Disrupting ectomycorrhizal symbiosis: Indirect effects of an annual invasive plant on growth and survival of beech (Fagus sylvatica) saplings

Accepted Manuscript Title: Disrupting ectomycorrhizal symbiosis: Indirect effects of an annual invasive plant on growth and survival of beech (Fagus sylvatica) saplings Author: Regina Ruckli Hans-Peter Rusterholz Bruno Baur PII: DOI: Reference:

S1433-8319(16)30005-1 http://dx.doi.org/doi:10.1016/j.ppees.2016.01.005 PPEES 25301

To appear in: Received date: Revised date: Accepted date:

27-2-2015 26-11-2015 12-1-2016

Please cite this article as: Ruckli, R., Rusterholz, H.-P., Baur, B.,Disrupting ectomycorrhizal symbiosis: Indirect effects of an annual invasive plant on growth and survival of beech (Fagus sylvatica) saplings, Perspectives in Plant Ecology, Evolution and Systematics (2016), http://dx.doi.org/10.1016/j.ppees.2016.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Highlights

Highlights - Effects of Impatiens glandulifera on Fagus sylvatica saplings were studied. - The invasive plant reduced EM colonization rate of Fagus sylvatica saplings. - The invasive plant reduced both EM morphotype richness and diversity of saplings.

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- The invasive plant influences forest regeneration after disturbance.

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- The invasive plant releases allelochemicals affecting EM fungal community.

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*Manuscript

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Disrupting ectomycorrhizal symbiosis: Indirect effects of an annual

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invasive plant on growth and survival of beech (Fagus sylvatica)

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saplings

5 Regina Ruckli, Hans-Peter Rusterholz*, Bruno Baur

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Section of Conservation Biology, Department of Environmental Sciences, University

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of Basel, St. Johanns-Vorstadt 10, CH-4056 Basel, Switzerland

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*Corresponding author: E-mail: [email protected]

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Tel.: +41-61 267 08 30; fax +41-61 267 08 32

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ABSTRACT

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Understanding how invasive plants modify symbiotic interactions between

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ectomycorrhizal (EM) fungi and native host trees is a central goal in invasion biology.

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We examined the effect of the annual invasive plant Impatiens glandulifera on the

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EM association and performance of Fagus sylvatica saplings in a controlled field

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experiment at three sites in a deciduous forest in Switzerland. A total of 1188 one-

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year-old F. sylvatica saplings were planted either in plots invaded by I. glandulifera,

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in plots from which the invasive plant had been manually removed or in plots which

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were not yet colonized by the invasive plant. The 54 (3 x 18) plots were equally

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distributed over three sites. Saplings, including their full root systems were harvested

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after 3, 6 and 15 months. Exudates of the invasive plant were extracted from resin

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bags buried in the soil during the seedling, flowering and senescent stage of I.

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glandulifera. EM colonization on F. sylvatica saplings growing in invaded plot was

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33% lower after 3 months and 66% lower after 15 months than saplings growing in

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plots from which I. glandulifera had been removed and in uninvaded plots. Survival

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and biomass of saplings were reduced by 16% and 30% after 15 months in plots

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invaded by I. glandulifera. Analysis of the internal transcribed spacer region of fungal

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rDNA (ITS) showed that the number of EM species was highly correlated with the

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number of EM morphotypes, indicating that the latter can be considered as a surrogate

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for EM species richness on roots of F. sylvatica saplings. EM morphotype richness on

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saplings was 32% lower after 15 months in invaded plots as compared to control

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plots. Chemical analysis revealed a high amount of naphthoquinones in plots with I.

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glandulifera suggesting that this putative allelochemical can be responsible for the

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reduction in both EM colonization and morphotype richness. Our findings

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demonstrate the negative impact of an annual invasive plant on the ectomycorrhizal

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symbiosis and performance of native F. sylvatica saplings.

40 Key-words: allelopathy, deciduous forest, disturbance, naphthoquinones, plant-soil

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feedback, resin bag

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Introduction

45 The intentional and unintentional introduction of non-native species is

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considered as a major threat to native biodiversity (Pimentel et al. 2005; Pejchar and

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Mooney, 2009). Non-native species have the potential to affect ecosystems by

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changing species diversity, community structure and interactions between organisms,

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sometimes leading to the local extinction of native species (Vila et al. 2011; Pŷsek et

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al. 2012). The invasion of non-native species is frequently facilitated by

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environmental disturbance (Davis et al. 2000; Moles et al. 2012). Other factors

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including mechanisms that change the composition and abundance of microbial soil

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biota increase the success of certain invasive plant species (Reinhart and Callaway,

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2006; Weidenhamer and Callaway, 2010). Some invasive plants are also able to

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modify symbiotic interactions between soil microbial organisms and native host

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plants to their own advantage in the introduced habitats as explained by the degraded

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mutualist hypothesis (Rudgers and Orr, 2009; Barto et al. 2011). This hypothesis

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states that some invasive plants have the potential to inhibit native symbiotic

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communities leading to an indirectly reduced fitness of native plant species (Stinson

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et al. 2006; Vogelsang and Bever, 2009).

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Along with competition and herbivory, arbuscular mycorrhizal symbiosis can be

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an important factor determining the diversity of plant communities and their

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succession dynamics (van der Heijden et al. 1998; Klironomos et al. 2011; Johnson et

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al. 2012). Within an ecosystem, the mycorrhizal network plays a crucial role for the

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transport of resources (Smith and Read, 2009; Johnson et al. 2012, Simard et al. 2012).

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Ectomycorrhiza (EM) symbiosis is the key factor for the establishment and growth of

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numerous deciduous and coniferous tree species (Smith and Read, 2009; Itoo and

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Reshi, 2013). EM symbiosis increases both soil nutrient and water uptake of host trees,

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strengthens pathogen and drought resistance and thus increases the stability of forest

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ecosystems (Simard and Durall, 2004).

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Non-native plants invading natural communities have the potential to disrupt mycorrhizal symbiosis and/or to alter the associated soil fungal community (Mummey

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and Rillig, 2006; Wolfe et al. 2008; Rudgers and Orr, 2009). The invasive plants’

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impact on EM may differ from that on AM, because the two groups of mycorrhizal

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fungi are phylogenetically and functionally divergent (James et al. 2006; Smith and

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Read, 2009). For example, the invasion of the non-native Douglas fire tree

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(Pseudotsuga menziesii) or the exotic tamarisk (Tamarix sp.) into native habitats

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differently affected EM and AM mycorrhizal associations of native tree species

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(Meinhardt and Gehring, 2012; Salamón et al. 2013).

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Here our focal invasive species was Impatiens glandulifera (Himalayan

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balsam), a herbaceous annual plant native to the western Himalaya. It was first

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introduced to Europe and North America as garden ornamental plant in the middle of

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the 19th century (Beerling and Perrins, 1993). After some years, the plant became

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naturalized and began to invade riparian and disturbed habitats (Hejda and Pysek,

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2006). In the last decades, I. glandulifera has increasingly invaded deciduous and

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coniferous forests disturbed by wind throws and/or intensive forest management in

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Europe (Nobis, 2008), becoming the dominant understory species at many places. I.

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glandulifera reduces species richness of native plants and causes shifts in plant

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species composition in riparian habitats (Maule et al. 2000; Hejda and Pŷsek, 2006).

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The novel weapon hypothesis, assumes that some invasive plant species

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produce secondary metabolites (allelochemicals) that are novel in their non-native

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ranges and that this novelty provides advantages to the invasive plant in interactions

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with native plants (Callaway and Ridenour, 2004; Inderjit et al. 2011).

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Naphthoquinones, putative allelochemicals of I. glandulifera, leached from its leaves

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and exuded from the roots into the soil, inhibit the growth of mycorrhizal fungi and

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germination of seeds of native herbaceous plant species (Ruckli et al. 2014a). In a

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recent field experiment, I. glandulifera reduced the colonization rate of arbuscular

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mycorrhiza (AM) on maple saplings (30 to 43%; Ruckli et al. 2014b). However, the effect of I. glandulifera on EM fungi under natural conditions is so far unknown.

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In the present study, we examined the potential impact of the invasive I.

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glandulifera on the symbiosis between native Fagus sylvatica (European beech)

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saplings and EM fungi. Fagus sylvatica is the most abundant deciduous tree species in

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natural forest communities in Central Europe and has an obligate symbiosis with EM

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fungi (Bolte et al. 2007). Seedlings and saplings of F. sylvatica are shade-tolerant, but

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also adapted to high irradiation showing considerable phenotypic and

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ecophysiological plasticity (Parelle et al. 2006; Petritan et al. 2007).

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Based on both the degraded mutualist and the novel weapon hypotheses, we

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predicted a time-dependent reduction of both EM colonization and EM diversity on

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roots of F. sylvatica saplings in the presence of I. glandulifera. The reduction is

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expected to be a result of the increasing amount of putative allelochemicals released

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by the invasive plant in the course of the vegetation period. Furthermore, we predicted

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a reduction in survival and biomass of the F. sylvatica saplings growing in plots with

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the invasive plant as a result of both decreased EM colonization rate and morphotype

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richness on roots of the saplings.

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Methods

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Study sites and field experiment

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The experiment was carried out in three sites (each measuring 50 m x 180 m) in a mixed deciduous forest 15 km south of Basel, northern Switzerland (47°26' N,

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7°33' E). In this region, the annual temperature averages 9.6 oC and the annual

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precipitation is 1021 mm. The three forest sites (situated within 1 km2) were

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differently affected by the windstorm Lothar in 1999. Eight years after this storm, the

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canopy closure of the remaining forest trees was measured at ten randomly chosen

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points in each site using a spherical crown densitometer (Forest suppliers Inc., US;

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Table A.1). Impatiens glandulifera started to invade the sites shortly after the storm in

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spring 2000.

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In spring 2008 (8 years after invasion), six homogenous patches of I.

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glandulifera measuring 15 m x 15 m were randomly chosen in each of the three sites.

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The patches were situated 5–10 m apart from each other. In each patch, two 5 m x 5 m

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plots with similar I. glandulifera cover adjacent to each other were installed. In one of

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the two plots, all I. glandulifera individuals with their adhering roots were removed

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by hand (hereafter referred to as removed) every spring in the years 2008–2010. The

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other plot was left invaded by I. glandulifera (hereafter referred to as invaded). As an

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additional control, six 5 m x 5 m control plots were selected that were not yet invaded

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by I. glandulifera in close proximity (7–20 m) to the experimental plots in each site

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(referred to as uninvaded). These plots served as a control for the slight mechanical

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disturbance of the soil by removing I. glandulifera. To prevent colonisation of I.

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glandulifera in the not yet invaded plots, all invasive plants growing wthin a distance

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of up to 7 m to these plots were removed five times both in 2009 and 2010. Thus, the

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experimental set-up consisted of 54 plots (3 sites x 3 treatments x 6 plots).

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A total of 1188 one-year-old Fagus sylvatica saplings obtained from a commercial forest nursery (Forstbaumschule Ingold AG; seed origin: Bützberg,

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Switzerland) were planted in the plots (22 saplings in each plot) on 18–19 April 2009.

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To minimize potential edge effects, the saplings were planted in a uniform pattern

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with an area of 1 m2 at least 1 m apart from the plot edge. The distance between

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saplings was 20–25 cm. Fine roots of the F. sylvatica saplings were pruned prior to

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planting to avoid transplanting EM from the nursery into the soil of the experimental

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plots. Saplings of similar height (mean ± SE; invaded: 25.4 ± 0.5 cm, removed: 26.7 ±

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0.9 cm, uninvaded: 23.8 ± 0.8 cm) were planted in the different plots.

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Soil chemical characteristics

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To evaluate any potential influence of soil characteristics on EM colonization

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rate, EM morphotype richness, biomass and survival of the F. sylvatica saplings, five

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randomly chosen soil samples were collected in each plot using a metal cylinder (soil

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depth: 5 cm; soil volume: 100 cm3) in October 2009. The five soil samples of a plot

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were mixed and sieved (mesh width 2 mm) and dried for 48 h at 60 oC. Soil water

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content (%) was determined using the fresh to dry weight ratio. Soil pH was measured

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in distilled water (1: 2.5 soil: water; Allen, 1989). Total soil organic matter content

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(SOM) was assessed as loss on ignition of oven-dried soil at 700 oC for 24 h (muffle

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furnace K114, Sorvall Heraeus, Kendro Laboratory, Switzerland). Total soil organic

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nitrogen content was determined following the standard method of Kjeldahl using a

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SpeedDigester K-439 and KjelFlex K-360 (Büchi Labortechnik AG, Switzerland).

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Total organic phosphorus content was measured following the protocol of Sparks et

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al. (1996) using a SPEKOLR spectrophotometer (Analytik Jena, Germany).

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Sampling of plant exudates in the soil

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The amount of naphthoquinones released by I. glandulifera into the soil was determined using resin bags (Ens et al. 2010; Ruckli et al. 2014a). Two resin bags (7.5

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x 5 cm; filled with 10 g Amberlite® XAD4) were buried 10–15 cm below ground

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surface in three randomly chosen plots per treatment in each of the three sites. Bags

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were exposed for 20 days either during the seedling/juvenile stage (17 May to 6 June),

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the flowering stage (17 July to 7 August) or during the senescent stage (18 October to

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7 November). After each period, the bags were transported on ice to the lab and stored

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by −80 oC. A total of 162 resin bags (54 in invaded plots, 54 in plots with removed

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invasive plants and 54 uninvaded plots) were used. The content of the two resin bags

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exposed in each plot were pooled for the extraction of the plant exudates. The

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analyses of naphthoquinones were conducted following the procedure of Ruckli et al.

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(2014a). Data on plant exudates are presented per bag.

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EM colonisation and sapling performance

To assess the effect of I. glandulifera on EM colonization and root and shoot

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biomass of F. sylvatica saplings, three saplings were harvested in each plot after 3

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months (20 June 2009), 6 months (20 September 2009) and 15 months (21 June 2010)

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yielding a total of 54 saplings for each treatment plot at each sampling date. The

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saplings were carefully dug out with the entire root system and put in plastic bags

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together with soil from the corresponding plot. The number of viable and dead F.

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sylvatica saplings within each plot was recorded at each sampling date. At the same

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time, the number of I. glandulifera individuals were counted in one subplot measuring

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0.25 m2 in each plot invaded by the invasive plant and their biomass was assessed (n

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= 18 subplots, Table A.1).

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In the laboratory, the saplings were stored at 4 oC until further processing. Roots were separated from the shoots and the roots were washed free of soil and

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organic debris. Any adhering material was removed with forceps. A subsample of five

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living fine roots per sapling (diameter < 2 mm, length 7–9 cm) was randomly chosen

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to assess EM colonization and EM morphotype abundance. In this subsample, the

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number of mycorrhized and non-mycorrhized root tips was counted using a binocular

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microscope (16 x magnification). The dry weights of the shoots and coarse roots (> 2

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mm) were determined after oven drying (48 h, 60 oC) and that of the fine roots after

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lycophilization (48 h, −56 oC).

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Two different approaches were applied to examine the effects of I. glandulifera on EM diversity. First, morphological typing of the EM root tips on all

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saplings harvested was quantified using a microscope (magnification: 10–60). EM

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morphotypes were determined based on macroscopic features (colour of mantle, type

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of ramification, presence of rhizomorphs and extramatricial hyphae) following Agerer

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(1987–2002; Table A.2). Second, the internal transcribed spacer (ITS) region of

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fungal rDNA was analyzed to determine the genetic diversity of EM fungi and to

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assess the association between morphotype diversity and EM species. One to 15 root

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tips of each EM morphotype determined in a subsample of the F. sylvatica saplings (n

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= 36) harvested after 3 months were kept individually in Eppendorf tubes in a

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cetyltrimethyl ammonium bromide buffer (2% CTAB) at room temperature.

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Total DNA was extracted from 1–3 root tips of each morphotype using the DNeasy Plant Mini Kit (Qiagen, Hombrechtikon, Switzerland). In the few cases this

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method failed, total DNA was extracted using the miniprep method developed by

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Gardes and Bruns (1993). For EM analyses, the ITS region of fungal rDNA was

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amplified using the primer pair ITS1/ITS4 (Gardes and Bruns, 1996). PCR reaction

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consisted of the following components (for 25 µl PCR mix): 2 µl template DNA, 2.5

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µl 1 x Buffer B (Solis BioDyne, Estonia), 1 µl MgCl (20 µM), 0.5 µl d-NTPs mix (20

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µM), 0.5 µl Primers (ITS1 and ITS4; 10 µM), 0.25 µl Taq DNA polymerase

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(FIREPol, Solis BioDyne, Estonia) and 17.75 µl sterile water. Amplification was

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achieved in a Eppendorf Mastercycler Pro (Vaudaux-Eppendorf AG, Schönenbuch,

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Switzerland) under the following conditions: initial denaturation at 94°C for 5 min,

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followed by 45 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec

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and extension at 72°C for 30 sec, with a final extension at 60°C for 10 min. The

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presence of successfully amplified PCR products was checked by analysing 5 µl PCR

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products on a 1.5% agarose gel, stained with gel Red (Roth AG, Switzerland) and

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visualized under UV light. Depending on the abundance of morphotypes on fineroots,

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we analysed one to eight samples of each morphotype. PCR products were purified

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and sequenced by Macrogen Inc. (Amsterdam, the Netherlands). Using the BLASTn

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tool, DNA sequences were aligned with sequences deposited in NCBI/GenBank

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(http://www. ncbi.nlm.nih.gov) and UNITE (http://www. unite.ut.ee) databases.

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Sequences with at least 97% similarity were defined as one operational taxon unit and

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regarded as a species (Table A.3).

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Statistical analyses

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All statistical analyses were conducted using the statistical package R, version 2.12.2 (R Development Core Team 2012). To avoid spatial pseudoreplication,

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analyses were performed with the mean values of each plot. First, we applied

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regression analyses to examine the relationships between EM colonization rate and

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fineroot biomass (R2= 0.26, n = 162, P < 0.0001) and between EM morphotype

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richness and fineroot biomass (R2= 0.030, n = 162, P = 0.014). Spearman-rank

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correlations analysis was applied to assess the relationship between EM morphotype

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richness and EM species richness. Linear mixed models for temporal

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pseudoreplication (LME) were used to analyse the effect of harvesting time,

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treatment, site, assessed soil characteristics, total biomass and biomass of F. sylvatica

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shoots and main roots on the residuals of EM colonization rate and EM morphotypes.

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Harvesting time and treatment (invaded, removed, uninvaded) nested within site were

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included as fixed factors, plot nested in time as random factor, total biomass of F.

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sylvatica and soil characteristics as cofactors. The same structured LME was used to

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examine whether the invasion of I. glandulifera affects Fisher’s alpha diversity of EM

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morphotypes. To assess the effect of the invasive plant on survival of F. sylvatica

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saplings and biomass data, we used a LME as described above, but included EM

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colonization rate and number of EM morphotypes as additional cofactors to the

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model. If necessary, data were sqrt, log- or arsine-transformed to obtain normally

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distributed residuals. The linear mixed models were stepwise reduced as

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recommended by Crawley (2007). Linear regression analyses were applied to evaluate

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potential effects of I. glandulifera density on EM colonization rate, root and shoot

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biomass and survival rate of F. sylvatica saplings.

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Permutational multivariate analysis of variance (PERMANOVA; Anderson,

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2005) was used to test whether the invasion of I. glandulifera affected the EM

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morphotype community. A preliminary analysis revealed that EM communities on F.

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sylvatica roots differed among the harvesting times. Therefore, data obtained after 3,

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6 and 15 months were analyzed separately. In these analyses, site and treatment

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(invaded, removed, uninvaded) nested within site were considered as fixed factor.

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Differences in the structure of EM morphotype communities were calculated using

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Bray-Curtis dissimilarity index. Treatment effects were examined with a posteriori

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pairwise comparison using the PERMANOVA t-statistic (Anderson, 2005).

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274 Results

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Invasion of I. glandulifera resulted in a lower EM colonization rate on the fine roots of F. sylvatica saplings than on fine roots of saplings growing in plots from

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which the invasive plant had been removed and in uninvaded plots (Fig. 1 and Table

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1). This reduction increased with the time saplings were growing in I. glandulifera

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plots. After 3 months, EM colonization was 33% lower in invaded plots than both in

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plots from which I. glandulifera had been removed and in uninvaded plots (Fig. 1).

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After 6 and 15 months, the reduction of EM colonization was 61% and 66%,

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respectively (Fig. 1). The significant interactions between site x harvesting time and

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treatment[site] x harvesting time indicate that EM colonization was differently

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reduced after 3, 6 and 15 months (Fig. 1). However, the density of I. glandulifera did

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not affect the EM colonization on F. sylvatica saplings (linear regression, R2 = 0.01,

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F1,52 = 0.17, P = 0.68).

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Relationship between EM morphotypes and EM species

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The molecular ITS analysis revealed that the 17 different EM morphotypes consisted of 19 EM species (Table A.3) and that the number of EM morphotypes

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determined per F. sylvatica saplings was closely related to the number of EM species

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(rs = 0.813, n = 36, P < 0.0001; Fig. 2). The morphotype diversity can therefore be

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considered as a surrogate for the EM species richness on the roots of F. sylvatica

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saplings.

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EM morphotype diversity

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A total of 54 EM morphotypes were identified on the fineroots of F. sylvatica

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saplings (Table A.2). Considering the different harvesting times, total number of EM

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morphotypes increased from 19 after 3 months to 51 after 15 months. Overall, the

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presence of I. glandulifera reduced the number of EM morphotypes (Fig. 3). Saplings

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growing in invaded plots harboured 16–43% fewer EM morphotypes than those

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growing in the plots with removed I. glandulifera and in uninvaded plots (Fig. 3).

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Fisher’s alpha diversity of EM morphotypes per sapling was also reduced by the

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invasion of I. glandulifera (Table 1). Considering all plots, EM diversity per sapling

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decreased with time (2.48 ± 0.10 after 3 months, 2.25 ± 0.10 after 6 months, 1.62 ±

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0.07 after 15 months; Table 2).

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The number of EM morphotypes on the fineroots of single saplings decreased

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from 6.2 ± 0.2 (mean ± SE) after 3 months, to 5.2 ± 0.2 after 6 months, and 4.4 ± 0.2

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after 15 months. The significant treatment[site] x harvesting time interaction indicates

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that the number of EM morphotypes in the three treatments was differently influenced

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after 3, 6 and 15 months (Table 1 and Fig. 3).

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Naphthoquinones in the soil and their effects on EM mycorrhiza

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The chemical analysis revealed that the putative allelochemical 2-methoxy1,4-naphthoquinone could be detected in moderate quantities in the soil of plots

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invaded by I. glandulifera and in very low quantities in plots from which the invasive

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plant has been removed (Table 3). In contrast, no naphthoquinones could be found in

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soil of uninvaded plots (Table 3). Furthermore, the amount of naphthoquinones

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measured in the soil of plots invaded by I. glandulifera slightly increased from the

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seedling stage to the senescent stage of the invasive plant (Table 3). Moreover, the

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results of non-linear regression analyses revealed that EM colonization rate and EM

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morphotype richness both decreased with increasing amounts of naphthoquinones

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recorded in the soil of different plots (EM colonisation: R2 = 0.61, F2,24 = 18.62, P <

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0.001; EM morphotype richness: R2 = 0.32, F2,24 = 7.03, P = 0.004; Fig. A.4).

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EM community

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The presence of I. glandulifera changed the composition of EM morphotypes

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on roots of F. sylvatica saplings (Table 4). Furthermore, the EM morphotype

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composition differed between the three sites (Table 4). Pairwise post hoc tests

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indicated that the EM morphotype composition after 15 months tended to differ

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between saplings growing in invaded plots and those in plots with removed I.

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glandulifera (Table 4).

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Survival and biomass of F. sylvatica saplings

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F. sylvatica saplings had a lower survival rate in plots invaded by I. glandulifera than saplings growing in plots from which I. glandulifera had been

346

removed and in uninvaded plots (Table 1). The survival of the saplings was also

347

influenced by EM colonization rate, EM morphotype richness, the fineroot biomass

348

and the total organic phosphorus concentration in the soil (Table 1). After 15 months,

349

the survival rate of saplings growing in invaded plots was 19% lower than those of

350

saplings growing in plots in which I. glandulifera had been removed and in uninvaded

351

plots (Table 1 and Table 2).

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352

ip t

345

The total biomass of F. sylvatica saplings and the biomass of their roots and shoots were lower in plots invaded by I. glandulifera (46–71%) compared to those in

354

plots from which the plant had been removed and in uninvaded plots (Table 5 and

355

Table A.5). However, the density of I. glandulifera did not seem to affect the survival

356

of saplings (linear regression, R2 = 0.01, F1,52 = 0.65, P = 0.425), the total biomass of

357

saplings (R2 = 0.04, F1,52 = 1.89, P = 0.180), their shoot biomass (R2 = 0.040, F1,52 =

358

2.13, P = 0.151) and their root biomass (R2 = 0.01, F1,52 = 1.42, P = 0.240).

360 361 362

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Discussion

Our field experiment showed that both the colonization rate and diversity of

363

EM fungi on fineroots of F. sylvatica saplings were reduced in plots with the invasive

364

annual plant I. glandulifera compared to saplings growing in control plots.

365

Furthermore, F. sylvatica saplings growing in plots with the invasive plants showed a

366

reduced biomass and lower survival rate than control saplings. These induced changes

367

increased with the time elapsed since the saplings were planted.

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368

There is increasing evidence that non-native plants invade forests (Essl et al. 2011; Godoy et al. 2011; Rusterholz et al. 2012) and have the potential to cause

370

changes in forest structure by disrupting mycorrhizal associations of native tree

371

species (Wolfe et al. 2008; Meinhardt and Gehring, 2012; Ruckli et al. 2014b). The

372

magnitude of the reduction in EM colonization rate on fineroots of F. sylvatica

373

saplings (33–66%) recorded in our study is similar to that reported in EM associations

374

of other native tree species affected by biennial and perennial invasive plants (40–

375

55%; Yamasaki et al. 1998; Wolfe et al. 2008; Meinhardt and Gehring, 2012; Boudiaf

376

et al. 2013). In contrast, Urgenson et al. (2012) showed that the invasion of knotweed

377

did not affect EM colonization of Sitka spruce seedlings. However, differences in

378

study design and sampling schedule should be taken into account when different

379

studies on the effects of invasive species on EM fungi of tree species are compared.

380

The experimental approach used in the present study rules out potential differences in

381

plant performance as well as in historical and initial conditions between invaded and

382

uninvaded sites (Hulme and Bremner, 2006; Lopezaraiza-Mikel et al. 2007; Nienhuis

383

et al. 2009).

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384

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369

We found a time-dependent effect of I. glandulifera on EM colonization rate on

385

fineroots of F. sylvatica saplings (Fig. 1). The low EM colonization rate of beech

386

saplings recorded in the initial phase of the experiment (after 3 months) might be a

387

result of planting saplings with pruned roots. After 6 and 15 months, F. sylvatica

388

saplings growing in control plots showed similar EM colonization rates as reported on

389

young or mature F. sylvatica trees (Druebert et al. 2009; Goicoechea et al. 2009;

390

Beniwal et al. 2011). In plots with I. glandulifera, however, saplings harvested after 6

391

and 15 months had a significantly reduced EM colonization rate compared to those

392

sampled after 3 months (Fig. 1).

17

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393

Most studies assessed the impact of invasive plants on arbuscular mycorrhizal and not on ectomycorrhizal communities. For example, Alliaria petiolata reduced the

395

AM colonization on maple seedlings (Acer saccharum), but the invasive plant did not

396

alter the AM richness (Barto et al. 2011). Other invasive plants may even cause a

397

higher AM diversity in native plant species (Lankau and Nodurft, 2013; Lekberg et al.

398

2013). These inconsistent results may be due to species-specific responses of the plant

399

species involved and/or differences in environmental conditions between invaded and

400

control sites. However, the potential influence of invasive plants on EM morphotype

401

richness of forest trees has so far not been examined, an exception being the study of

402

Castellano and Gorchov (2012), which showed that dense stands of Alliaria petiolata

403

reduced EM diversity on planted Quercus rubor seedlings. We found a similar

404

decrease in EM morphotype diversity on F. sylvatica saplings, which might be a

405

result of the reduced EM colonization on saplings growing in invaded plots. Different

406

EM morphotypes vary in their ecological functions, e.g. in uptake of specific nutrients

407

(Baxter and Dighton, 2001; Erland and Taylor, 2002). A high diversity of EM

408

morphotypes can therefore be beneficial for the nutrient uptake, water supply and thus

409

for tree growth (Baxter and Dighton, 2005; Diagne et al. 2013). For example, biomass

410

production of Betula pendula saplings was positively related to EM diversity under

411

low fertility conditions (Jonsson et al. 2001) and the growth rate and nitrogen fixation

412

of Acacia mangium seedlings increased with increasing EM diversity (Diagne et al.

413

2013). A reduced EM diversity and altered morphotype composition could therefore

414

influence the nutrient and water supply of beech saplings and their fitness. However,

415

the composition of EM morphotypes differed only marginally between beech saplings

416

growing in invaded plots than those in plots with removed I. glandulifera. This

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417

finding suggests that the reduced EM colonization rate might contribute to the

418

reduction in biomass and survival of the beech saplings in invaded plots.

419

EM fungi were found to react sensitively to allelochemical active compounds found in leaf litter, leaves and roots of Rubus idaeus (Cote and Thibault, 1988),

421

Imperata cylindrica (Hagan et al. 2013) and Solidago canadensis (Yuan et al. 2014)

422

as well as in I. glandulifera (Ruckli et al. 2014a). Shoots and roots of I. glandulifera

423

contain the putative allelochemical 2-methoxy-1,4-naphthoquinone with antimicrobial

424

and antifungal effects (Lobstein et al. 2001; Ruckli et al. 2014a). In our field

425

experiment, this naphthoquinone was detected in moderate quantities in the soil of

426

plots invaded by I. glandulifera and in extremely low quantities in plots from which

427

the invasive plant had been removed (Table 3). In soil from uninvaded plots this

428

compound could not be detected. Furthermore, in soil with naphthoquinones both EM

429

colonization rate and EM morphotype richness were negatively related to the amount

430

of exudates (Fig. A.4). Bioassays showed that naphthoquinones obtained from I.

431

glandulifera reduce the growth of EM fungi mycelium (Ruckli et al. 2014a). One

432

explanation for these findings is that allelopathic effects of I. glandulifera inhibit EM

433

symbiosis. Evidence from other invasive plants confirms that allelopathic compounds

434

disturb EM and AM mycorrhizal symbiosis with native species (e.g. Cantor et al.

435

2011; Grove et al. 2012; Ruckli et al. 2014b).

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436

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420

Dense stands of plants change the light conditions in the ground vegetation

437

layer. Thus, a reduction in the light availability could reduce EM colonization rate and

438

EM morphotype richness on F. sylvatica saplings as well as their survival. Light

439

availability is considered a crucial factor for the establishment, survival and growth of

440

tree species in forests (Perry et al. 2008). Several studies demonstrated a positive

441

relationship between light availability and colonization of arbuscular mycorrhizal

19

Page 20 of 42

fungi (Pearson et al. 1991; Gehring, 2004; Füzy et al. 2014). However, it is so far

443

unknown whether a light-driven increase in productivity also affects the abundance,

444

diversity and community structure of ectomycorrhiza (Druebert et al. 2009). EM

445

colonization of birch and coniferous tree species were not affected by differences in

446

light conditions (Dehlin et al. 2004, Cheng et al. 2005). Furthermore, light availability

447

did not influence EM colonization of shade tolerant tree species including F. sylvatica

448

(Turner et al. 2009; Clark and St. Clair, 2011; present study). In contrast, light

449

availability seems to positively affect EM colonization of shade intolerant tree species

450

(Urgenson et al. 2012). The observed reduction in EM colonization rate and EM

451

richness can therefore not be explained by differences in light conditions between

452

plots and forest areas. Furthermore, the fact that the density of I. glandulifera had

453

neither an impact on EM colonization rate nor on the number of EM morphotypes on

454

saplings supports this interpretation. However, other factors including soil nutrients

455

that were not examined in this study and soil bacterial communities may differ

456

between invaded and uninvaded plots and could influence the success of the invading

457

plant. The contribution of these additional factors could not be assessed in this field

458

study.

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459

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442

The reduced survival rate of F. sylvatica saplings recorded in invaded plots

460

might be a result of the reduced EM colonization rate (Table 1). Saplings with a low

461

EM colonization rate might be more vulnerable to pathogen infection resulting in a

462

reduced survivorship (Smith and Read, 2009). Indeed, in the presents study, F.

463

sylvatica saplings growing in invaded plots showed a lightly increased fungal

464

infection rate relative to control saplings (data not shown). Interestingly, after 15

465

months, the biomass of saplings was lower in invaded plots than in control plots, but

466

the saplings' survival rate was not related to their biomass. In contrast, Ammer et al.

20

Page 21 of 42

(2011) reported that I. glandulifera did not affect growth and survival of Abies alba

468

und Pinus abies seedlings. The authors explained this unexpected finding by the fact

469

that the removal of Rubus fructicosus caused higher growth and survival rates of the

470

tree seedlings (Ammer et al. 2011). Furthermore, the different findings could also be a

471

result of species-specific responses to the invasive plant.

472

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Although the impact of I. glandulifera on native plant species seemed to be less severe than those of Fallopia and Solidago species (Hejda et al. 2009; Stoll et al.

474

2012), we provide experimental evidence that also an annual invasive plant reduces

475

the survival of F. sylvatica saplings. This effect was similar in the three sites with

476

different degrees of disturbance. A possible explanation for this finding is that the

477

invasive plant negatively affects EM symbiosis of F. sylvatica saplings. It has to be

478

established whether this is a widespread phenomenon in forests frequently disturbed

479

by logging. Then the spread of I. glandulifera in mixed deciduous forests could be

480

another serious threat to forest regeneration. Several other tree species are connected

481

to the same EM network as F. sylvatica (Lang et al. 2011). Thus, further tree species

482

could be negatively affected by I. glandulifera. In the long-term, the negatively

483

affected EM symbiosis may destabilize forest ecosystems.

485 486

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484

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Acknowledgements

487

We thank S. Egli and S. Hutter for advice concerning EM fungi determination and J.

488

Küng, A. Lenz, E. Lischer, M. Mühlebach, V. Ruckli and M. Schefer for field

489

assistance. G. Glauser (Chemical Analytical Service of the Swiss Plant Science Web,

490

University of Neuchâtel) conducted the chemical analysis of the plant exudates. We

491

are grateful to A. Baur, D. Blumenthal, S. van der Linde and two anonymous

21

Page 22 of 42

492

reviewers for their constructive comments on the manuscript. Financial support was

493

received from the Velux Foundation, Zürich.

494 495

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32

Page 33 of 42

Fig. 1. EM colonization (%) on fineroots of Fagus sylvatica saplings growing in plots

752

invaded by Impatiens glandulifera, in plots from which the invasive plant had been

753

removed and in uninvaded plots in the three sites. Saplings were harvested after 3

754

months, 6 months and 15 months. Means ± SE (n = 6) are shown. Different letters

755

indicate significant differences among treatments (invaded, removed, uninvaded)

756

within each site and for each harvesting time (Tukey’s HSD, P < 0.05).

ip t

751

cr

757

Fig. 2. Relationship between the number of EM morphotypes and the number of EM

759

species revealed by rDNA (ITS) analysis found on fineroots of Fagus sylvatica

760

saplings (n = 36) sampled after 3 months.

an

us

758

761

Fig. 3. Number of EM morphotypes on fineroots of Fagus sylvatica saplings growing

763

in plots invaded by Impatiens glandulifera, in plots from which the invasive plant had

764

been removed and in uninvaded plots in the three sites. Saplings were harvested after

765

3 months, 6 months and 15 months. Means ± SE (n = 6) are shown. Different letters

766

indicate significant differences among treatments (invaded, removed, uninvaded)

767

within each site and for each harvesting time (Tukey’s HSD, P < 0.05).

Ac ce pt e

d

M

762

33

Page 34 of 42

Ac ce p

te

d

M

an

us

cr

ip t

Figure 1

invaded removed uninvaded

Site 1

EM colonization (%)

100

100

80 b

b

b

b

Site 2

80

b

60

a

a

a

6 months

15 months

Harvesting time

0

b b

60 40

b

b b

a

a

20

3 months

0 6 months

15 months

Harvesting time

b

a

a

20 3 months

Site 3

80

b 40

a

20 0

b

b

b b a

b b

60 40

100

Page 35 of 42 3 months

6 months

15 months

Harvesting time

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ip t

Figure 2

us

10

an M

8

te

d

7 6

Ac ce p

Number of EM species

9

5 4 3 2 1 1

2

3

4

5

6

7 Page 36 of 42

Number of EM morphotypes

8

Ac ce p

te

d

M

an

us

cr

ip t

Figure 3

Number of EM morphotypes

Site 1

10

10

8 6

b b

b b a

4

a

2 0

Site 2

10

b

8

b b

a

invaded removed

6

a

8

b b

a

a b

a 4

b

a

0 6 months 15 months Harvesting time

6

b b

a a

b b

4

a

a

2

2 3 months

uninvaded

Site 3

3 months

0 6 months 15 months Harvesting time

3 months

Page 37 of 42 6 months 15 months Harvesting time

cr

ip t

Table 1

us

Table 1

Summary of LME analyses on EM colonization, number of EM morphotypes, EM diversity (Fisher’s alpha) and survival of F. sylvatica

EM colonization (%)

an

saplings.

Number of EM morphotypes

F

P

d.f.

F

Site

2,118

11.54

<0.0001

2,122

Treatment[site]

6,118

34.56

<0.0001

Harvesting time

2,10

15.01

0.0001

EM colonization (%)

†

†

Number of EM morphotypes

†

†

Total biomass

–

–

†

†

1,118

6.00

–

–

1,118

7.25

0.008

4,118

8.14 3.22

Soil moisture (%) Total organic matter (%) -1

Total organic phosphorus (μg PSO4 g soil)

Ac c

Site x harvesting time Treatment[site] x harvesting time

d.f.

F

P

d.f.

F

P

34.58

<0.0001

2,135

22.15

<0.0001

2,133

9.40

0.001

6,118

30.69

<0.0001

6,135

5.84

<0.0001

6,133

3.43

0.004

2,10

20.27

<0.0001

2,10

34.86

<0.0001

2,10

14.08

0.001

†

†

†

†

†

†

†

1,133

34.78

<0.0001

†

†

†

†

†

†

†

–

–

–

–

–

–

–

–

1,133

†

†

†

–

–

–

1,135

4.08

d

ep te

Fineroot biomass (mg)

12,118

Survival of F. sylvatica (%)

P

M

d.f.

EM diversity

†

0.016 –

1,118 –

9.36

0.003

–

–

18.26

<0.0001

–

–

–

0.045

–

–

–

–

–

–

–

–

–

–

–

1,118

10.11

0.002

–

–

–

1,133

<0.0001

4,118

0.06

0.99

–

–

–

–

–

–

0.001

12,118

2.20

0.016

–

–

–

–

–

–

4.85

0.029

Significant P-values (< 0.05) are in bold – cofactor was excluded from the model † not considered in the model

Page 38 of 42

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ip t

Table 2

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Table 2

Mean ± SE of EM diversity (Fisher’s alpha) and survival of F. sylvatica saplings growing in different plots (invaded, removed, uninvaded) in the

1

After 6 months EM diversity F. sylvatica survival (%)

uninvaded

invaded

removed

uninvaded

invaded

removed

uninvaded

2.24 ± 0.27

3.32 ± 0.22

2.90 ± 0.19

2.41 ± 0.26

2.40 ± 0.27

3.06 ± 0.33

1.87 ± 0.17

1.60 ± 0.11

2.46 ± 0.29

73.5 ± 1.4

75.8 ± 6.9

84.9 ± 3.7

74.2 ± 4.6

90.9 ± 3.1

87.1 ± 3.2

65.2 ± 4.3

68.9 ± 5.3

90.2± 3.0

1.84 ± 0.08

2.83 ± 0.12

2.84 ± 0.15

2.17 ± 0.29

2.59 ± 0.31

2.61 ± 0.26

1.48 ± 0.30

1.67 ± 0.01

2.18 ± 0.31

72.0 ± 1.8

74.2 ± 7.6

81.8 ± 4.8

66.7 ± 5.5

90.2 ± 3.8

78.0 ± 6.2

60.6 ± 2.5

65.2 ± 5.5

78.8 ± 3.8

EM diversity

Ac c

After 15 months

F. sylvatica survival (%)

3

removed

ep te

F. sylvatica survival (%)

2

invaded After 3 months EM diversity

Sites

d

Harvesting time

M

an

three sites. Data from three harvests are shown (n = 6, in each case).

1.54 ± 0.14

1.95 ± 0.16

1.97 ± 0.27

1.44 ± 0.17

1.86 ± 0.12

1.67 ± 0.30

1.26 ± 0.22

1.41 ± 0.11

1.50 ± 0.16

62.1 ± 3.5

70.5 ± 6.5

67.4 ± 4.6

59.1 ± 4.4

81.8 ± 5.6

75.8 ± 5.7

53.0 ± 9.8

64.4 ± 5.3

70.5 ± 6.3

Page 39 of 42

Table 3

Table 3 Amount of 2-methoxy-1,4-naphthoquinone (µg per bag) determined in resin bags buried for 20 days in the soil of plots invaded by Impatiens glandulifera, in plots from which the invasive plant had been removed and in uninvaded plots. Sampling was

means ± SE (n = 6) are shown.

Removed

Uninvaded

Seedling/juvenile

0.606 ± 0.187

not detected

not detected

Flowering

0.741 ± 0.161

0.013 ± 0.013

not detected

Senescent

1.021 ± 0.261

an

us

Invaded

0.007 ± 0.007

not detected

Ac ce pt e

d

M

Plant age

cr

ip t

repeated three times considering I. glandulifera plants of different age. In each case,

Page 40 of 42

Table 4

Table 4 Results of PERMANOVAs testing the effects of site and treatment (invaded, removed, uninvaded) on the composition of EM morphotypes on F. sylvatica root tips after 3, 6 and 15 months (a), and pairwise post hoc tests between treatments (b).

3 months P

F

P

Site Treatment[site]

2

1.57

0.042

2.47

0.002

6

2.01

0.002

1.65

0.002

Residual

45 t

P

t

removed – invaded

0.88

0.546

1.06

uninvaded – invaded

1.72

0.002

2.11

uninvaded – removed

1.91

0.002

b) Pairwise post hoc tests between treatments

1.97

P

2.61

0.002

1.61

0.004

P

t

P

0.332

1.40

0.056

0.002

2.39

0.002

0.002

2.05

0.026

Ac ce pt e

d

M

Significant P-values (< 0.05) are in bold

F

cr

F

us

d.f.

15 months

an

Source

6 months

ip t

a) PERMANOVA

Page 41 of 42

cr

ip t

Table 5

Site

Treatment[site]

d.f 2,133 6,133 2,10 1,133 1,133 1,133 -

Root biomass F P 35.93 <0.0001 5.25 0.0001 15.00 0.001 4.91 0.028 2.87 0.115 4.48 0.036 -

an

Shoot/root F P 15.99 <0.0001 1.67 0.132 11.87 0.002 4.78 0.031 2.28 0.064 -

Ac c

Harvesting time EM colonization (%) Number of EM morphotypes Soil moisture (%) Total organic matter (%) Total organic phosphorus Site x harvesting time Treatment[site] x harvesting time

d.f. 2,131 6,131 2,10 1,131 4,131 -

Shoot biomass F P 12.72 <0.0001 4.13 0.001 19.95 <0.001 6.68 0.011 3.70 0.057 -

M

Harvesting time EM colonization (%) Number of EM morphotypes Soil moisture (%) Total organic matter (%) Total organic phosphorus Site x harvesting time Treatment[site] x harvesting time

d.f. 2,134 6,134 2,10 1,134 1,134 -

d.f. 2,135 6.135 2,10 1,135 -

Mainroot F P 38.05 <0.0001 4.60 0.001 5.36 0.026 4.28 0.041 -

Fineroot d.f. F P 2,118 9.52 0.0001 6,118 9.87 <0.0001 2,10 91.52 <0.0001 1,118 11.64 0.0001 1,118 5.26 0.024 4,118 2.11 0.084 12,118 2.71 0.003

d

Treatment[site]

Total biomass F P 25.28 <0.0001 5.61 <0.0001 17.99 0.001 6.24 0.014 -

Mainroot/fineroot d.f. F P 2,119 1.82 0.167 6,119 3.92 0.001 2.10 45.56 <0.0001 1,119 2.95 0.088 4,119 2.28 0.064 12,119 1.91 0.039

ep te

Site

d.f. 2,135 6,135 2,10 1,135 -

us

Table 5 Summary of LME analyses on different components of biomass and the proportions of shoot/root and mainroot/fineroot of F. sylvatica saplings.

Significant P-values (< 0.05) are in bold – cofactor was excluded from the model

Page 42 of 42