Attributing Functions to Ectomycorrhizal Fungal Identities in Assemblages for Nitrogen Acquisition Under Stress

The ISME Journal (2014) 8, 321–330 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Attributing functions to ectomycorrhizal fungal identities in assemblages for nitrogen acquisition under stress

Rodica Pena and Andrea Polle Forest Botany and Tree Physiology, Georg-August Universita¨tGo¨ttingen, Bu¨sgenweg 2, Go¨ttingen, Germany

Mycorrhizal fungi have a key role in nitrogen (N) cycling, particularly in boreal and temperate ecosystems. However, the significance of ectomycorrhizal fungal (EMF) diversity for this important ecosystem function is unknown. Here, EMF taxon-specific N uptake was analyzed via 15N isotope enrichment in complex root-associated assemblages and non-mycorrhizal root tips in controlled experiments. Specific 15N enrichment in ectomycorrhizas, which represents the N influx and export, as well as the exchange of 15N with the N pool of the root tip, was dependent on the fungal identity. Light or water deprivation revealed interspecific response diversity for N uptake. Partial taxon- specific N fluxes for ectomycorrhizas were assessed, and the benefits of EMF assemblages for plant N nutrition were estimated. We demonstrated that ectomycorrhizal assemblages provide advantages for inorganic N uptake compared with non-mycorrhizal roots under environmental constraints but not for unstressed plants. These benefits were realized via stress activation of distinct EMF taxa, which suggests significant functional diversity within EMF assemblages. We developed and validated a model that predicts net N flux into the plant based on taxon-specific 15N enrichment in ectomycorrhizal root tips. These results open a new avenue to characterize the functional traits of EMF taxa in complex communities. The ISME Journal (2014) 8, 321–330; doi:10.1038/ismej.2013.158; published online 12 September 2013 Subject Category: Microbe-microbe and microbe-host interactions Keywords: 15N labeling; beech (Fagus sylvatica); drought; modeling; mycorrhiza; shade

Introduction Although it has been well established that EMF have key roles in plant nutrition, much less is The roots of most plant species are associated with known about the functions of distinct fungal taxa mycorrhizal fungi that mediate nutrient exchange within complex ectomycorrhizal assemblages for between the plants and soil and thus have a central nutrient acquisition and host supply. Ectomycorrhizal role in biogeochemical cycles (Finlay, 2008). In communities are usually composed of a diverse temperate and boreal forests, fungi that form flora consisting of several dominant and many ectomycorrhizas are the dominant symbiotic life infrequent EMF species (Bue´e et al., 2007; Courty form. Ectomycorrhizal fungi (EMF) encase colonized et al., 2010; Pena et al., 2010; Lang et al., 2011; root tips with a dense hyphal net, termed the Tedersoo et al., 2012a; Danielsen et al., 2013). EMF mantle, and forage the soil for nutrients by extend- community structures are strongly influenced by N ing extraradical hyphae or hyphal cords (Finlay, deposition (Lilleskov et al., 2011; Kjøller et al., 2008). As nitrogen (N) is a major limiting nutrient in 2012). Stable isotope studies have revealed that EMF many forest ecosystems (LeBauer and Treseder, species differ in their abilities to exploit different N 2008), the role of EMF in the N nutrition of trees sources (Hobbie and Ho¨gberg, 2012). Furthermore, has received considerable attention (Hobbie and in situ ectomycorrhizal communities exhibit strong Hobbie, 2008; Hobbie and Ho¨gberg, 2012). In temporal differences in the capability of different addition to N delivery, recent studies have suggested EMF taxa to access litter-derived N (Pena et al., that EMF may also limit N transfer to host trees 2013a). The experimental manipulation of EMF under N-limiting conditions (Na¨sholm et al., 2013). diversity has shown context-dependent effects for fungal mixtures on plant biomass production and N nutrition (Chu-Chou and Grace, 1985; Jonsson et al., Correspondence: A Polle, Forest Botany and Tree Physiology, 2001). As the mechanistic concepts that explain Georg-August Universita¨tGo¨ttingen, Bu¨sgenweg 2, 37077 the interactions between different EMF taxa in Go¨ttingen, Germany. E-mail: [email protected] complex assemblages are still missing, the func- Received 28 April 2013; revised 25 July 2013; accepted 8 August tional relevance of EMF identities for tree nutrition 2013; published online 12 September 2013 remains enigmatic. Elucidating functional diversity Response diversity of ectomycorrhizal fungi R Pena and A Polle 322 is important for understanding the role of ectomy- (Pena et al., 2013b). Ah horizon soil (20 cm depth) corrhizal fungi in biogeochemical cycles in a collected in the Tuttlingen beech forest (latitude fluctuating environment. 471590N, longitude 81450E) was used. The germi- Our study aimed to attribute functions for N nated seedlings were maintained in a greenhouse acquisition to ectomycorrhizal species identities in under ambient conditions (20 1C, 55% air humidity) root-associated assemblages and uncover taxon- with additional light to achieve a 16-h photoperiod specific responses to environmental stress factors. with 200 mmol PAR m À 2 s À 1 at plant height (lamps We used young beech (Fagus sylvatica L.) trees, series 3071, Schuch, Worms, Germany). After 4 which are the major tree species of the natural months, eight seedlings per treatment were evalu- vegetation in Central European temperate forests ated for their mycorrhizal status showing that (Ellenberg and Strutt, 2009). The current beech roots of seedlings in untreated soil were 40±4% forest distribution range is endangered as a result colonized by EMF, whereas seedlings in sterilized of drought stress because of climate change (Weber soil were non-mycorrhizal. A total of 120 mycor- et al., 2013). Beech trees are tolerant of deep shade rhizal and 120 non-mycorrhizal beech seedlings in the youth phase (Ellenberg and Strutt, 2009); were transplanted without adherent soil individu- however, shade-induced carbon limitations have a ally into 660 ml pots with a sand-peat mixture negative impact on EMF colonization (Druebert and supplied daily with 56 ml of nutrient solution þ et al., 2009). Here, we conducted controlled experi- containing 0.4 mM NH4 as the sole N source. ments with beech seedlings cultivated in natural This concentration was chosen because it was forest soil to develop characteristic EMF communities. similar to that in forest soil of the Tuttlingen site The mycorrhizal trees were subsequently grown in (Dannenmann et al., 2009), well above Km values for þ sand to permit analysis of intact root systems and NH4 uptake of various EMF (0.005–0.25 mM; þ supplied with ammonium (NH4 ) concentrations Jongbloed et al., 1991; Eltrop and Marschner, 1996) similar to those found in beech forest soils (median and because preceding analysis with attached þ À 1 þ 0.5 mmol NH4 kg soil; range: 0.05–2 mmol NH4 beech roots at the Tuttlingen site showed saturation À 1 þ kg soil; Gessler et al., 2005; Go¨ransson et al., of NH4 uptake at concentrations above 0.05 mM 2006; Dannenmann et al., 2009; Andreasson et al., (Gessler et al., 2005). The seedlings were grown 2012). Subsets of the plants were exposed to either in full light (200 mmol PAR m À 2 s À 1) or in the full light or shade according to the characteristic shade (35–40 mmol PAR m À 2 s À 1 at plant height). light climate in beech forests (median: 150 mmol The light climate was chosen according to the PAR m À 2 s À 1, range 25–255 mmol PAR m À 2 s À 1; conditions in thinned and unthinned beech plots Kreuzwieser et al., 1997; Lemoine et al., 2002; in the Tuttlingen forest with mean seasonal light Mayer et al., 2002; Fotelli et al., 2003; Gessler levels of 176 and 25 mmol PAR m À 2 s À 1, respectively et al., 2005; Hertl et al., 2012). Light and shade (Gessler et al., 2005). After 2 months, the irrigation treatments were combined with sufficient irrigation solution was reduced to 37% for half of the plants or water shortage to mimic typical environmental grown under each light regime. After 16 days, when stresses. N acquisition was measured after the the well-irrigated plants had a predawn leaf water application of 15N in root tips associated with potential of À 0.36±0.02 MPa and those subjected to distinct EMF species and non-mycorrhizal root tips. a limited water supply had a predawn leaf potential We tested the hypotheses that (i) ectomycorrhizal of À 1.34±0.06 MPa, the plants were harvested assemblages show taxon-specific differences for (Pena et al., 2013b). During the last 3 days before þ NH4 acquisition and (ii) environmental stress harvest, each beech seedling received a total of results in functional shifts in EMF species for N 1.864 mg of N in a solution of either non-labeled 15 acquisition. Partial fluxes for EMF-associated root NH4Cl or NH4Cl (99 atom %, Cambridge Isotope tips were assessed with whole-plant N uptake Laboratories, Inc., Hampshire, UK). Biomass and for mycorrhizal and non-mycorrhizal plants. whole plant 15N data were determined (Pena et al., We provide evidence that the taxon-specific 15N 2013b). Ten 15N labeled and three non-labeled plants enrichments in EMF-associated root tips can be used per treatment were randomly selected and used for to predict net N flux into the host plant. These the analyses. results provide a basis for testing functional redundancy and response diversity of EMF assemblages in future field studies. EMF identification and quantification For each growth regimen, the whole root system of 10 beech seedlings per treatment was inspected Materials and methods using a binocular microscope (Leica M205 FA, Leica Microsystems, Wetzlar, Germany). Within each Plant cultivation and experimental treatments sample, the root tips were assigned to one of the Fungicide-treated beech nuts (Fagus sylvatica L., following fractions: vital ectomycorrhizal (EM), vital provenance: Forstsaatgutstelle Oerrel, Niedersach- non-ectomycorrhizal (NM), dead ectomycorrhizal sen, Germany) were grown in sterilized or untreated (DM) and dead non-ectomycorrhizal (DR). Live and forest soil in individual pots as described previously dead EM or NM root tips were distinguished as

The ISME Journal Response diversity of ectomycorrhizal fungi R Pena and A Polle 323 previously described (Downes et al., 1992; Winkler where the biomass represents the mean dry mass of et al., 2010). Vital EM root tips were classified using one root tip as determined for each EM fungal species, the morphotyping key of Agerer (1987–2012). and 15N atom % excess ¼ 15N atom % labeled À 15N The abundance of each morphotype was quantified atom % unlabeled, where labeled and unlabeled refer in the whole root system. The morphotypes to samples obtained from plants exposed to 15Nand were photographed (Leica DFC 420 C, Leica unlabeled nutrient solutions, respectively. Microsystems), and a scale bar was used to deter- The calculation of partial fluxes and prediction of mine ectomycorrhizal lengths. For anatomical total N flux is described in the Supplementary analysis, the morphotypes were embedded in information SI1. styrene-methacrylate (Ducic et al., 2008). Cross-sec- tions with a thickness of 1 mmwerecutwithan autocut microtome (Ultracut E, Reichert-Jung, Vienna, Statistical analysis Austria) and used to measure mantle thickness (Pena Statistical analysis was performed using Statgraphics et al., 2013a). Plus 3.0 (StatPoint, Inc., St Louis, MO, USA). When Approximately 20 root tips from each morphotype necessary, data were logarithmically or square root were collected, and aliquots were used for the transformed to satisfy the criteria of normal distribu- molecular identification of fungal species by ITS tion and homogeneity of variance. When transforma- sequencing as previously described (Lang et al., tion of the data did not meet these requirements, the 2011). The sequences obtained were assigned to Kruskal–Wallis and Mann–Whitney U-tests were fungal taxa by BLAST searches carried out against applied instead of analysis of variance. Means or public databases (National Center for Biotechnology medians were considered to be significantly different from each other when Pp0.05. Information (NCBI), http://www.ncbi.nlm.nih.gov/ 0 and UNITE, http://unite.ut.ee). The sequences were The Shannon–Wiener index of diversity (H ) was deposited in NCBI GenBank under the accession calculated with the following equation (Shannon and Weaver, 1949): numbers HM748636–HM748643 (Supplementary Ps 0 Figure S1). H ¼ pi ln pi i¼1 where S is the number of species in the sample and pi is the proportion of species i in the sample. C, N and 15N measurements in EMF-colonized root tips Evenness was calculated as follows: Among the fungal species colonizing the beech 0 roots, five were sufficiently abundant for each E ¼ H /H(max) treatment to be collected for isotope analysis. with Hmax ¼ ln (S). Depending on fungal morphology, 20 (Tomentella The effects of stress treatments on EMF community punicea) to 60 root tips (Cenococcum geophilum) composition were analyzed by principal component were required for a suitable sample for isotope analysis using the free PAST software package 2.17c analysis. One replicate comprised the root tips (http://folk.uio.no/ohammer/past/, Hammer et al., obtained from one individual plant. EM morpho- 2001). A variance–covariance matrix, which centers types, NM, DM and DR root tips of each plant were the data and in which the treatments were incorpo- cut under the binocular microscope. Two different rated as categorical nominal variables, was used to sets of instruments were used to handle non-labeled perform principal component analysis (Jolliffe, 1986). and labeled plants to avoid cross contamination. EM The analysis was followed by a multiple analysis of tips were excised at the last lateral root ramification variance to detect differences between species. ensheathed by the hyphal mantle; NM tips were sampled at the youngest and active ‘white’ zone (Evert and Eichhorn, 2007). Dead root tips with Results a shrunken and dry appearance were cut at the The EM community structure in response to irradiance same position as the NM tips. The samples were and water availability freeze-dried. The vital root tips of beech seedlings were 42±4% Freeze-dried root tips were weighed (0.2–1.1 mg) colonized with 10 different EMF species, regardless using a super-micro balance (S4, Sartorius, of light (F ¼ 2.22, P ¼ 0.144) or drought treatment Gottingen, Germany). Total C, N and 15N concentra- ¨ (F ¼ 2.16, P ¼ 0.150). The most abundant species tions in root tips were determined with an isotope (T. punicea, Cenococcum geophilum, Tuber rufum, ratio mass spectrometer (IRMS Deltaplus, Thermo Tuber sp.1 and Tuber sp.2) were associated with Finnigan Mat, Bremen, Germany) coupled to an approximately 95% of colonized root tips, whereas elemental analyzer (EA 1108, Fisons, Rodano, Italy). the other detected fungal species (Tomentella badia, The relative 15N abundance was expressed as the unknown EMF (Telephoraceae), Sebacina sp., following ratio: 15NðÞ¼ atom % 15N Â100 14N þ 15N Cortinariussp.,Tomentella viridula) colonized o5% 15N content per root tip was calculated as follows: of the EM root tips (Supplementary Figure S2). All 15N content (ng) ¼ (biomass (g) Â N concentration fungal species were previously identified in natural (ng g À 1) Â 15N atom % excess)/100, assemblages of beech EMF in the same forest from

The ISME Journal Response diversity of ectomycorrhizal fungi R Pena and A Polle 324 which the present soil inoculum was used (Pena et al., Fungal species identities determine ectomycorrhizal N 2010). acquisition in response to environmental stress Principal component analysis revealed shifts in We reasoned that specific N enrichment (measured the EMF community structure under light or shade as 15N atom–% above natural abundance) is the with sufficient or limited water availability result of N influx and export, as well as the (Figure 1). The first two components of the exchange of 15N with the N pool of the root tip. principal component analysis represented 96% Therefore, specific N enrichment is an indicator of of the variation in EMF species abundance in the physiological activity of N metabolism in a given response to drought and shade (Supplementary type of root tip if it exceeds the 15N enrichment of Table S1a). All variables (LC, LD, SC and SD) were dead root tips (DR and DM) and natural abundance highly loaded onto PC1 (0.850oro0.951), with of 15N. In DM and DR, the 15N signature was vectors of similar lengths and directions suggest- significantly higher than the natural 15N abundance ing that all sets of variables were correlated and (0.3661±0.0002; Figure 2). This unexpected enrich- equally important in explaining variation in EMF ment in DR or DM might have been caused by abundance. LC treatment was the main source soaking with the nutrient solution, by very low of variation for PC2 (r ¼ 0.472). The minimum physiological activity of root tips or root tips that hulls revealed the contrasting influence of were initially active but died during the labeling light and shade on fungal abundance. Multiple phase or by activities of microfungi and bacteria analysis of variance confirmed differences between colonizing the mycorrhizosphere (Heinonsalo et al., fungal species (F ¼ 60.5, Pspecieso0.001) and 2001; Calvaruso et al., 2007; Izumi and Finlay, revealed significant interaction between light 2011). Only 15N enrichment that was above the DR and EMF species (F ¼ 2.7, P(species x light) ¼ 0.005, and DM threshold was considered to reflect active Supplementary Table S1b); T. rufum increased 15N uptake. in full light (LC), whereas T. punicea and The specific 15N enrichment of vital NM root tips Tuber sp. 1 increased in response to shade of non-mycorrhizal beech trees was similar to that of (Figure 1). The abundance shifts in EM fungal NM root tips of inoculated beeches (P ¼ 0.228, species composition did not affect the Shannon means shown in Figure 2) and well above that of diversity index H0 or Evenness of the assemblages dead root tips (Figure 2). Specific 15N enrichment in (Supplementary Table S2). NM root tips decreased in response to drought and shade compared with well-irrigated and irradiated

Figure 2 The specific 15N enrichment of young beech root tips (RT) colonized by different ectomycorrhizal fungal species Figure 1 Biplot of the principal component analysis (PCA) (Cenococcum geophilum, Tuber rufum, Tomentella punicea, ordination for similarities in the relative abundances of ectomy- Tuber sp 1 and Tuber sp 2). Data show the means (n ¼ 5±s.e., corrhizal fungal species associated with the roots of young beech except n ¼ 4 for T. rufum and Tuber sp.2 under SC, n ¼ 3 for trees (Fagus sylvatica). LC ¼ full light, well-irrigated; LD ¼ full T. rufum under SD and n ¼ 10 for NM and DR). Different letters for light, drought; SC ¼ shade, well-irrigated; SD ¼ shade, drought. NM-RT indicate significant differences at Pp0.05 between The abbreviations refer to the following fungi: Cg, Cenococcum treatments. The asterisks indicate significant differences geophilum; Tp, Tomentella punicea;Tr,Tuber rufum; Tsp1, Tuber (*Po0.05, **Po0.01, ***Po0.001) compared with NM-RT. sp.1; Tsp2, Tuber sp.2; Csp, Cortinarius sp.; Ssp, Sebacina sp.; DM, dry ectomycorrhizal; DR, dry non-mycorrhizal; NM, non- Tesp, Telephoraceae; Tb, Tomentella badia;Tv,Tomentella mycorrhizal; LC, full light, well-irrigated; LD, full light, drought; viridula. Species clusters are indicated by minimum hulls. SC, shade, well-irrigated; SD, shade, drought; NS, not significant.

The ISME Journal Response diversity of ectomycorrhizal fungi R Pena and A Polle 325 plants (Figure 2), indicating that the physiological formation of new root tips and death of old root tips activity of NM root tips is highly sensitive to were negligible within our 3-day labeling period. environmental stress. Based on the total number of vital root The specific 15N enrichment of root tips colonized tips and whole plant 15N content, we calculated with C. geophilum was similar to that of NM root mean fluxes of 3.3 and 1.6 ng N h À 1 root tip À 1 for tips under LC conditions but was unresponsive to well-irradiated and shaded plants, respectively stress and therefore exceeded the 15N enrichment of (Supplementary Table S3). However, the previous NM root tips under low light and low water analyses of specific 15N enrichment (Figure 2) availability (Figure 2). implied that not all vital root tips were actively Notably, the specific 15N enrichment of root tips involved in N metabolism to the same extent under from well-irrigated beech plants colonized with different experimental treatments. We reasoned that T. punicea was even lower than that of NM root it should be possible to predict total plant 15N tips, regardless of the light level, and almost as low content by assessing the contributions of different as in DR (Figure 2). This finding suggests that EMF categories of root tips to total uptake if the specific formed with T. punicea did not significantly 15N enrichment of a given root tip category was participate in active N acquisition under LC and proportional to the N flux through this type of root SC conditions (Figure 2). However, in drought- tip. Based on this assumption, we introduced stressed beech plants, 15N accumulation in activity coefficients for each root tip category, which T. punicea-colonized root tips was increased were normalized relative to the 15N enrichment of moderately in LD root tips and strongly in SD root the root tips of NM plants (Supplementary tips and therefore significantly exceeded the 15N Information SI1). With the NM plants, independent enrichment of dead or NM root tips (Figure 2). flux measurements were obtained and used to Root tips colonized with Tuber sp. 1 and Tuber predict the fluxes of EM plants. Assessment of the sp. 2 behaved similarly to those colonized with relative contributions of the different root tip T. punicea (Figure 2). In shaded, well-irrigated categories to the total flux clearly revealed that plants, the specific 15N enrichment of the Tuber- EMF contributed less to N flux than NM root tips in colonized root tips was similar to that of DR but was well-irrigated beeches, but their relative importance significantly enriched when the shaded plants were increased strongly under drought conditions drought stressed (Figure 2). (Figure 3a). The relative contribution of EMF to N The strongest specific 15N enrichment was found flux was highest under SD conditions (Figure 3a). in root tips of light-exposed beech plants colonized The 15N content in EM plants was predicted by the with T. rufum, particularly when the plants were sum of the partial fluxes for each experimental exposed to drought stress (Figure 2). However, in treatment (LC, LD, SC and SD) (Supplementary contrast with the other two Tuber species, the Information SI1). Using a complementary approach, physiological activity of T. rufum-colonized root the 15N of NM plants was predicted based on the tips was very sensitive to shade (P ¼ 0.03). data for EM plants (Supplementary Information We repeated the experiment under stronger SI1). As the predicted 15N in EM plants was based drought stress and confirmed the behavior of the on measured 15N values in NM plants and vice EMF species with regard to the 15N enrichment in versa, the two data sets yielded independent response to the stress treatments (Supplementary predictions of 15N and therefore could be used to Figure S3). validate each other. A curvilinear relationship To test whether the 15N enrichment of ectomyco- established for the N uptake of NM plants predicted rrhizas was related to fungal biomass, the fungal the calculated N uptake of EM plants with a highly mantle volumes of distinct EMF species were significant correlation (P ¼ 0.006, R(adjusted) ¼ 92%). calculated and related to the total amount of 15Nin The measured values for the 15N content of the colonized root tips. A negative relationship was plants were plotted against the calculated values for found between the amount of 15N in root tips and the total 15N uptake (Figure 3b). A linear regression fungal mantle volume, suggesting that thicker fungal model with a slope of 1 was highly significant mantle structures led to a dilution rather than an (Figure 3b). This result supports the notion that the enrichment of the 15N label (Supplementary specific enrichment of 15N in root tips is an indicator Figure S4). Greater 15N enrichment is therefore not of flux through the root tip. a result of greater ectomycorrhizal biomass.

Discussion Modeling plant N uptake by partial N flux assessment for different ectomycorrhizal taxa EMF assemblages show interspecific differences for As the beech plants received 15N-labeled ammonium N acquisition and response diversity to stress as the sole N source during the last 3 days before We documented clear differences in the N acquisition harvest, the 15N content of the entire plant was the by ectomycorrhizas of different identities and result of the net N flux through all active root provided evidence that uptake through ectomy- tips during this time period. We assumed that the corrhizas increased under drought stress and

The ISME Journal Response diversity of ectomycorrhizal fungi R Pena and A Polle 326 emanating hyphae and known saprotrophic capa- cities, such as Cortinarius sp. and Tomentella viridis, ultimately accumulated more N from degrad- ing leaf litter compared with EMF with short extraradical mycelium, indicating spatiotemporal differentiation for access to a complex organic N source (Pena et al., 2013a). In our study, the aforementioned species were also present but were rare, suggesting that beech trees may foster fungal species without immediate benefits because the þ supply with NH4 does not require the degradation of organic matter. Furthermore, long distance transport of N via fungal rhizomorphs is carbohydrate demanding (Ekblad et al., 2013). Therefore, long distance EMF are probably less favored by small plants with limited light resources than short distance EMF. þ In temperate forests, NH4 is an important soluble inorganic N source, rapidly taken up by beech trees (Gessler et al., 1998). In our experiment, we used an þ NH4 concentration similar to that found in the Tuttlingen forest (Dannenmann et al., 2009), where the soil for beech mycorrhizal inoculation was sampled. The beech trees in the Tuttlingen forest are colonized by a characteristic EMF flora, includ- ing all species present in this study (Bue´e et al., 2005; Pena et al., 2010; Lang et al., 2011). Intact þ roots of these trees showed saturation of NH4 þ uptake above a threshold of 50 mM NH4 when þ exposed to feeding solutions with increasing NH4 concentrations (Gessler et al., 2005). As the plants in þ our experiment were acclimated to NH4 concentra- Figure 3 Relative contribution of different root tip categories to tions well above this threshold, the pronounced the total N flux (a) and correlation of the predicted and measured þ N uptake of young beech (Fagus sylvatica) trees (b). EM, interspecific differences in EMF NH4 accumulation ectomycorrhizal plants; NM, non-mycorrhizal plants; LC, full were unexpected. T. rufum and T. punicea exhibited light, well-irrigated; LD, full light, drought; SC, shade, well- the greatest contrast in N acquisition. T. punicea irrigated; SD, shade, drought. Data indicate means (n ¼ 5). forms rhizomorphs (Agerer, 2001), which have typically been associated with water transport and decreased in strongly shaded plants, with pro- improvement of host water status (Duddridge et al., nounced interspecific differences. These results 1980; Brownlee et al., 1983; Plamboeck et al., 2007). support our initial hypotheses and may have wider Here, we showed that in addition to possible ecological implications for ecosystem functions functions in water transport, T. punicea is important when anthropogenic disturbances lead to species for nutrient supply under stress in shaded plants. In loss. For example, strong dominance of the uni- contrast, T. rufum, which displayed the highest versal species C. geophilum in drought-stressed specific N enrichment under full light, was highly environments may be less beneficial with regard to sensitive to shade. Similar to our results, this N nutrition than associations with T. punicea. species was frequently found on the root tips of Previous analyses of natural carbon and N isotope sun-exposed beech trees and completely lost after discrimination have mainly focused on functional long-term shade exposure (Druebert et al., 2009). As differences of EMF species for the utilization of T. rufum has a thin mantle, it is unlikely that different resources (Zeller et al., 2007; Ho¨gberg carbohydrate demand for colonization and main- et al., 2008; Tedersoo et al., 2012b). In contrast, tenance is responsible for this behavior (Markkola studies investigating the utilization of the same et al., 2004). resource by distinct taxa in the same environment The ascomycetes, C. geophilium and three truffle þ are scarce, but this information is important for our species actively participated in NH4 uptake but understanding of the functional redundancy of EMF showed divergent behavior for N acquisition in as contributors to ecosystem resilience. For exam- response to drought and shade. Although the small ple, in a forest community, most ectomycorrhizas, number of species precludes numerical analyses, regardless of the fungal species, showed early our results support the notion that lineage-specific enrichment of litter-derived N, likely from released classification is unsuitable as a proxy for functional solutes (Pena et al., 2013a). However, EMF with traits (Tedersoo et al., 2012b). In ecological terms,

The ISME Journal Response diversity of ectomycorrhizal fungi R Pena and A Polle 327 our findings reflect functional redundancy and (Meinen et al., 2009). EMF colonization delays response diversity (Mori et al., 2013). However, the drought-induced decrease of the plant water more extensive analyses, particularly under field potential (Beniwal et al., 2010; Pena et al., 2013b). conditions and along environmental gradients, are Although the high N flux rates of unstressed NM required to characterize distinct EMF traits in in situ plants imply that EMF is not necessary for plant N assemblages and their adaptive importance for plant nutrition, the susceptibility of NM root tips to water nutrition. limitation underlines the ecological significance of EMF in buffering plant nutrition against fluctuating environmental conditions. The underlying physio- 15N-specific enrichment in root tips as a proxy for N logical or molecular mechanisms of this advantage flux and the buffering functions of ectomycorrhizas in are unclear but may be related to improved water response to environmental stress retention by hyphae (Lehto and Zwiazek, 2011) or The high concordance of measured and predicted fungal-induced activation of osmotic solutes in roots uptake with activity coefficients lends support to (Luo et al., 2009a, b). Furthermore, fungal stress the suggestion that specific N enrichment is a tolerance is important. Among the fungi in our proxy for N transport activities at the level of the study, C. geophilum is known as a drought-tolerant root tip. This is an important result because it species (Coleman et al., 1989; Di Pietro et al., 2007). implies that stable isotope applications under It is abundant on beech roots in dry habitats (Jany field conditions can be used to assess the relative et al., 2003; Pena et al., 2010), but its function for taxon-specific contributions of EMF in assem- forest tree N nutrition has been disputed (Herzog blages to plant N nutrition after correction for et al., 2012; Kipfer et al., 2012). The current results unspecific N enrichment. Whether this model is indicate a buffering function for C. geophilum also valid for other N sources, such as nitrate, against varying environmental conditions but also amino acids or complex compounds, must be suggest that other EMF species provide greater host further investigated. benefits under water-limiting conditions. Ectomycorrhizas did not increase N uptake or In conclusion, this study shows that the analyzed þ biomass compared with non-mycorrhizal plants ectomycorrhizal taxa use NH4 in in situ assem- þ under propitious environmental conditions (Pena blages to strongly diverging degrees. As NH4 is an et al., 2013b). The EMF colonization rate and fungal important N source across temperate forest ecosys- species richness of the beech trees in our study were tems (Gobert and Plassard, 2008), its uptake must be similar to those reported in other studies with granted under different environmental conditions. young trees (Bledsoe et al., 1982; Wilson and We provide evidence that the benefits of the Harley, 1983; Ho¨gberg, 1989; Dahlberg, 2001; Izzo ectomycorrhizal assemblage for N uptake and host et al., 2006) but were much lower than in old- transfer are mainly realized under environmental growth forests, where usually 95% to 99% of the constraints. The physiological and molecular bases vital root tips are colonized by EMF (Pena et al., for the diverging responses within and between 2010; Lang and Polle, 2011; Na¨sholm et al., 2013). fungal species are currently unknown. With the Therefore, the mean N fluxes, which are similar to advent of mycorrhizal genome projects (Marmeisse those reported by others (for example, Ho¨gberg, et al., 2013), novel opportunities to uncover the 1989: approximately 4 ng N h À 1 RT À 1), may be molecular processes that are involved in response confounded by NM root tips. Our data suggest that diversity are expected. The use of molecular tools fully mycorrhizal, well-irrigated and irradiated together with stable isotope incorporation will trees would even exhibit lower N uptake than that unravel the functions of EMF assemblages, and we observed because of the low activity coefficients for expect great progress in expanding our understand- þ NH4 of the prevailing EMF. This reasoning con- ing of ecosystem resilience, which depends on both trasts with the widely accepted beneficial implica- diversity and functional redundancy. tions of EMF for plant nutrition but supports results obtained in a boreal forest (Na¨sholm et al., 2013). The authors concluded that when N but not carbon was the limiting factor, EMF could aggravate N Conflict of Interest limitation in trees (Na¨sholm et al., 2013). EMF control of the host N supply may also be a reason for The authors declare no conflict of interest. the decreased N/C ratios commonly found in EM compared with NM plants (Ducic et al.,2008; Druebert et al.,2009;Joneset al., 2009; Pena Acknowledgements et al., 2013b). We thank M Fastenrath, M Franke-Klein and T Klein for Notably, the impact of EMF on host N supply is their excellent technical assistance. We are grateful to mitigated by moderate drought. Episodes during J Dyckmans for measuring 15N (Centre for Stable Isotopes, which roots are exposed to water limitations KOSI, University of Go¨ttingen). We gratefully acknowledge frequently occur in the upper soil layer (Holst financial support by the Deutsche Forschungsgemeinschaft et al., 2009), where the majority of root tips reside (DFG, Po362/19).

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