richness of the whole fungal community was reduced in the N-replicates compared with the control- and unsampled-replicates 1 (1998) and 2 yr (1999) after the start of fertilization (Fig. 1). Total number of sporocarps of all macromycetes already showed a reduction in the N-replicates compared with the control- and unsampled-replicates in 1997, after only 3 to 4 months of fertilization. If mycorrhizal and saprobic species were viewed separately, a prominent effect of fertilization on ectomycorrhizal species was observed, whereas the saprobic community was not affected (Fig. 1). Simpson’s index of diversity of the ectomycorrhizal community was reduced in the N-plot in 1998 and 1999 compared with the previous years, whereas the saprotrophs were not affected (Fig. 1). Compared with the control- and unsampled replicates, ectomycorrhizal species richness in the N-replicates were lower in 1998 with a mean of 1.5 ± 0.3 species compared to 5.0 ± 0.9 and 4.7 ± 0.9 species in the control- and unsampled replicates, respectively, and in 1999 with 2.5 ± 0.3 species in the N-replicates compared to 9.3 ± 0.8 and 9.0 ± 2.1 species in the control- and unsampled replicates, respectively (mean ± standard error). The proportion of ectomycorrhizal to saprobic species frequencies, measured as the number of 1 m × 1 m subplots per replicate in which a species was found, drastically decreased in the N-plot from a mean of 64% ± 6% in 1994 to 18% ± 4% in 1999. This was not observed in the control- and unsampled-plot. In repeated measures ANOVA as well as in contrasts analysis of data of 1999, significant changes of species richness, number of sporocarps, and Simpson’s index of diversity in response to N addition were observed mainly in the ectomycorrhizal community (Table 1). Simpson’s index of diversity showed a significant treatment-by-time interaction also in the saprobic community. However, this was not caused by N addition, but because of reduced diversity in the N-plot in 1994 (cf. Fig. 1).
Table 1. ANOVA table of effects of nitrogen addition on species richness, number of sporocarps and Simpson’s index of diversity (1 − D) based on sporocarp data. Only treatment × time interactions and linear contrasts of data from 1999, respectively, are given. Treatment × time interaction of repeated measures (1994–99) comparing progressions of the respective parameter in N-replicates (N), control-replicates (C) and unsampled-replicates (UR)
Overall repeated measures
(treatment × time)
N vs C, UR
C vs UR
, P < 0.05;
, P < 0.01;
, P < 0.001; ns, not significant. df, degrees of freedom; ss, sum of squares; F, variance ratio; Sig, significance.
Ordination of relevé scores over time confirmed that N addition strongly affected the aboveground fungal community (Fig. 2). All four N-replicates showed a prominent shift on the first axis in 1998 and 1999. This change exceeded spatial variability. Spatial variability within treatment-replicates was used as a reference measure to estimate the level of statistical noise (Legendre & Legendre, 1998). The similarity over time of all four N-replicates fell below the lower limit in 1999, which was not observed in the control-and unsampled-replicates.
Four ectomycorrhizal species accounted for most of the difference between relevés of the N-plot of 1998 and 1999 and all other relevés (Fig. 3). These ‘best differentiating’ species were determined by Jancey’s Ranking of F-Values in the MULVA-5 program. Sporocarp production of Amanita aff. submembranacea, Inocybe grammata, Russula fuscorubroides, and Russula laricina was clearly reduced or even absent in these relevés. The effect of N addition on other ectomycorrhizal species is less obvious since they fruited spatially and temporally less regularly. Even so, a negative effect of N addition probably existed for some of them. For instance, sporocarps of Cortinarius parvannulatus and Thelephora palmata were formed in all four control-replicates and in two unsampled-replicates in 1999, but were not formed (T. palmata) or were formed in only one replicate (C. parvannulatus) of the N-plot (Fig. 3). No ectomycorrhizal species fruited for the first time in the N-replicates 1 or 2 yr after the start of fertilization. No effect of N addition was found on Hygrophorus pustulatus and Clavulina cristata, which were among the most abundant ectomycorrhizal species in the experimental area, forming sporocarps every year. None of the saprobic species showed a reduction in sporocarp production caused by N fertilization, but neither were positive reactions apparent. Five saprobic species fruited only in the N-plot in 1999 (Mycena epipterygia, Clitocybe gibba, Lepista gilva, Lepista inversa, Postia caesia (on wood)), however, all only in one of the four replicates.
Effects of N addition on belowground diversity (ectomycorrhizas)
In total, 1536 root tips were subjected to DNA analysis, 1017 of which were successfully amplified (66%). Twenty percent of these ectomycorrhizal samples showed two or more PCR products of different length. If they contained one strong and one faint product, they were further analysed and interpreted as two species on the same root tip. The restriction fragments of the two PCR products were separated on the basis of peak height in the GeneScan Analysis program. In total, we obtained RFLP patterns of 903 root tips. Sixty-eight different RFLP-types were distinguished (Table 2). No differences were noted in mean numbers of RFLP-types in the relevés among the control- and N-replicates in all 3 yr of survey (Table 2). We found a mean number of nine to eleven different RFLP-types in the replicate plots of 8 m × 8 m when rarefaction for a sample size of 26 mycorrhizas was applied. Simpson’s index of diversity was not affected by fertilization (Table 2).
Table 2. Diversity of belowground ectomycorrhizal communities in the fertilized plot (N) before (N(control)), and 1 resp. 2 yr after treatment and in the control plot. The figures represent mean frequencies (n = 4) of the RFLP-types in a replicate plot of 8 m × 8 m. The number of replicate plots in which the RFLP-type could be found is shown in parenthesis. Species or species groups which produced conspicuous sporocarps between 1994 and 1999 in the control-or N-plot are written in boldface
RFLP-types found in only one relevé: RFLP-type 27 – RFLP-type 46, Cenococcum geophilum type 2, Cenococcum geophilum type 3, Cortinarius laniger, Laccaria montana, Russula xerampelina, Thelephora palmata, Tylospora fibrillosa.*Before fertilization.**Determined with analysis of the ML5/6 region of the mitochondrial large subunit rRNA gene (Bruns et al., 1998).***Species richness of mycorrhizas using rarefaction (Hurlbert, 1971) for a standardized sample size of 26 mycorrhizas.
The ordination of relevé scores indicated that N addition did not have a prominent effect on the belowground community after 1 or 2 yr of fertilization (Fig. 4). There was no distinct shift on either of the first two axes which would exceed spatial variability. However, in 1999 all four N-replicates showed higher scores on the second axes than in 1997 or 1998. This was not observed in the control-replicates. Van der Maarel’s index of similarity among control-replicates and N-replicates in the subsequent years also indicated a possible change in the belowground community (Fig. 5). In 1999, unlike the two previous years, mean similarity among control-and N-replicates (0.47 ± 0.03) was lower than mean similarities among N-replicates (0.56 ± 0.04) or among control-replicates (0.54 ± 0.02). This difference was significant in contrasts analysis (Table 3).
Table 3. ANOVA table of effect of nitrogen addition on van der Maarel’s similarity based on RFLP-types within and between treatments. Only the treatment × time interaction and linear contrasts of data from 1999 are given. Treatment × time interaction of repeated measures (1997–99) comparing progressions of similarity within N-replicates (N), within control-replicates (C), and among N- and control-replicates (N-C)
Overall repeated measures
(treatment × time)
N-C vs N, C
N vs C
, P < 0.05; ns, not significant. df, degrees of freedom; ss, sum of squares; F, variance ratio; Sig, significance.
Five RFLP-types showed significant changes in abundance which might be due to N fertilization (Table 2). Abundance of Russula laricina, which was one of the three most common species at the root level, was significantly reduced in the N-replicates in 1999 (1.3% ± 0.7%) compared with its abundance in 1997 (14.7% ± 1.5%). A small reduction was also present in the control-replicates (1997: 14.9% ± 5.9% 1999: 11.6% ± 3.0%), but was not significant. Russula integra showed a significant difference in abundance in the N-plot (0.6% ± 0.6%) compared to the control-plot (5.6% ± 1.1%) after 2 yr of N addition, which was not present in the previous years. This difference was mainly due to an increase of this species in the control-replicates. The abundance of Tylospora asterophora did not change prominently in either treatment plots. It was more frequent in the N-replicates (means 21–24%) than in the control-replicates (means 13–14%) on all three sampling occasions. However, this difference was significant only in 1998 and 1999, and was mainly due to an increase of frequency in one of the four N-replicates. Mean frequencies of RFLP-type 6 and RFLP-type 9 were increased in the N-plot 2 yr after the start of N addition. The abundances of RFLP-type 6 were significantly different between the N-plot in 1997 and 1999 (1997, 0%; 1999, 5.3% ± 2.7%) and between the N-plot and the control-plot in 1999 (C, 0.6% ± 0.6%). RFLP-type 9 showed a significant difference of frequencies in 1999 between the N-plot and the control-plot (C, 0%; N, 2.6% ± 1.0%). Frequency of Hygrophorus pustulatus was increased in 1999 in the N-plot, which was not present in the control plot, but no significant differences were noted. If total frequencies of all identified macromycetous, ectomycorrhizal species are considered (Table 2, species written in boldface), a continuous reduction in the N-plot from 1997 to 1999 can be noted. Mean frequency decreased from 25.7% ± 3.5% in 1997 to 20.9% ± 2.4% in 1998 to 13.3% ± 2.7% in 1999. The frequencies in 1997 and 1999 are significantly different from each other. However, this reduction was mainly caused by one species, Russula laricina. In the control plot, a clear but not significant reduction of frequency in 1998 compared with 1997 and 1999 can be noted (1997, 27.1% ± 5.8%; 1998, 19.4% ± 4.7%; 1999, 29.3% ± 6.4%). Total frequency of species belonging to the Thelephoraceae and Corticiaceae forming inconspicuous sporocarps increased in the N-plot from 46.0% ± 1.6% in 1997 to 50.0% ± 4.1% in 1998 to 57.2 ± 3.5% in 1999. Frequency in the control-plot did not change (1997, 36.5%± 4.9%; 1998, 35.0% ± 8.3%; 1999, 38.0% ± 2.1%).
Identification and comparison between above- and belowground data
Twenty-five ectomycorrhizal species formed sporocarps in the N-plot or the control-plot between 1994 and 1999 (Fig. 3). Twelve of these 25 species (48%) were also found on the root-system (Table 2, species written in boldface). In total, 22 of the 68 different RFLP-types (32%) were identified with the help of the existing reference database. Seven of them did not form conspicuous, epigeous sporocarps (e.g. Tuber sp., Cenococcum geophilum types and species of the Thelephoraceae and Corticiaceae). Four of them did not fruit in the experimental plots in the 6 yr of assessment, but sporocarps were found in the vicinity (Russula xerampelina, Cortinarius privignorum, C. laniger, Cortinarius group 3). Fifty-eight percent of the ectomycorrhizal samples (519 of 903 samples), consisting of 46 different RFLP-types, remained unknown. Thirteen of these unknown types (Table 2) were placed into family or subfamily groups by preliminary sequence analyses of the ML5/ML6 region of the mitochondrial large subunit rRNA gene. For this purpose, we used the database published in Bruns et al. (1998) and performed a heuristic analysis with parsimony in PAUP 3.1.1 (Swofford, 1993). Six of the unknown species were placed into the group of Thelephoraceae, among them the most common RFLP-type no. 1. These thelephoroid types accounted for 44% of all root tips. Four types belong to the Russulaceae, two types were placed into the cantharelloid group and one type, which was one of the five most frequently found patterns, was placed to the Hygrophoraceae. ITS-RFLP clustering supported most of these findings.
In terms of sporocarp frequencies (measured as the number of 1 m × 1 m subplots per replicate in which the species was found), the following descending order of species abundances (cumulated over 6 yr of survey; in percentage of all species; ≥ 1%) was observed in the experimental area: Russula laricina (34%) > Amanita aff. submembranacea (26%) > Hygrophorus pustulatus (11%) > Russula fuscorubroides (11%) > Inocybe grammata (6%) > Clavulina cristata (5%) > Lactarius deterrimus (2%) > Cortinarius parvannulatus (1%). In terms of RFLP frequencies, the order of types which were identified as belonging to the macromycetous species was as follows (see Table 2): Russula laricina (10%) > Hygrophorus pustulatus (5%) > Russula integra (2%) > Cortinarius group 1 (including C. parvannulatus; 2%) > Cortinarius group 2 (1%) > Cortinarius privignorum (1%) > Lactarius deterrimus (1%). The two most common RFLP-types do not form conspicuous sporocarps and belong to the Thelephoraceae (RFLP-type 1; 19%) and Corticiaceae (Tylospora asterophora; 18%). Species from which sporocarps and ectomycorrhizal root samples were identified, showed the following spatial and temporal distribution: Russula laricina was common in terms of sporocarp production and RFLP frequency in both treatment plots before N addition (Fig. 3) and decreased drastically in the N-plot 2 yr after N addition, both at the root level and in sporocarp frequency. In the N-plot, sporocarp production was already reduced half a year after N addition (Fig. 6, 1997). In the control-plot, sporocarp frequency varied among the years of observation, which was seen at the root level as well (Fig. 6). The abundance of Hygrophorus pustulatus in terms of sporocarp frequency as well as RFLP frequency was not affected by N addition. Lactarius deterrimus was found, both above- and belowground, only in 1997 irrespective of treatment. Russula integra showed the same RFLP frequencies in 1997 and 1998 in both treatments, frequency in 1999 increased in the untreated plot and decreased in the fertilized plot. During the 6 years of sporocarp survey, only eight sporocarps of this species were noted (Fig. 3), which makes comparisons difficult. During the six years of sporocarp survey, Cortinarius parvannulatus was more frequent in the control-plot than in the N-plot. This was also reflected in the root data (Cortinarius group 1). Ectomycorrhizas of Cortinarius group 2 (including C. gentilis) were found both in control-and N-plots, but sporocarps were only formed in the control-plot. Root tips with Cortinarius privignorum were found at all sampling occasions in either or both treatment plots, but no sporocarps were formed in the plots during the 6 yr of observation.
Soil sampling had no effect on sporocarp production. This was seen in contrasts analysis with data of 1999 (Table 1). In repeated measures ANOVA of similarities, species richness, sporocarp abundance and diversity, control-replicates and unsampled-replicates showed no treatment × time interaction (data not shown).
Sporocarp production of most ectomycorrhizal species decreased drastically after only 1 yr of N addition, whereas the saprobic community showed no distinct change. These results are in accordance with previous data on the effect of increased N input on sporocarp formation of fungi. The present study also supports the findings that the effect of N addition is much less pronounced in the belowground diversity, in that no change in the number of taxa forming ectomycorrhizas and in Simpson’s index of diversity was observed. However, a clear change of the ectomycorrhizal composition at the root level in response to increased N input was evident at the species level. Our data indicate that the reaction of single species to increased N input, as seen in sporocarp surveys, was reflected in their abundances at the root level. Even yearly fluctuations of sporocarp production were expressed in the frequencies of the ectomycorrhizas in some species. Species which produced large sporocarps in and close to the experimental plots in the 6 years of survey accounted for 23% of all sampled root tips. Such a relatively low degree of representation of the fruiting species on the root-system has also been seen in previous studies (Mehmann et al., 1995; Gardes & Bruns, 1996a; Dahlberg et al., 1997; Kårén & Nylund, 1997; Erland et al., 1999, Jonsson L. et al., 1999; Jonsson T. et al., 1999; Mahmood et al., 1999). We identified the symbiont of 44% of all root tip samples, comprising nine different RFLP-types, as belonging to the Thelephoraceae and Corticiaceae, taxa which produce inconspicuous sporocarps. Our data suggest that the proportion of these species on the root-system increases at higher N levels, whereas the proportion of species which produced fewer sporocarps after N input decreases at the root level.
Sporocarp inventories revealed a distinct change of the fungal community after only 1 yr of fertilization due to a decrease of ectomycorrhizal diversity. The sporocarp production of four of the six most dominant ectomycorrhizal species decreased drastically or even ceased. They comprise two species from the genus Russula (R. laricina, R. fuscorubroides), which has been reported to be sensitive to increased N levels in several N-deposition studies (Rühling & Tyler, 1991; Brandrud, 1995; Lilleskov & Fahey, 1996) and one species each of the genera Amanita and Inocybe (A. aff. submembranacea, I. grammata). Two of the common fruiters, Hygrophorus pustulatus and Clavulina cristata, showed no fruiting decrease after N addition. According to Arnolds (1991) and Arnolds & Jansen (1992), ectomycorrhizal fungi which mainly associate with coniferous species have shown the greatest reduction in abundance during the 20th century. These authors suggest that generalist species, forming mycorrhizas with a wide range of tree species, are less affected by increased N availability than are host-specific species. To some extent, our data confirm these findings: The two N-sensitive Russula and the Amanita species occur mainly in coniferous forests in the subalpine zone (Romagnesi, 1967; Einhellinger, 1987; Breitenbach & Kränzlin, 1991), and one of the N-insensitive species, C. cristata, is a very common species throughout Switzerland, occurring in coniferous and deciduous forests. However, I. grammata, which did reveal reduced sporocarp production, is associated with conifers as well as birch and beech (Stangl, 1989). The N-insensitive H. pustulatus presents an ambivalent situation: it is widespread throughout Switzerland, but restricted to Norway spruce stands (Breitenbach & Kränzlin, 1991), and thus is host-specific but within a broad geographical area. It might, therefore, be true that species inhabiting a more closely defined ecological niche, like the Russula and the Amanita species mentioned above, are more sensitive to N input, but a generalization is certainly not possible.
The saprobic community showed no change in response to N addition in terms of species richness and abundance. None of the species showed a clear decrease or increase of sporocarp formation in response to N addition. Rühling & Tyler (1991) noted an increase in the sporocarp production of most leaf litter and humus decomposers after N addition, particularly Clitocybe gibba and Lepista inversa. In the present study, these species occurred in the experimental area for the first time in the N-plot 2 yr after the start of N addition, which indicates that they might have benefited from higher N levels.
Mycorrhizal colonization seems to be much less sensitive to increased N input than sporocarp formation, as seen in ordination analyses. This is in accordance with other studies in which above- and belowground aspects of ectomycorrhizal fungi were taken into account (Menge & Grand, 1978; Ritter, 1990; Termorshuizen, 1993; Brandrud, 1995; Kårén & Nylund, 1997). Nevertheless, the present study confirms the findings provided by morphotype analyses that N addition has an effect on the ectomycorrhizal composition at the root level. Some RFLP-types showed significant changes in abundance 2 yr after the start of N addition. The most distinct shift can be noted for Russula laricina, which was one of the most abundant species at the root level before N supply and drastically decreased in the fertilized plots 2 yr after the start of N addition. Even more so, its sporocarp production was significantly reduced already 1 yr after starting N addition. Reduced sporocarp production seems, therefore, to be connected with or followed by decreased colonization potential at higher N levels. Laboratory and field investigations show that the infection potential of some species is decreased in response to N addition (Newton & Pigott, 1991; Arnebrant & Söderström, 1992; Arnebrant, 1996; Egli, 1996). Reduced mycelial growth is supposed to be one of the reasons for this phenomenon (Arnebrant & Söderström, 1992; Wallander & Nylund, 1992; Arnebrant, 1994).
The mechanisms by which increased N availability depresses growth processes of ectomycorrhizal fungi are not fully understood. The main hypothesis is that it affects C allocation in both partners of the symbiosis. On the one hand, C supply from the plant to the fungus can be reduced (Vogt et al., 1993) since the provision of C skeletons for N assimilation is enhanced by higher N levels in both roots and leaves (review in Champigny, 1995; Björkman, 1942). On the other hand, an additional consumption of sugars for N assimilation in the fungus itself can reduce the amount of C provided for fungal growth (Wallander, 1995). This hypothesis might also explain why saprobic species, which are not dependent on carbohydrates provided directly from living plant cells, are less affected by N addition. In the present study, N concentrations in roots and needles were significantly increased from 1.1% to 1.5% and 1.2% to 1.3%, respectively, 2 yr after the start of N addition (M. Peter, unpublished). According to the hypothesis described earlier in this section, a decrease in C supply to the ectomycorrhizal fungi caused by N addition therefore seems to be probable and might be the reason for reduced above- and belowground abundances of some species. Beside a direct impact of increased N availability, a change in the pH of the forest soil, which is usually associated with N input, may affect ectomycorrhizal fungi. In the fertilized plots, mean pH significantly decreased in the organic layer from 4.5 to 4.1 (pH in H2O; M. Peter, unpublished). Impacts of acidification are, however, thought to play a minor role in the observed decrease of ectomycorrhizal fungi in Europe (Arnolds & Jansen, 1992). Field invest-igations as well as laboratory studies show either increasing or decreasing ectomycorrhizal colonization or no clear relationship between these two variables, and it is suggested that effects of pH may differ between sites (for review see Cairney & Meharg, 1999). Since species in coniferous stands are adapted to the prevailing acidic soil conditions, it is unlikely that the relatively small decrease of pH observed in the present study was directly responsible for the decrease of ectomycorrhizal sporocarp production and root colonization of some species. A possible interaction cannot, however, be entirely excluded.
Not all ectomycorrhizal species show the same sensitivities as seen in the present study and in other field and laboratory experiments. Some species apparently can cope better with, or even profit from, increased N availability (for review see Wallenda & Kottke, 1998). In the present study, species belonging to the Thelephoraceae and Corticiaceae increased in frequency at the root level 2 yr after N addition. Similar observations, based on morphotype analyses, were obtained by Taylor & Read (1996) and Boxman et al. (1998), who noted an increase in Tylospora-like morphotypes in sites with higher N levels. Different sensitivities of species to N input might be caused by several factors. Since it is thought that a shortage of carbohydrates provided from plants for fungal growth under high N levels plays an important role, the ability to utilize other C sources might explain a better adaptation of some species. Saprophytic capabilities of ectomycorrhizal fungi have been demonstrated in several experiments (Dighton et al., 1987; Abuzinadah & Read, 1989; Durall et al., 1994; Perez-Moreno & Read, 2000). For example, Tylospora fibrillosa was shown to have decomposing and proteolytic abilities (Ryan & Alexander, 1992; Cairney & Burke, 1994). Sporocarps of resupinate ectomycorrhiza formers, which include most members of the Thelephoraceae and Corticiaceae, are commonly developed on litter and soil debris and on well-decayed wood, perhaps suggesting saprophytic abilities (Erland & Taylor, 1999).
We do not know if the observed reduction of ectomycorrhizal frequencies of some species in response to N addition will persist and if species might even be out-competed by better adapted ones. Since previous long-term studies about the impact of increased N levels on belowground species composition have been conducted by studying morphotypes only, information at the species level is scarce. Experiments in which existing N levels were decreased by sod-cutting (Baar, 1996) or by means of a roof and application of clean throughfall water (Boxman et al., 1998) have shown that species richness measured by sporocarp formation can be restored. This indicates that species might persist at the roots in unfavourable environments. Lilleskov et al. (1998), however, found a reduced number of ectomycorrhizal species at the roots of trees growing in a forest close to a NH3 production facility and receiving high inputs of N for 28 yr. On the long run, it might therefore be possible that some species will disappear at continuing N addition. In the present study, the N input was quite high (150 kg N ha−1 yr−1). The question arises whether the observed decrease of sporocarp formation of ectomycorrhizal fungi in lower N-input studies (Brandrud, 1995; Boxman et al., 1998) is also reflected at the roots at the level of species. Morphotype analyses indicated no shift in the belowground species composition in these low N-level experiments with inputs of 30–40 kg N ha−1 yr−1 (Brandrud, 1995; Brandrud & Timmermann, 1998, Wallenda & Kottke, 1998), but it is possible that changes were overlooked by this method. The functional significance of a change in species composition at the fungus–root interface and probably also in the abundance of mycelia in the soil for the trees and the whole forest ecosystem is not known. A deeper insight into the physiological properties of different ectomycorrhizal species, which result in unequal sensitivities to the available amount of inorganic N, is necessary and could possibly provide answers to this field. In the present study, belowground ectomycorrhizal diversity was not reduced by N addition, but a shift in abundance at the root level from species forming large sporocarps to species which produce no or resupinate sporocarps took place and might get more pronounced at a continuing input. Even if ectomycorrhizal functions for the trees are still provided with these species, an irreversible loss of fungal species may occur in the long run. Considering this, present N-input levels up to a mean of 35 kg N ha−1 yr−1 as found in Swiss forests (Flückiger & Braun, 1998) might therefore be problematic.
We noted a good correlation between changes in above- and belowground abundances for some species. Even yearly fluctuations of sporocarp production were reflected in the belowground frequencies. The same correlation was observed in previous studies (Laiho, 1970; Agerer, 1990). This supports the hypothesis of Dahlberg et al. (1997) who assumed that a part of the among-year variation of sporocarp production not accounted for by annual variation in temperature and precipitation (Dahlberg, 1991), may be due to fluctuations in the mycorrhizas. Some species (Amanita aff. sumbembranacea, Inocybe grammata, Russula fuscorubroides, Clavulina cristata), however, were common in terms of sporocarp production but were either never found on roots or, in case of R. fuscorubroides, identified only in two root samples. For Clavulina cristata, the reason for the mismatch might be intraspecific variation in the ITS region. The unknown RFLP-type 4 of the present study clustered with the cantharelloid types from the ML5/ML6 database of Bruns et al. (1998). This group is closely related to Clavulina cristata (Burns et al., 1998). In fact, the location of the root tips on which we detected this RFLP-type corresponded exactely to the observed patches of Clavulina sporocarps. Furthermore, ITS-RFLP patterns were identical in one restriction enzyme and similar in the other two. For the other species, a possible explanation for the mismatch between above- and belowground abundances could be that they might be mainly present on roots at lower depth than the sampled layer (approx. upper 5 cm). Egli (1981) studied the vertical distribution of ectomycorrhizal morphotypes and noticed some types to be restricted to specific vertical soil layers. In relation to the study reported here, we took some soil cores and sampled mycorrhizas which occurred at lower depth in the organic layer (between approx. 5–10 cm) and showed an unfamiliar morphotype. Among these samples, we were able to identify I. grammata, which had not been found in the previously. No RFLP pattern matched the one of A. aff. submembranacea, however, so this species might occur at even lower depth. Gardes & Bruns (1996a), who also found a mismatch between above- and belowground frequencies of some species, suggested that the discrepancy might result from different patterns of resource allocation among species. The efficiency in acquiring carbon from the host plant may vary among ectomycorrhizal species or additional access of saprobic sources of carbohydrates may result in different proportions of mycorrhizas for the same amount of sporocarps. If additional saprobic capabilities were the reason for the observed mismatch between above- and belowground abundances, one would assume that these species might be less affected by increased N input, which, however, was not the case in the present study. Insights into vertical stratification of species, together with information about the effect of N on single species, might therefore provide better knowledge of nutrient and C allocation of different species.
Ectomycorrhizal species richness is markedly higher below- than aboveground in the present study, where 68 different RFLP-types compared with 25 fruiting species were found. Of the sampled ectomycorrhizas > 75% were formed by species which did not produce conspicuous sporocarps in the investigated plots. This is in accordance with previous studies (Mehmann et al., 1995; Gardes & Bruns, 1996a; Dahlberg et al., 1997; Kårén & Nylund, 1997, Jonsson et al., 1999a; Jonsson et al., 1999b; Mahmood et al., 1999; Taylor & Bruns, 1999). In all these investigations, mycorrhizas formed by species of the Corticiaceae and Thelephoraceae accounted for a large proportion of ectomycorrhizal root tips. In the present study, these resupinate taxa as well as the Russulaceae were dominant on root systems in untreated plots, which seems to be a general pattern of EM communities in California (Taylor & Bruns, 1999). Our data indicate that N addition has an influence on the ratio of these two groups of species in favour of the resupinate taxa. The functional significance of these until recently overlooked resupinate species thus deserves even more attention in the light of their role in N-polluted regions.
We are grateful to Simone Falcato and Urs Büchler for laboratory assistance and appreciate the constructive comments and statistical advice of Rosmarie Honeggerr, Gérald Achermann, and Felix Gugerli, which greatly improved the manuscript. We also thank the Hauert Dünger company for placing the fertilizer at our disposal, Tom Bruns and colleagues for making their database available at http://plantbio.berkeley.edu/~bruns, and Ursula Eberhardt for providing samples of Tylospora spp. Financial support was provided within the scope of the Long-term Forest Ecosystem Research LWF by the Swiss Agency for the Environment, Forests and Landscape (SAEFL/BUWAL).