Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests

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Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests Tessa Camenzind a, b, *, Jürgen Homeier d, Karla Dietrich c, Stefan Hempel a, b, Dietrich Hertel d, Andreas Krohn a, b, Christoph Leuschner d, Yvonne Oelmann c,
rez f, Matthias C. Rillig a, b Pål Axel Olsson e, Juan Pablo Sua €t Berlin, Altensteinstr. 6, 14195 Berlin, Germany Institute of Biology, Freie Universita Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), 14195 Berlin, Germany c Geoecology, University of Tübingen, Rümelinstraße 19-23, 72070 Tübingen, Germany d €ttingen, Untere Karspüle 2, 37073 Go €ttingen, Germany Albrecht von Haller Institute of Plant Sciences, University of Go e Department of Biology, Lund University, Box 118, 22100 Lund, Sweden f Departamento de Ciencias Naturales, Universidad T
ecnica Particular de Loja, Loja, Ecuador a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 July 2015 Received in revised form 5 November 2015 Accepted 18 November 2015 Available online xxx

Studies in temperate systems provide evidence that the abundance of arbuscular mycorrhizal fungal (AMF) depends on soil nutrient availability, which is mainly explained in the context of resource stoichiometry and differential plant biomass allocation. We applied this concept to an understudied ecosystem e tropical montane forest e analyzing root and AMF abundance along an elevational gradient with decreasing nutrient availability, combined with responses to nitrogen (N) versus phosphorus (P) additions. At three sites from 1000 to 3000 m above sea-level we analyzed fine root length, AMF root colonization as well as extraradical AMF biomass (neutral lipid fatty acid 16:1u5, hyphal length and spore counts) in a nutrient manipulation experiment. We found a significant increase in root length as well as intra- and extra-radical AMF abundance with elevation. Overall, P additions significantly increased, whereas N additions decreased AMF abundance, with differential though nonsystematic changes along the elevational gradient. Strongest effects were clearly observed at the intermediate site. These findings suggest a general dependency of roots and AMF on nutrient availability, though responses to N and P additions differed from previous studies in temperate systems. In the context of future nutrient depositions, results suggest diverging responses of AMF abundance depending on site characteristics. © 2015 Published by Elsevier Ltd.

Keywords: Altitudinal gradient Arbuscular mycorrhizal fungi Root length Southern Ecuador Tropical montane forest

1. Introduction Arbuscular mycorrhizal fungi (AMF) represent an ancient group of plant symbionts, associated with 80% of land plants (Wang and Qiu, 2006). These monophyletic (Glomeromycota) and obligately biotrophic plant symbionts are regarded as keystone mutualists in terrestrial ecosystems (Rillig, 2004) and distributed worldwide € across biomes (Treseder and Cross, 2006; Opik et al., 2010). Beside pathogen protection, improved water uptake and positive effects

* Corresponding author. Institute of Biology, Freie Universit€ at Berlin, Alten-

Q1,2 steinstr. 6, 14195 Berlin, Germany. Tel.: þ49 30 838 53145; fax: þ49 30 838 53886. E-mail address: [email protected] (T. Camenzind).

on soil structure, the main function of the symbiosis is improved uptake of soil nutrients in exchange for photosynthetic carbon (Smith and Read, 2008). Intraradical structures like coils and arbuscules allow direct plant-fungus nutrient exchange, and a large extraradical mycelium expanding beyond the root depletion zone enables efficient nutrient uptake (Parniske, 2008). Traditionally, AMF have been associated with plant P nutrition (Koide, 1991; Read, 1991), but a growing amount of studies indicates a similarly important role in the uptake of N (Hodge et al., 2010; Veresoglou et al., 2012) and other nutrients (Marschner and Dell, 1994; Lehmann et al., 2014). Consequently, soil fertility affects AMF abundance and occurrence: if carbon costs at high nutrient levels exceed the mycorrhizal benefits, plants may down-regulate AMF

http://dx.doi.org/10.1016/j.soilbio.2015.11.011 0038-0717/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Camenzind, T., et al., Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/ j.soilbio.2015.11.011

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abundance as has been shown experimentally (Carbonnel and Gutjahr, 2014 and citations therein), whereas infertile soil conditions strengthen the reliance on mycorrhizae (Johnson et al., 2015). In this context, the functional equilibrium model, which assumes that plants invest more into structures associated with the acquisition of a limiting resource, predicts that plant allocation is shifted towards belowground structures like roots and mycorrhizae in low fertility soils (Ericsson, 1995; Johnson, 2010), which has been demonstrated repeatedly: Increases in AMF abundance with declining nutrient availability along fertility gradients have been shown experimentally (Abbott et al., 1984; Bolan et al., 1984; Breuillin et al., 2010), but also along natural gradients comparing different sites (Anderson et al., 1984; Bohrer et al., 2001), successional gradients (Zangaro et al., 2014) and chronosequences (Treseder and Vitousek, 2001; Dickie et al., 2013). In contrast, available studies on altitudinal gradients, which are also characterized by decreasing nutrient availability with elevation due to lower temperatures and slow nutrient cycling (Soethe et al., 2008; Unger et al., 2010), report the opposite trend: AMF abundance decreases towards higher altitudes (Read and Haselwandter, 1981; Wu et al., 2004; Kessler et al., 2014), often associated with increased abundance of dark septate endophytes (DSE) or ericoid mycorrhizae, but also non-mycorrhizal plants (Gardes and Dahlberg, 1996; Schmidt et al., 2008; Urcelay et al., 2011). These findings were explained by decreasing temperature in parallel to declining nutrient availability, negatively influencing nutrient use efficiency and the mutual benefit of both partners (Gardes and Dahlberg, 1996; Ruotsalainen et al., 2002; Averill and Finzi, 2011). However, regarding belowground structures in general, a shift in plant investment towards root biomass was indeed observed along € rner and Renhardt, 1987; Girardin several altitudinal transects (Ko et al., 2010; Hertel and Leuschner, 2010). Additionally, multiple observational studies in altitudes up to 5200 m report high AMF root colonization values in certain areas (Schmidt et al., 2008; Liu et al., 2011; Soteras et al., 2015). Thus, patterns in mycorrhizal abundance along elevational gradients might be far more complex and variable depending on transect characteristics. Effects of soil fertility on AMF abundance can also be tested in nutrient manipulation experiments. In the light of increased nutrient deposition expected in the future due to anthropogenic activities (Galloway et al., 2008; Mahowald et al., 2008; Wilcke et al., 2013), it is important to understand impacts on this crucial symbiotic association. Field studies in temperate areas have revealed a net negative effect of N and/or P additions on AMF abundance (reviewed in Treseder, 2004) in accordance with the functional equilibrium model (Johnson, 2010), though especially the effects of N but also P additions have been shown to be heterogeneous and context dependent (e.g. Johnson et al., 2003; Blanke et al., 2005; Garcia et al., 2008). This divergence has been mainly explained by potential direct nutrient addition effects on AMF by elimination of nutrient deficiencies of the fungus itself (Abbott et al., 1984; Treseder and Allen, 2002) or changes in soil conditions (Dumbrell et al., 2010). Thus, in very low fertility soils the fungus itself might be nutrient-limited and increase in abundance following nutrient additions (Bolan et al., 1984; Treseder and Allen, 2002; Alguacil et al., 2010). Additional complexity is given by the variation of limiting nutrients among sites e mainly N, P or both in terrestrial ecosystems (Elser et al., 2007; Vitousek et al., 2010) e resulting in potential differences in AMF responses to N versus P additions (Eom et al., 1999; Johnson et al., 2003; Blanke et al., 2012). Furthermore, other factors like plant or microbial community changes may directly or indirectly influence AMF abundance (Johnson et al., 2004; Bradley et al., 2006). In this study, within the framework of a nutrient manipulation experiment in Southern Ecuador (Homeier et al., 2012, 2013), we

examined the effects of N and P additions on extra- and intraradical AMF abundance along an elevational gradient. In tropical rainforests, AMF are known to represent the dominant mycorrhizal type (Torti et al., 1997; Shi et al., 2006; McGuire et al., 2008; Averill et al., 2014), which is also reported for our study area (Kottke et al., 2004; Camenzind and Rillig, 2013). Nonetheless, most of our knowledge on AMF functionality and ecology is based on studies conducted in temperate areas (Alexander and Selosse, 2009). Available data from another nutrient manipulation experiment in the tropics (Hawaii) partly revealed predictable changes in the response of AMF abundance to N and P additions along the tested soil chronosequence, switching from N- to P-limitation (Vitousek et al., 1995), with additional divergence among the intra- and extraradical phases (Treseder and Vitousek, 2001; Treseder and Allen, 2002). In the case of elevational gradients, besides a decline in overall nutrient availability (Soethe et al., 2008; Unger et al., 2010), theory predicts a shift from P limitation at lower altitudes towards N-limited systems at high altitudes according to soil age: P originates primarily from soil weathering, thus, old systems (at lower altitudes) become depleted in P, whereas N enters the system mainly via fixation from the atmosphere and accumulates over time (Walker and Syers, 1976; Tanner et al., 1998). Though this pattern has been confirmed in general (Vitousek et al., 1995; Meir et al., 2001; Fisher et al., 2013), simple ecosystem limitation of a single nutrient (Liebig's law) was also questioned by findings of N/P co-limitations at low and intermediate altitudes (Kaspari et al., 2008; Fisher et al., 2013), as also reported from our study area (Graefe et al., 2010; Homeier et al., 2012), and the potential concomitant influence of other nutrients (Townsend et al., 2008; Wright et al., 2011; Baribault et al., 2012). We tested the hypotheses that: (1) mycorrhizal abundance (measured as the percentage of root colonization, colonized root length and extraradical AMF abundance) as well as root length will increase with elevation along the elevational gradient concurrently with decreasing soil fertility, though there might be a shift in mycorrhizal types (e.g. towards ecto- or ericoid mycorrhizae); (2) AMF abundance will generally decrease following nutrient additions, as already short-term effects were visible at least for N additions at one altitudinal level (Camenzind et al., 2014); (3) effects of N versus P additions will shift in relation to the previously described gradient of N/P- towards N-limitation with elevation (Graefe et al., 2010; Moser et al., 2011; Wolf et al., 2011; Homeier, 2013). 2. Materials and methods 2.1. Study area The study sites are located within or adjacent to the Podocarpus National Park in the Cordillera Real, the eastern range of the South Ecuadorian Andes (Beck et al., 2008; Bendix et al., 2013). This area is part of the “Tropical Andes” hotspot of biodiversity (Myers et al., 2000) and characterized by its high number of plant species (Homeier et al., 2008) as well as other organism groups (Brehm et al., 2005; Orme et al., 2005). Samples were taken at three sites along an altitudinal gradient: (1) the 1000 m site with premontane forest (Bombuscaro; 1000e1140 m, 4110 S, 78 960 W), (2) the 2000 m site with lower montane forest (Reserva San Francisco, 2050e2150 m, 3 980 S, 79 080 W) and (3) the 3000 m site with upper montane forest (Cajanuma; 2880e2960 m, 4120 S, 79170 W). Among these sites there is a complete turnover of tree species, with less than five shared species between the 2000 m site and either of the two other sites (Homeier et al., 2013). For details on plant communities see Homeier et al. (2008). The altitudinal gradient is accompanied by an increase in rainfall (2230, 1950 and

Please cite this article in press as: Camenzind, T., et al., Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/ j.soilbio.2015.11.011

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4500 mm yr1 at the three sites, respectively) and decreasing air temperature (19.4, 15.7 and 9.4  C mean annual air temperature) (Moser et al., 2007). Regarding soil characteristics, sites differ in bedrock material (for details see Martinson et al., 2013) as well as thickness of the organic layer: at 1000 m the mineral soil is only covered by litter, whereas at 2000 and 3000 m a thick organic layer exists (up to 40 cm). 2.2. Sampling design At each elevational level, a two-factor fertilization experiment with N (as urea, 50 kg ha1 yr1) and P (as NaH2PO4$2H2O, 10 kg ha1 yr1) was set up in a fully randomized block design: four blocks per site, each including a 20  20 m plot per treatment (unfertilized control, N, P, and NP). Within these plots six subplots of 2  2 m were marked randomly (Homeier et al., 2012). Fertilization started in February 2008, with applications twice per year. We randomly included three subplots per plot in our analyses (see Fig. S1). We took soil cores within the subplots (5 cm diameter, 15 cm in depth) in February 2013 at all three sites. Samples were dried at 40  C, transported to Freie Universit€ at Berlin and stored at room temperature. These samples were used for quantification of AMF root colonization as well as extraradical AMF abundance. For root length analyses, additional samples were taken with a soil corer (3.5 cm diameter, 40 cm in depth) adjacent to cores designated for AMF analyses, stored at 4  C and transferred to € t Go € ttingen. Georg-August Universita In order to enable the direct comparison of elevational levels samples were taken from the upper part of the soil, which represents the main “rooting area” (Vance and Nadkarni, 1992; Soethe et al., 2006), though it encompasses the mineral soil layer at 1000 m and the organic layer at 2000 and 3000 m. Thus, at the 2000 and 3000 m sites only roots extracted from the organic layer were included in further analyses. To allow for broader conclusions and increase the robustness of observed results we included a time series with data on intraradical AMF abundance originating from consecutive years at the 2000 m site. This extensive sampling effort was only conducted at 2000 m, since the project is mainly focused there (Wullaert et al., 2010; Homeier et al., 2012) and it is most easily accessible. We included samples from January/February 2009, 2010 and 2011 (for dataset of 2009 see Homeier et al., 2012) and an additional dataset from October 2010 (only % root colonization, see Camenzind et al., 2014) (Fig. S1). Analyses were conducted after transport of soil/root samples to Freie Universit€ at Berlin in the respective year. Fertilization effects were not expected to be confounded by seasonal variation, though minor variation in overall colonization rates might be observed, since there is no pronounced seasonality in this area but discontinuous precipitation (Bendix et al., 2006). In January/February 2009, 2010 and 2011, methodology slightly differed from the procedure described here: AMF root colonization was directly assessed on live roots classified as described below. 2.3. Root length and intraradical AMF abundance For root length analyses, samples were soaked in water and adhering soil material was removed using a 0.25 mm sieve. Live fine roots were separated under the stereomicroscope based on color, root elasticity, and the degree of cohesion of cortex, periderm and stele (Persson, 1978; Leuschner et al., 2001). Root length (of live roots) was analyzed using WinRhizo (version 2007, Regent Instrument Inc., Quebec, Canada). Cumulative fine root length per soil volume was calculated, subsequently referred to as “root length”.

3

The majority of analyzed roots belongs to trees which represent the dominant life form in this forest. For the quantification of intraradical AMF abundance, roots were extracted with tweezers from a standardized subset of soil (5 g for 2000/3000 m sites and whole soil samples for 1000 m site, dependent on the amount of roots in soil and differences in bulk densities). A representative subsample (approximately 30 root pieces of 1e2 cm) was stained with 0.05% Trypan Blue according to a modified staining protocol (Phillips and Hayman, 1970; Camenzind et al., 2014). The percentage of AMF root colonization was quantified at 200 magnification using the magnified intersections method by McGonigle et al. (1990). Different intraradical AMF structures were recorded separately, including coils, arbuscules, intercellular hyphae and vesicles. The percentage of roots colonized by AMF, as well as separate AMF structures, was calculated according to McGonigle et al. (1990). Additionally, the ratio of individual AMF structures as a proportion of total AMF structures found in roots was quantified. In order to calculate AMF-colonized root length combining AMF root colonization rates with root length data of live roots only, during the microscopic AMF quantification live and dead root intersects were differentiated by cell form and structure and only live root intersects were included. A comparison with dead/live root length data (separated under the stereomicroscope as described above) for six samples revealed a strong correlation of the relative amounts of dead and live roots identified by the two different methods (Fig. S2). Furthermore, basing AMF root length colonized on live roots only yields more reliable and comparable estimates. Both, the percentage of AMF root colonization (in %) and colonized root length by AMF (in cm3 soil) were chosen as response variables since both variables are valid measurements of AMF intraradical abundance: the latter is more accurate in representing actual AMF abundance estimates in a system, though percentage values are more often presented and provide values independent of root length effects (Hart and Reader, 2002; Treseder, 2004). 2.4. Extraradical AMF abundance For the quantification of extraradical AMF biomass, roots were removed from 10 g (at 1000 m)/2 g (at 2000 m and 3000 m) soil samples and remaining soil material was used for fatty acid analyses. Soil was then milled and 0.5 g (for soil from 2000 m to 3000 m sites) or 5.0 g (for soil from 1000 m site) used for the extraction. Unequal amounts of soil material were used for extraction due to the large difference in organic material and bulk density between sites. Lipids were extracted as described by van Aarle and Olsson (2003). Neutral lipids were purified by silica column fractionation (Bond Elut, Varian Inc., Palo Alto, CA, USA) with chloroform (van Aarle and Olsson, 2003). The resulting fractions were subjected to mild alkaline methanolysis and analyzed on a gas chromatograph with a flame ionization detector and a 50 m HP5 capillary column, according to Frostegård et al. (1993). NLFA 16:1u5 was quantified as an estimate of AMF biomass. PLFA 16:1u5 is also represented in high levels in AMF (Olsson and Johansen, 2000; Olsson and Wilhelmsson, 2000), however, in natural systems it is only of limited use since there is a strong background by bacterial derived PLFA 16:1u5 (Olsson et al., 1999; Ngosong et al., 2012). In order to test for the significance of the biomarker NLFA 16:1u5 in this system, we included spore abundance and hyphal length as additional measurements of extraradical AMF abundance for a subset of samples (control and P fertilized plots at 1000 and 2000 m). These data were used for comparisons among methods, since the applicability of the biomarker NLFA 16:1u5 in natural

Please cite this article in press as: Camenzind, T., et al., Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/ j.soilbio.2015.11.011

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systems has rarely been evaluated (Olsson et al., 1997; Ngosong et al., 2012) (for a detailed discussion and comparison of methodological approaches see Supplementary Materials). In the mineral soil at 1000 m standard methods were applied: hyphae were extracted by a modified aqueous filtration extraction method (Bardgett, 1991; Camenzind and Rillig, 2013) and spores were isolated by wet-sieving and decanting (Gerdemann and Nicolson, 1963) followed by centrifugation in a 20/60% sucrose gradient (Daniels and Skipper, 1982). In the organic layer at 2000 m methods had to be adapted to this substrate: hyphae were quantified based on a method described for hyphal quantification in tropical leaf litter (Aristizabal et al., 2004; Camenzind and Rillig, 2013), counting AMF hyphae directly in stained material of the organic layer. For spore quantification, the method was modified to allow for effective detachment of spores from organic material and centrifugation avoiding large litter fragments (for detailed methodological descriptions see Supplementary Materials). 2.5. Soil properties For the analysis of soil properties additional soil samples were taken in February 2013. Samples were obtained from all six subplots (taken with a soil corer of 3.5 cm diameter), pooled separately for each plot and transferred to University of Tübingen and Freie €t Berlin for further analyses. Here, the soil material was Universita dried at 40  C. Total concentrations of P, Al, Ba, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Sr and Zn were determined on milled soil using a microwave (Start 1500, MLS, Germany) pressure digestion with HNO3 and H2O2 as digestion agents. Base elements were determined with an Element Analyzer (ICP-OES 5300 DV, Perkin Elmer, Germany). Total soil C and N contents were determined on milled soil with an Elemental Analyzer (EuroEA, HekaTech, Germany). Nutrient storage per cm3 of substrate was calculated based on soil bulk densities 5 cm below the surface, i.e. a mixture of mineral soil and a thin layer of organic material at 1000 m and the organic layer only at 2000 and 3000 m (see Martinson et al., 2013). Soil pH was analyzed in a 1:5 (v:v) suspension of soil in deionized water.

For the analyses of nutrient additions affecting intraradical AMF abundance within consecutive sampling times at 2000 m, we also used a two-way linear mixed effects model with N and P as fixed factors including sampling time (months since the beginning of the NUMEX experiment) as covariate. As response variables percentage of root colonization and colonized root length by AMF were tested, but also root colonization rates by individual AMF structures (coils, intercellular hyphae, vesicles and arbuscules). In case of AMF structures, prior to univariate analyses overall significance was tested by two-way multivariate analyses of variances (MANOVA) incorporating sampling time and Block as covariates. Besides individual root colonization rates by AMF structures, we also analyzed the relative proportion of every structure in total AMF root colonization in order to assess changes in colonization strategies (Johnson et al., 2003; Dickson et al., 2007). All models were tested for the underlying assumptions of normality and homogeneity of variances. In case these were not met, data were log, square root or box-cox transformed. 3. Results 3.1. Elevational gradient Along the elevational gradient soil nutrient storage generally decreased with increasing elevation. Total P storage linearly decreased from 1000 to 3000 m and C/N values gradually increased with elevation. Soil pH was significantly reduced towards 3000 m (Table 1). Intra- and extraradical AMF biomass as well as root length increased with elevation (Fig. 1, Table 2). The percentage of root colonization by AMF linearly increased from an average of 25 (±5) % at 1000 m to 61 (±5) % at 3000 m. Since root length also showed an increasing trend from 0.76 (±0.16) cm3 soil at 1000 m to 1.51 (±0.24) cm3 soil at 3000 m, colonized root length by AMF likewise showed a significant increase with elevation. The amount of NLFA 16:1u5 as a proxy for extraradical AMF abundance was highly significantly increased at 2000 and 3000 m (6.63 ± 0.5 and 6.10 ± 0.6 nmol cm3 soil, respectively), with lipid contents tripled compared to at 1000 m (1.92 ± 0.2 nmol cm3 soil).

2.6. Statistical analyses All statistical analyses were conducted in R version 3.1.2. (R Core Team, 2014). For the analyses of variation in soil characteristics among sites only control plots were included, and differences analyzed by univariate analyses of variances (ANOVA) followed by Tukey's ‘Honest Significant Difference’ post hoc test. For statistical comparisons we only included relevant soil depths for this dataset (referring to the upper soil layers representing the main “rooting area”), encompassing mineral layer at 1000 m in contrast to organic layer at 2000 and 3000 m. In order to account for the underlying block design of the nutrient manipulation experiment, in the following analyses linear mixed effects models were applied including Plot nested within Block as random effects, using the function lme() implemented in the package “nlme” (Pinheiro et al., 2014). For the analysis of N and P addition effects on root length and intra- and extraradical AMF abundance along the altitudinal gradient, a three-way model including the fixed effects N, P and altitudinal level (coded as factor) was used. Interaction terms of N, P and altitude were used to test for differential effects among elevational levels. For graphical illustrations and further interpretations we analyzed effects at every elevational level separately by one-way analyses.

3.2. Effects of nutrient additions on AMF abundance along the altitudinal transect We found significant changes in abundances following N and P additions after five years of nutrient applications, with the strongest effects observed at 2000 m (Fig. 2ael, Table 2). N additions caused a significant decrease in the percentage of AMF root colonization, whereas P additions had an overall significant positive effect (Table 2). These effects were consistent at all elevational levels, also indicated by the absence of significant interaction terms of N and P with altitude (Table 2). Root length as well as AMF-colonized root length was not significantly affected by nutrient additions (Table 2). Only at 2000 m a marginal significant positive effect of N additions on root length values was detected (Fig. 2b). However, we found a significant three way interaction term of N:P:altitude in the case of AMFcolonized root length (Table 2). The amount of NLFA 16:1u5 in soil was significantly positively affected by P additions, although to different extents among the elevational levels as also indicated by the significant interaction term of P with altitude (Table 2). Strongest effects were detected at 2000 m (Fig. 2k). N additions did not cause an overall change in the amount of NLFA 16:1u5, though at 2000 m a significant decrease by N additions was observed (Table 2, Fig. 2k).

Please cite this article in press as: Camenzind, T., et al., Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/ j.soilbio.2015.11.011

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Table 1 Soil characteristics of the study sites across the elevational gradient. Nutrient contents are expressed as mg cm3 soil, mean ± standard error. Values marked with different online letters are significantly different among sites (one-factor ANOVA; P < 0.05). Soil parameter

1000 m

2000 m

3000 m

Thickness organic layer (cm)a Bulk density (g/cm3)a pH N C C/N P Ca Fe Al K Mg

e 0.91 ± 0.06 4.42a 2.86 ± 0.44a 41.5 ± 5.4b 14.8 ± 0.6c 0.165 ± 0.023a 0.387 ± 0.030a 12.13 ± 2.16a 40.38 ± 4.88a 2.31 ± 0.38b 0.93 ± 0.45a

10e30 0.18 ± 0.05 3.57b 3.13 ± 0.11a 74.5 ± 1.2a 23.9 ± 0.7b 0.088 ± 0.006b 0.108 ± 0.004b 0.21 ± 0.03b 0.82 ± 0.09b 0.28 ± 0.03b 0.12 ± 0.03b

10e40 0.11 ± 0.01 3.47c 1.77 ± 0.14b 49.5 ± 2.9b 28.3 ± 1.8a 0.055 ± 0.007c 0.146 ± 0.028b 0.11 ± 0.02c 0.44 ± 0.05c 0.17 ± 0.03c 0.09 ± 0.02b

a

Values are based on data presented in Martinson et al., 2013.

contrast, had no significant influence when analyzing the whole time series, however, as described above there was a sudden increase observed in the P treatment after five years. AMF-colonized root length did not decrease following N additions, but P additions resulted in a marginally significant positive effect (Fig. 3b, Table 3). Regarding individual AMF structures, results of the MANOVA indicated significant overall effects of N additions (P < 0.001). In separate analyses we observed significant negative effects of N additions with the strongest impact on the percentage of vesicles and intercellular hyphae (Fig. 4, Table 3). Looking at the ratio of abundances of single structures in the roots, the relative amount of coils even increased from 66 (±4) % in the control to 74 (±3) % in the N treatment (P ¼ 0.06), whereas all other structures significantly decreased (data not shown).

Fig. 1. Changes in arbuscular mycorrhizal abundance as well as root length along an elevation gradient. Mean values of standardized data (scaling the variables in the range [0,1]) are presented, including only samples from control plots.

In contrast to extraradical AMF abundance measured by NLFA 16:1u5 contents, we did not find an effect of P additions on either soil hyphal length or spore abundance at 1000 and 2000 m (Table S1). 3.3. Temporal progression of nutrient impacts at the 2000 m site By analyzing intraradical AMF abundance at 2000 m for the entire time series between 2009 and 2013 we generally confirmed results found in the fifth year of nutrient applications at this elevational level (see above), namely N decreased AMF abundance whereas P led to an increase (Fig. 3a and b, Table 3): Testing the impact of nutrient additions on the percentage of AMF root colonization we found an overall significant negative effect of N additions, even though it appeared to attenuate after five years of continuous nutrient applications (Fig. 3a, Table 3). P additions, by

4. Discussion 4.1. Patterns amongst different elevational sites The observed elevational gradient is characterized by decreasing nutrient availability with increasing altitude, which is also indicated by our data on soil nutrient storage. Total nutrient storage may not correlate with nutrient availability, but this pattern has been found as well in previous studies focusing on plantavailable nutrients (Soethe et al., 2007; Moser et al., 2011; Wolf et al., 2011; Baldos et al., 2015). This trend is most likely associated with lower temperatures, more frequent soilewater logging and high concentrations of phenolic compounds in decomposing litter that slow down decomposition and mineralization processes at higher altitudes (Kirschbaum, 1995; Wilcke et al., 2002, 2008). Furthermore, along elevational gradients a shift from P- towards N-limitation is predicted (Walker and Syers, 1976; Fisher et al., 2013). First results of NUMEX on aboveground biomass suggested N/P co-limitation at 1000 and 2000 m, switching towards N-limitation at 3000 m (Homeier et al., 2012; Homeier, 2013; Baldos et al., 2015). This pattern received additional support by leaf N:P ratios

Table 2 Effects of nitrogen (N) and phosphorus (P) additions on root length and AMF abundance along an elevational gradient. F-values are presented, asterisks indicate significant effects (linear mixed effects model; *P < 0.05, **P < 0.01, ***P < 0.001).

Degrees of Freedom Root length (cm3 soil) Colonized root length by AMF (cm3 soil) AMF root colonization (%) Extraradical AMF biomass (NLFA 16:1u5 nmol cm3 soil)

N

P

N:P

Altitude

N:Altitude

P:Altitude

N:P:Altitude

1.27 1.4 0 4.4* 1.3

1.27 0.1 0.7 4.5* 46.2***

1.27 0.1 0.5 0 0.2

2.9 1.7 6.0* 43.9*** 99.2***

2.27 2.2 0.7 1.4 0.9

2.27 0.2 0.7 2.3 21.7***

2.27 2.6 3.6* 2.2 2.9

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Fig. 2. Responses of AMF and root abundance to nitrogen (N) and phosphorus (P) additions. Changes in root length (aec), colonized root length by AMF (def), AMF root colonization rates (gei) and AMF biomass in soil (jel) following five years of nitrogen (N) and phosphorus (P) additions at three altitudinal levels are presented. Bars illustrate mean values, error bars the respective standard error. Asterisks indicate significant differences compared to the control treatment (linear mixed-effect model; P < 0.1; *P < 0.05, **P < 0.01, ***P < 0.001). Bargraphs of respective control plots are marked by reduced color intensity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

measured for several plant species (Güsewell, 2004; unpublished data). As hypothesized we observed an altitudinal increase in root length, a trend that has been reported previously (Leuschner et al., 2007; Girardin et al., 2010; Hertel and Leuschner, 2010). Contrary to previous studies on altitudinal transects (e.g. Read and Haselwandter, 1981; Wu et al., 2004) we also found an increase in AMF abundance regarding both the intra- and extraradical phase. In line with root abundance this pattern supports the hypothesis of increased plant investment in roots and mycorrhizae with decreasing nutrient availability. Distinct causal agents, however, cannot be identified by this study design, since there is strong colinearity of nutrient availability with e.g. moisture, temperature or atmospheric pressure along the altitudinal gradient (see Moser et al., 2011), which potentially affect plant productivity, enzymatic rates and plant water balance, and thereby also plant biomass

€ rner, 2007; Graefe et al., 2011). Additionally, sites allocation (Ko differ in plant community composition and soil types, and changes cannot be exclusively related to elevational effects (Homeier et al., 2013; Martinson et al., 2013). Nevertheless, our results for AMF abundance along the elevational gradient are inconsistent with available literature. Potential reasons for these deviations in results from other altitudinal transects might be (i) the comparatively moderate altitudes tested here e studies conducted by Read and Haselwandter (1981), Haselwandter (1987) as well as by Schmidt et al. (2008) tested gradients including far more extreme habitats; (ii) the methodological approach we used to quantify AMF at the community level allows more general conclusions about the whole system in comparison to analyses of single plant species (Wu et al., 2004; Schmidt et al., 2008) or plant groups (Kessler et al., 2014); (iii) site-specific characteristics independent of altitude might have influenced the

Please cite this article in press as: Camenzind, T., et al., Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/ j.soilbio.2015.11.011

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observed gradients e in most transect studies explanatory power is constrained by adequate replication of altitudinal levels. A shift towards other mycorrhizal types as described previously (Gardes and Dahlberg, 1996; Schmidt et al., 2008; Urcelay et al., 2011) was not detected; microsclerotia characteristic for DSE were occasionally found at all altitudes and ectomycorrhizal Hartig nets only at 2000 m (on average 5%), since here the dominant tree Graffenrieda emerginata harbors both mycorrhizal types (Haug et al., 2004). Ericoid mycorrhizae were only observed infrequently at 3000 m, though at other sites at this elevation they are slightly more abundant (personal observation). 4.2. Effects of nutrient additions on AMF abundance

Fig. 3. Responses in AMF abundance to nutrient additions measured over time. AMF root colonization rates (a) and colonized root length (b) in response to nitrogen (N) and phosphorus (P) additions measured in five consecutive years at the 2000 m site only are shown. Absolute differences compared to the control treatment (average control values are illustrated by dotted line, the respective standard error as grey area) are displayed. Little asterisks indicate significant differences compared to the control within sampling times (linear mixed-effect model; P < 0.05).

Table 3 Effects of nitrogen (N) and phosphorus (P) additions on intraradical AMF abundance and root length at 2000 m, including samples taken in consecutive years. F-values are presented, asterisks indicate significant effects (linear mixed-effects model; *P < 0.05, **P < 0.01, ***P < 0.001).

Degrees of Freedom Root length (cm3 soil) Colonized root length by AMF (cm3 soil) Total AMF root colonization (%) Root colonization by coils (%) Root colonization by intercellular hyphae (%) Root colonization by vesicles (%) Root colonization by arbuscules (%)

N

P

N:P

1.9 0.8 0.0 9.5* 5.5* 14.9*** 14.5*** 6.9*

1.9 4.4 4.2 1.5 0.0 3.5 0.9 4.6

1.9 0.3 0.0 0.6 0.2 0.3 0.5 1.1

Contrary to our hypothesis that nutrient additions will decrease AMF abundance, we observed an overall increase in AMF abundance following P additions, whereas N additions indeed had a negative effect. Few studies previously also reported increases following moderate P additions, mostly associated with soils characterized by very low P concentrations (Olsson et al., 1997; Treseder and Allen, 2002; Alguacil et al., 2010). But a combination with negative N effects is unexpected, since this kind of interaction has to our knowledge never been observed in temperate studies. Interestingly, as part of a fertilization experiment in the lowland tropical forest in Panama, the same pattern was revealed by Wurzburger and Wright (in press), though changes in AMF abundance were less pronounced than responses in roots. In the latter study aboveground productivity was shown to be co-limited by K, N and P (Wright et al., 2011; Santiago et al., 2012). Here, underlying mechanisms may on the one hand be interpreted from a plant perspective according to the functional equilibrium model (Johnson et al., 2003; Johnson, 2010): in this context the observed pattern can only be explained by increased plant N demand following P additions, favoring plant allocation towards AMF. This explanation implies a predominant role of AMF in N uptake, since AMF are simultaneously reduced in response to N additions. Indeed, AMF potentially play an important role in N uptake (Hodge et al., 2010; Veresoglou et al., 2012), especially in acidic soils with less mobile ammonium being the dominant N form (Guether et al., 2009; Martinson et al., 2013; Baldos et al., 2015). However, root length tending to increase at 2000 m in the N treatment contradicts the presumed reduction in plant allocation towards belowground structures, at least at this elevational level. Furthermore, at 2000 m a reduction in root colonization was associated with a proportional increase in coils, the mycorrhizal structures which are directly involved in nutrient exchange in

Fig. 4. Responses of differential intraradical AMF structures to nutrient additions. Root colonization rates of intercellular hyphae (a), coils (b), arbuscules (c) and vesicles (d) at 2000 m in response to nitrogen (N) and phosphorus (P) additions are displayed, including data from consecutive sampling times. Bars illustrate mean values, error bars the respective standard error. Asterisks indicate significant differences compared to the control (linear mixed-effect model; *P < 0.05, **P < 0.01, ***P < 0.001).

Please cite this article in press as: Camenzind, T., et al., Opposing effects of nitrogen versus phosphorus additions on mycorrhizal fungal abundance along an elevational gradient in tropical montane forests, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/ j.soilbio.2015.11.011

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Paris-type AMF (Smith and Smith, 1997; Cavagnaro et al., 2003), lending support to the notion that plant investment in AMF was not reduced (Johnson et al., 2003; Blanke et al., 2012). On the other hand, an underlying mechanism of direct fungusresponses to nutrient additions might be more likely: in this case an increase in AMF abundance in response to moderate P additions would result from P-limitation of the fungus itself (Abbott et al., 1984; Olsson et al., 1997; Treseder and Allen, 2002). Decreases following N additions in this case might be explained by detrimental treatment effects independent of N availability, as also indicated by negative effects on soil microbial biomass (Homeier et al., 2012; Baldos et al., 2015; Castillo-Monroy, personal communication). We have no indications on potential detrimental mechanisms in the N treatment based on available data, but a meta-analysis by Treseder (2008) indicated likewise net negative effects of N additions on microbial biomass, mainly discussed in the context of detrimental biochemical soil processes following N additions and reduced carbon availability. Incorporating the analysis of temporal shifts in long-term nutrient manipulation experiments as well as treatment effects on different components of the AMF symbiosis adds robustness and reliability to observed results (Johnson et al., 2003; Garcia et al., 2008), in this case especially concerning the consistency of responses to N additions across several sampling times. The time lag, though, in response to P additions points to a certain threshold of P contents in soil (or plants) which provokes responses in AMF abundance. Nevertheless, exact mechanisms causing these patterns can only be determined by follow-up experimental approaches, especially since there are additional factors to consider. For example changes in AMF community composition, which has been observed at the 2000 m site (Camenzind et al., 2014), may affect AMF abundances since AMF species differ in colonization intensities (Johnson, 1993; Helgason et al., 2002; Egerton-Warburton et al., 2007). Furthermore, the physiology of AMF in tropical forests is only poorly understood and some studies indicate potential differences in AMF traits compared to temperate systems, which need to be addressed in detail to enable a more profound interpretation of these results (Moyersoen et al., 1998, 2001; Aristizabal et al., 2004; Camenzind and Rillig, 2013). 4.3. Nutrient response shifts in AMF abundance along the elevational gradient We did not clearly confirm our third hypothesis, proposing a systematic shift in N versus P effects on AMF abundance along the elevational gradient in parallel to shifts from N/P towards N-limitation (Treseder and Allen, 2002; Johnson et al., 2003; Graefe et al., 2010; Homeier, 2013). However, significant interaction terms of N and P with altitude in the case of AMF-colonized root length and extraradical AMF biomass show differential responses among elevational levels. Interestingly, at the 1000 and 2000 m sites, which are both presumably N/P co-limited, the main effects of N and P were similar, but much more pronounced at 2000 m. This might be associated with higher root colonization intensity at lower soil fertility, making the symbiotic association more susceptible even to moderate nutrient amendments (Olsson et al., 1997; Johnson et al., 2015). The underlying mechanisms, however, might differ among these two sites due to differences in nutrient availability (Treseder and Allen, 2002; Johnson, 2010). At 3000 m, by contrast, there was no clear effect of nutrient additions on AMF abundance. However, combining the observed trend of lower root length and AMFcolonized root length in the N treatment with previously observed increased investment in aboveground biomass (tree diameter growth and fine litter production, see Homeier, 2013)

points towards shifts in plant allocation in response to N additions. This pattern would confirm the proposed N-limitation at this site (Walker and Syers, 1976; Moser et al., 2011). Thus, the predicted gradual response shift in AMF abundance along the elevational gradient might be masked by simultaneous shifts in plant allocation, but also by differences in underlying mechanisms. Additionally, it has to be considered that also other limiting nutrients may play a role (Santiago et al., 2012; Wullaert et al., 2013), whereas the relevant nutrients might even differ between plants and soil microbes (Krashevska et al., 2012). Previous nutrient manipulation experiments along fertility gradients showed gradual changes, though with quite heterogeneous site-specific effects: along the Hawaiian chronosequence Treseder and Allen (2002) verified a differential N and P effect on AMF hyphal length depending on the respective nutrient limitation of the fungus at N- versus P-limited sites, whereas Treseder and Vitousek (2001) showed at the same site rather consistent effects on AMF root colonization at another sampling time. Gradual shifts reported by Johnson et al. (2003) for an N:P gradient of grassland sites also confirmed predictable gradients, though only effects of N additions were considered. Sundqvist et al. (2014) found no clear patterns along an elevational gradient in arctic tundra, but AMF abundance was measured by PLFA 16:1u5 as the only signature, and not by using the more specific NLFA 16:1u5 (Olsson, 1999). This large variation between experiments highlights the complexity of the studied relationships most likely driven by differences in underlying mechanisms, but it also emphasizes the need of standardized experimental designs and methodologies to allow for comparisons among ecosystems and generalizations of expected outcomes. 4.4. Conclusions In summary, our data add new insights to understanding AMF functional roles by testing common theories in an understudied ecosystem type. Results confirm a general dependency of AMF and also roots on soil nutrient status in tropical montane forests. Interestingly, responses to N versus P additions differed from findings in temperate studies, whereas clear interpretations of underlying mechanisms are impeded by the lack of basic knowledge on AMF functionality in these systems. Nevertheless, our findings allow for explicit tests in future experiments to further expand our knowledge on this biome. We found differences in responses along the elevational gradient, though the predicted gradual shift were only partly observed. Most markedly, strongest effects were seen at the intermediate site, indicating that different susceptibilities in AMF abundance can be expected based on soil nutrient levels, an important factor to include in future models on the effects of increased nutrient depositions. Acknowledgments Financial support was provided by the Deutsche Forschungsgemeinschaft (DFG FOR816). We thank the Ministerio de Q3 Ambiente del Ecuador for the research permit and Nature and Culture International (NCI) in Loja for granting access to the San Francisco reserve and the research station. We thank Stavros Veresoglou for valuable support in the data analysis. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2015.11.011.

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