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Received Date : 20-Nov-2013 Accepted Date : 01-Apr-2014 Article type

: Primary Research Articles

Nitrogen and phosphorus additions impact arbuscular mycorrhizal abundance and molecular diversity in a tropical montane forest.

Running head: Mycorrhizal responses to nutrient additions

Tessa Camenzind1,2, Stefan Hempel1,2, Jürgen Homeier3, Sebastian Horn1,2, Andre Velescu4, Wolfgang Wilcke5, Matthias C. Rillig1,2*

1 Institute of Biology, Freie Universität Berlin, Altensteinstr. 6, D-14195 Berlin, Germany 2 Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), D-14195 Berlin, Germany 3 Albrecht von Haller Institute of Plant Sciences, University of Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany 4 Geographic Institute, University of Bern, Hallerstr. 12, 3012 Bern, Switzerland 5 Institute of Geography and Geoecology, Karlsruhe Institute of Technology, Kaiserstr. 12, 76131 Karlsruhe, Germany

*Corresponding author: Matthias C. Rillig; tel: +49 30 838-53165; fax -53886; mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/gcb.12618 This article is protected by copyright. All rights reserved.

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Keywords: Arbuscular mycorrhizal fungi; tropical montane forest; pyrosequencing; community; fertilization; biodiversity; NUMEX; Ecuador

Abstract Increased nitrogen (N) depositions expected in the future endanger the diversity and stability of ecosystems primarily limited by N, but also often co-limited by other nutrients like phosphorus (P). In this context a nutrient manipulation experiment (NUMEX) was set up in a tropical montane rainforest in southern Ecuador, an area identified as biodiversity hotspot. We examined impacts of elevated N and P availability on arbuscular mycorrhizal fungi (AMF), a group of obligate biotrophic plant symbionts with an important role in soil nutrient cycles. We tested the hypothesis that increased nutrient availability will reduce AMF abundance, reduce species richness and shift the AMF community towards lineages previously shown to be favored by fertilized conditions. NUMEX was designed as a full factorial randomized block design. Soil cores were taken after two years of nutrient additions in plots located at 2000m above sea level. Roots were extracted and intraradical AMF abundance determined microscopically; the AMF community was analyzed by 454-pyrosequencing targeting the large subunit rDNA. We identified 74 operational taxonomic units (OTUs) with a large proportion of Diversisporales. N additions provoked a significant decrease in intraradical abundance, whereas AMF richness was reduced significantly by N and P additions, with the strongest effect in the combined treatment (39% fewer OTUs), mainly influencing rare species. We identified a differential effect on phylogenetic groups, with Diversisporales richness mainly reduced by N additions in contrast to Glomerales highly significantly affected solely by P. Regarding AMF community structure we observed a compositional shift when analyzing presence/absence data following P additions. In conclusion, N and P additions in this ecosystem affect AMF abundance, but especially AMF species richness; these changes

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might influence plant community composition and productivity and by that various ecosystem processes.

Introduction Tropical forests are characterized by exceptionally high plant diversity, hold an estimate of 30% of terrestrial carbon stocks and regulate hydrological and climatic cycles. Nevertheless, they are endangered by human activities (Avissar & Werth, 2005; Brummitt & Lughadha, 2003; Dixon et al., 1994; Wright, 2010). Increased nitrogen (N) depositions by anthropogenic activities (Galloway et al., 2008; Phoenix et al., 2006; Wilcke et al., 2013) might alter these diverse ecosystems primarily limited by N and also phosphorus (P) (Fisher et al., 2013; Tanner et al., 1998; Wright et al., 2011). The tropical mountain rainforest in southern Ecuador has been identified as biodiversity “hotspot” based on the number of plant species (Homeier et al., 2008; Myers et al., 2000) and other macroorganisms (Brehm et al., 2008; 2005). In contrast, the function and diversity of microorganisms, including the ecologically important group of mycorrhizal fungi, remains largely hidden. Arbuscular mycorrhizal fungi (AMF) - a phylum of obligate biotrophic plant symbionts that are associated with the majority of land plants (Smith & Read, 2008) - are well known to represent the dominant mycorrhizal form in tropical forests (summarized in Averill et al., 2014; e.g. Gehring & Connell, 2006; Janos, 1980; Kottke et al., 2004; Moyersoen et al., 2001). However, compared to the broad range of studies investigating functionality, physiology and diversity of AMF in temperate systems knowledge on their role in the tropics remains scarce (Alexander & Selosse, 2009), though especially their influence on carbon sequestration (Zhu & Miller, 2003) might be highly relevant in an ecosystem regarded as major global carbon sink (Dixon et al., 1994). Besides that, AMF perform numerous important ecological functions: they contribute to plant nutrition and growth, water uptake and soil stability (Rillig, 2004; Smith & Read, 2008). Within this symbiosis, the fungal partner provides nutrients to the plant in exchange for This article is protected by copyright. All rights reserved.

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carbon: the large extraradical mycelium exploits the soil and intraradical fungal structures permit nutrient exchange between the partners. In this way, mainly P and N, but also other nutrients are delivered (Hodge et al., 2010). Effects of increased nutrient depositions by anthropogenic activities on AMF are likely because elevated nutrient availability reduces the benefit provided by these symbionts. According to the functional equilibrium model, a decrease in fine roots and mycorrhizal structures is expected when soil nutrients become less limiting (Johnson, 2010), since plants then allocate resources towards structures that acquire the next most limiting resource (Bloom et al., 1985; Ericsson, 1995). Beside plant-mediated changes the fungus itself can be nutrient-limited and sensitive to changes in soil properties (Guo et al., 2012; Treseder & Allen, 2002). These effects have been evaluated in field studies (e.g. Blanke et al., 2005; Eom et al., 1999) and pot experiments (e.g. Cavagnaro et al., 2003; Olsson et al., 1997) in temperate systems. A meta-analysis by Treseder (2004) confirmed an overall decrease of AMF abundance with increased nutrient availability across studies, but also showed that observed responses of AMF to changing nutrient availabilities are contextdependent and strongly related to the initial soil nutrient status (Johnson, 2010; Johnson et al., 2003; Treseder & Allen, 2002). In tropical forests, an ecosystem characterized by strong nutrient limitations (Tanner et al., 1998; Vitousek, 1984) but also great heterogeneity in terms of plant diversity, results of fertilization experiments on AMF abundance might well differ. In addition to changes in abundance, AMF community composition may be affected. Previous studies report lower AMF species richness because of N (Egerton-Warburton et al., 2007; Wang et al., 2011) and also P fertilization (Alguacil et al., 2010; Gosling et al., 2013). Furthermore, there might be a shift in species composition, often towards a community dominated by lineages within the Glomerales (Alguacil et al., 2010; Egerton-Warburton et al., 2007), a group known to include strong and rather disturbance-tolerant competitors (Helgason et al., 2002; Ijdo et al., 2010; Lekberg et al., 2007). AMF species have been reported to differ in traits such as colonization intensity, both in roots and soil and in nutrient uptake (Maherali This article is protected by copyright. All rights reserved.

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& Klironomos, 2012; Powell et al., 2009). Thus, changes in environmental conditions might favor specific AMF lineages. Except for one agricultural study (Lin et al., 2012), available datasets on changes in AMF diversity following fertilization applied either spore counts (e.g. Egerton-Warburton et al., 2007; Wang et al., 2011), terminal restriction length polymorphism (T-RFLP) (e.g. Gosling et al., 2013) or cloning and sequencing (e.g. Alguacil et al., 2010). The latter molecular methods improve biases introduced by spore measurements, which are constrained by seasonal and species-dependent patterns (Liu et al., 2012b; Sanders, 2004), though still compared to deep-sequencing methodologies the degree in precision to describe community shifts is low (Öpik et al., 2009).

AMF community changes following nutrient additions might strongly affect a system that is characterized by high diversity, since high species richness may point to more intense specialization (Johnson & Steiner, 2000; Wright, 2002) and functional redundancy amongst species cannot be directly assumed (Isbell et al., 2011; Lyons et al., 2005; van der Heijden et al., 1998). A steep increase in diversity towards the tropics, as described for plants (Myers et al., 2000), has not been shown for AMF to the same extent. Available molecular datasets basing on T-RFLP and cloning and sequencing (Aldrich-Wolfe, 2007; Haug et al., 2013; Husband et al., 2002) as well as spore-based approaches (e.g. Cuenca & Lovera, 2010; de Carvalho et al., 2012; Stürmer & Siqueira, 2011; Tripathi & Khare, 2012) report high AMF richness in tropical forests, but still in the upper range of AMF species richness detected in temperate zones (e.g. Davison et al., 2011; Gai et al., 2009; Montesinos-Navarro et al., 2012). However, because of a worldwide sampling scheme which cannot yet be considered as spatially representative at a global scale, major gaps still exist (Kivlin et al., 2011; Öpik et al., 2010), a circumstance true for fungi in general (Arnold et al., 2000; Setaro et al., 2012; Shearer et al., 2007).

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In order to test for potential impacts of future nutrient depositions, a nutrient manipulation experiment (NUMEX) was set up in an old-growth tropical montane forest in southern Ecuador (Homeier et al., 2012; Wullaert et al., 2010). AMF have been previously shown to represent the dominant mycorrhizal symbionts in the study area (Camenzind & Rillig, 2013; Kottke et al., 2004) with a relatively high species richness revealed by cloning and sequencing (Haug et al., 2013; Haug et al., 2010). As part of NUMEX we evaluated the effects of N and P additions on AMF at the stand scale. N and P were added in moderate amounts - in the case of N approaching anthropogenic nutrient inputs expected for the next 50 years (Galloway et al., 2008; Phoenix et al., 2006; Wilcke et al., 2013) – in a full factorial randomized block design. We analyzed intraradical AMF abundance as well as the intraradical community structure in order to obtain a comprehensive picture of potential effects. We hypothesize based on findings of previous experiments and the assumption of a tropical forest characterized by N and P co-limitation (Homeier et al., 2012; Wullaert et al., 2010) that (i) additions of N and P will decrease AMF abundance; (ii) nutrient additions will decrease AMF richness, with strong impacts on a community most likely characterized by high species richness, as might be revealed by 454-sequencing (Haug et al., 2013; Öpik et al., 2009); (iii) a shift towards AMF lineages previously shown to be favored by fertilization will occur (Alguacil et al., 2010; Egerton-Warburton et al., 2007), since AM fungal species are known to differ in morphological traits (Maherali & Klironomos, 2012), soil demands (Guo et al., 2012; Johnson, 1993; Lekberg et al., 2007) and nutrient uptake strategies (Kiers et al., 2011; Munkvold et al., 2004; Powell et al., 2009).

Methods Study area The study site is located at 2030 – 2120 m a.s.l. within the Podocarpus National Park, located in the Cordillera Real, an eastern range of the South Ecuadorian Andes (3°59`S, 79°05`W) This article is protected by copyright. All rights reserved.

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(Beck et al., 2008). This area represents a hotspot of biodiversity, with more than 280 tree species described, with Lauraceae, Melastomaceae and Rubiaceae as the dominant plant families. Graffenrieda emarginata Triana (Melastomaceae) is the most abundant tree species at the site, with on average one fourth of trees belonging to this species. The vegetation type is classified as evergreen lower montane forest (Homeier et al., 2008). The climate is warm humid with an average annual temperature of 15.2°C at 1950 m a.s.l. and an annual precipitation of approx. 2000 mm. Precipitation is particularly high from April to September without a pronounced dry season. The soil is a Stagnic Cambisol (IUSS Working Group WRB 2007) with a thick organic layer up to 35 cm (Wullaert et al., 2010). The pH of the organic layer ranges from 3.8 – 5.0 (Wullaert et al., 2010).

Sampling design The fertilization experiment was set up as a fully randomized two-factor block design with four blocks, each including a 20 x 20 m plot per treatment (unfertilized control, N, P and NP) (Homeier et al., 2012). Nutrient additions of 50 kg ha−1 yr−1 of N (as urea) and 10 kg ha−1 yr−1 of P (as NaH2PO4·2H2O) were applied on the respective plots, split into two applications per year. Fertilization started in February 2008. Within each plot 6 subplots of 2 x 2 m size were marked randomly. In October 2010 we sampled every subplot taking one soil core (10 cm in depth, 5 cm in diameter), resulting in a total of 96 soil samples (6 subplots x 4 treatments x 4 blocks). We have previously shown that there is no vertical change in the amount of intraradical structures in the depth profile (Camenzind & Rillig, 2013) and therefore the samples are an adequate representation of the organic layer. Right after sampling approximately 20 root pieces of 1 – 2 cm length were separated with sterile tweezers and stored in 97% ethanol at -20°C for further molecular analyses. Since we extracted roots from organic soil no clay particles adhered to root pieces and cleaning was not necessary. The

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remaining sample was oven-dried at 40°C. All samples were transferred to Freie Universität Berlin.

Environmental data We collected information on spatial coordinates, soil properties and tree communities related to the soil samples for inclusion in the analysis. Based on maps with exact subplot locations, a distance matrix with x/y coordinates was created. Data concerning total element concentrations in the organic layer were based on samples taken in June 2009. Total C, N, P, S, K, Na, Mg, Ca, Fe, Mn, Al and Zn concentrations as well as pH values were determined as described by Wullaert et al. (2010). Furthermore, we included litter leachate collected with zero-tension litter lysimeters and analyzed them for pH, total P, ortho-PO43--P, total N, NH4+-N, NO3--,N, DON (dissolved organic N), and Cl- concentrations as described by Wullaert et al. (2010). In addition, we determined electrical conductivity with a conductimeter (Cond 315i, WTW, Weilheim, Germany) and concentrations of Ca, Mg, and K with flame atomic absorption spectroscopy (AAS, FSAAS 400, Varian, Mulgrave, Australia or ZEEnit 640, Jena Analytik, Jena, Germany) and of TOC (total organic carbon) with a TOC Analyzer (varioTOC cube, Elementar Analysensysteme, Hanau, Germany). Maps with tree species locations (>9 m diameter at breast height) were used to generate a matrix of trees within 3 m radius around every sampling location.

Morphological analyses For the determination of root length and intraradical AMF abundance, roots were quantitatively extracted from a 5 g subsample of dried soil with tweezers. Root length was analyzed using WinRhizo (version 2007, Regent Instrument Inc., Quebec, Canada). For the analysis of AMF root colonization, a representative subsample of fine roots (at least 30 root This article is protected by copyright. All rights reserved.

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pieces of 1 – 2 cm) was stained with Trypan Blue according to a modified staining protocol (Camenzind & Rillig, 2013; Phillips & Hayman, 1970). After an extensive clearing step due to the thickness and dark color of roots (1-2 days in 10% KOH at 60°C, 20-30 minutes in 20% H2O2 at room temperature), they were acidified in 1M HCl and stained for 1.5 – 2.5 hours in 0.05% Trypan Blue at 60°C. The duration of staining depended on root diameter. AMF root colonization was quantified at 200x magnification using the line-intersect method (McGonigle et al., 1990). Different intraradical AMF structures were recorded separately, including coils, arbuscules, intercellular hyphae and vesicles.

DNA extraction and sequencing For DNA extraction from roots we used the PowerSoil DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, USA). We targeted the variable region of the large subunit rDNA (LSU), since it provides high resolution but also allows alignments across all Glomeromycota (Stockinger et al., 2010). As a first step, we conducted two nested PCRs using primers proposed by Krüger et al. (2009), spanning a DNA fragment of approx. 1300bp, including the SSU, ITS and LSU region. This primer set has been shown to effectively amplify all groups within the Glomeromycota (Krüger et al., 2009; Stockinger et al., 2010). We inserted approximately 40 ng of isolated DNA in the first PCR amplification and 5 µl PCR product in the second. The following PCR conditions were applied for all PCR reactions: 95°C for 2 min, following 30 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 40 sec, following 72°C for 2 min. The high number of PCR cycles is necessary since these primers represent a mix and fewer PCR cycles amplify templates insufficiently (Kohout et al., 2014; Krüger et al., 2009; Senés-Guerrero et al., 2013). In order to limit potential errors during amplification we used the proof-reading Kappa HiFi DNA polymerase (Kappa Biosystems, Woburn, USA). Applying the relatively unspecific primers LR3 and LR0R a short fragment (~720 bp) suitable for pyrosequencing was amplified in a third PCR (based on 50 ng DNA of gel-purified PCR This article is protected by copyright. All rights reserved.

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product) covering the highly variable regions D1 and D2 within the LSU (Hofstetter et al., 2002; Liu et al., 2012a; Stockinger et al., 2010; see also http://www.biology.duke.edu/fungi/mycolab/primers.htm). The forward primer LR3 was modified with adapter A as well as the sample-specific barcodes, the reverse primer LR0R with adapter B. PCR products were pooled in equimolar amounts, purified by agarose gel extraction and sequenced by the Göttingen Genomics Laboratory on a Roche FLX 454 pyrosequencing instrument.

Sequence processing and OTU delineation Raw sequence data were processed in MOTHUR (Schloss et al., 2009). Quality control included trimming to 360 – 720 flows, the maximum deviations of primer sequences were set to 2 bp, barcode errors to zero. A denoising and preclustering step (Quince et al., 2009) was applied to correct for pyrosequencing related errors and to constrain the overestimation of OTUs. Sequences were clustered into OTUs using CROP (Hao et al., 2011). In addition to the general CROP algorithm (here corresponding to approximately 95% similarity level) we implemented a similarity level of 97% (OTUs97), since there is no consensus so far on the similarity level allowing species delineation in the LSU (e.g. Guo et al., 2012; Lekberg et al., 2013; Li et al., 2010). By that we aimed to test for potential treatment effects at higher resolution and to check for robustness of results. Representative OTU sequences (center sequence of each cluster identified by CROP) were subjected to a Basic Local Alignment Search Tool (BLASTn) (Altschul et al., 1997) against GenBank at the National Center for Biotechnology Information (NCBI – http://www.ncbi.nlm.nih.gov). Non-AMF sequences were clearly identifiable with no Glomeromycota hits. OTU abundance tables were created using the clustering files created by MOTHUR and CROP during the preclustering and OTU clustering steps, respectively. OTU singletons were removed as putative pyrosequencing errors (Becklin et al., 2012; Dickie, This article is protected by copyright. All rights reserved.

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2010; Tedersoo et al., 2010). OTUs where the BLAST search indicated a chimeric structure were removed as well. In order to confirm the placement of OTUs within the Glomeromycota and to achieve a more precisely resolved taxonomic classification we built a maximum-likelihood tree in RAxML version 7.4.2, using GTRGAMMA implementation and 1000 bootstrap analyses. Glomeromycota reference data provided by Krüger et al. (2012) in addition to the respective ten closest BLAST hits of every OTU from GenBank were used as reference data. Four BLAST hits of non-Glomeromycota OTUs from Basidiomycota and Ascomycota were inserted as outgroup. We included a constraint tree, in this case a maximum-likelihood tree built by long reference sequences including the SSU, ITS and LSU region (Krüger et al., 2012), which provides a well supported phylogenetic backbone on which shorter sequences of BLAST hits and OTUs can be placed. Rough alignments were conducted in MAFFT version 7 (Katoh & Standley, 2013) and refined in MUSCLE version 3.8.31 (Edgar, 2004). The position and quality of OTUs was checked manually in the alignment. Some OTUs exhibited large deletions, probably due to chimera formation and were removed from further analyses. Because of varying read numbers, every sample was resampled with replacement to 850 reads, mean abundance values for each OTU in each sample were calculated based on 1000 permutations (Wehner et al., 2013). The applied threshold (one third more than the lowest number of reads obtained; see Table S1 and Fig. S1) minimizes the loss of data and largely avoids “upsampling”. Further analyses were conducted with resampled OTUs, as well as with resampled OTUs97 in the case of treatment effects on richness, diversity and overall community composition. All analyses were additionally validated with the help of original non-resampled data in order to survey the impacts of resampling and the applied threshold (no differences were found in comparison to the analysis of resampled data; results not presented).

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Statistical analyses All statistical analyses were conducted in R version 2.15.0 (R Development Core Team, 2012). Beside AMF richness, Shannon´s H was calculated as diversity index using the function diversity() in R package „vegan“ (Oksanen et al., 2013). Rarefaction curves were drawn with specaccum() and rarecurve() based on non-resampled OTUs. For detailed analyses of differential fertilization effects on taxonomic groups, OTUs were classified into the respective Glomeromycota order according to their placement in the maximum-likelihood tree. Tree-based approaches provide a more reliable OTU classification, especially in the analysis of samples from an understudied ecosystem. Nevertheless, to allow for comparability we included a more conservative approach: OTUs were classified to order level according to their BLAST results in MEGAN version 4.70.4 (Huson et al., 2011). Univariate analyses of treatment effects were based on a two-way linear mixed effects model using the function lmer() in package „lme4“ (Bates et al., 2012) including N and P additions as fixed effects and Plot nested within Block as random effects. P-values were generated with cftest() implemented in package “multcomp” (Hothorn et al., 2008). Model assumptions of normality and homogeneity were tested and if necessary data were log- or sqrt-transformed, respectively. For illustrations of single treatment effects in comparison to the control the described model was applied as one-way analysis. Fertilization effects on different AMF structures were tested by a two-way multivariate analysis of variance (MANOVA), including Block and Plot as covariates. To account for spatial autocorrelation in community data due to the study design we created spatial eigenvectors by Moran Eigenvector Mapping (Caruso et al., 2012; Dray et al., 2006) in the package “spacemakeR” (Dray, 2010) and included them in the multivariate analyses. The following analyses were conducted in the R package “vegan”. Multivariate effects of fertilization treatments were analyzed by a two-way permutational multivariate analysis of variance (perMANOVA; Anderson, 2001) using the function adonis() with 999 permutations, This article is protected by copyright. All rights reserved.

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including spatial eigenvectors as covariates. The use of pyrosequencing read numbers as a measure of abundance is still under debate (Amend et al., 2010), although this is commonly used (e.g. Becklin et al., 2012; Lekberg et al., 2012). Furthermore, the application of nested PCR with a large number of PCR cycles might overestimate the abundances of most abundant OTUs. Therefore, beside Bray-Curtis dissimilarity as a distance index of abundance data we also included Jaccard similarity coefficients based on presence/absence data. Both were created with vegdist(). Results were illustrated by plotting ordination structure of a redundancy analysis (RDA) dependent on N and P additions and spatial eigenvectors as conditional parameters.

Environmental factors, the most abundant tree species as well as spatial coordinates were plotted on the ordination using envfit(). Important independent environmental variables were pre-selected by principal component analysis (PCA), selecting for ecologically relevant variables and excluding collinear predictors (Caruso et al., 2012). In order to identify whether the community composition shifted towards a new species set or rather diminished to a subset of the preexisting species pool, we conducted a nestedness analysis as well as an indicator species analysis. The function nestedness() in the package “bipartite” (Dormann et al., 2009) was used, which implements the program binmatnest developed by Rogriguez-Girones & Santamaria (2006), calculating a matrix temperature in comparison to three different null models. The position of treatments in the resulting stacked matrix was analyzed using a non-parametric Kruskal-Wallis test, following pairwise comparisons by Wilcoxon signed-rank tests (Verbruggen et al., 2012). Potential treatmentrelated indicator species were identified by the function multipatt() in the package “indicspecies”, implementing 999 permutations (De Caceres & Legendre, 2009).

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Results AMF detected in the study system The morphological analyses revealed that across all treatments on average 37 ± 19 % (mean ± standard deviation) of the root system was colonized by AMF. Paris-type AMF were clearly the dominant morphological form with 20 ± 9 % of roots colonized with coils in contrast to very few (about 1 %) arbuscules. Concerning molecular root analyses, the nested PCR successfully amplified DNA from a total of 64 samples, including samples from every plot. Four samples were removed additionally after pyrosequencing with low read numbers (

Nitrogen and phosphorus additions impact arbuscular mycorrhizal abundance and molecular diversity in a tropical montane forest.

Increased nitrogen (N) depositions expected in the future endanger the diversity and stability of ecosystems primarily limited by N, but also often co...
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