# Shrub encroachment modifies soil properties through plant resource economics traits

Lucía Laorden-Camacho, Karl Grigulis, Elena Tello-García, Blandine Lyonnard, Marie-Pascale Colace, Christiane Gallet, Ulrike Tappeiner, Georg Leitinger, Sandra Lavorel

► To cite this version:

Lucía Laorden-Camacho, Karl Grigulis, Elena Tello-García, Blandine Lyonnard, Marie-Pascale Colace, et al.. Shrub encroachment modifies soil properties through plant resource economics traits. Plant and Soil, 2025, 514 (2), pp.2083-2104. 10.1007/s11104-025-07506-3. hal-05389328

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# Shrub encroachment modifies soil properties through plant resource economics traits

Lucía Laorden-Camacho* · Karl Grigulis · Elena Tello-García ·

Blandine Lyonnard · Marie-Pascale Colace · Christiane Gallet ·

Ulrike Tappeiner · Georg Leitinger · Sandra Lavorel

Received: 21 November 2024 / Accepted: 24 April 2025

© The Author(s) 2025

## Abstract

*Background and aims* Shrub encroachment alters ecosystem functions. Yet, changes in plant community traits and soil properties along succession from grassland to shrubland in European mountains are poorly understood.

*Methods* We used a trait-based approach to investigate the indirect effects of shrubs from community weighted means (CWM) of plant traits to soil properties along a gradient of encroachment in subalpine grasslands at two sites in the Alps. We hypothesized that increasing shrub density shifts plant communities towards more conservative traits, which nonlinearly increases carbon sequestration and impacts nutrient cycling. We tested our hypothesized model of indirect effects using structural equation models, which

accounted for biomass allocation to leaves and stems in CWM calculations.

*Results* As expected, CWM dry matter content (DMC) increased and CWM of nitrogen (N) and phosphorus (P) decreased with increasing shrub biomass. Increasing CWM DMC resulted in increasing soil C:N ratio and soil organic matter (SOM) concentration, and decreasing pH. Decreasing CWM P resulted in decreasing soil available P, but changes in CWM N had no effect on available N. There was no indication of nonlinear changes.

*Conclusion* This study demonstrates that with shrub encroachment plant communities gradually become more conservative with tougher and nutrient-poor tissues, which leads to soil acidification, SOM accumulation and lower P availability. We also demonstrate that DMC, an easy measurable trait, is a sufficient indicator for effects of plant tissue quality on soils of shrub encroached subalpine grasslands and could be used in future trait-based models, allowing projections under climate change scenarios.

**Keywords** Shrub encroachment · Plant functional traits · Plant economics spectrum · Plant-soil interactions

## Introduction

Shrub encroachment is a global process that profoundly impacts ecosystem functioning (Myers-Smith

Responsible Editor: Wei Sun.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s11104-025-07506-3.

L. Laorden-Camacho (✉) · E. Tello-García · U. Tappeiner · G. Leitinger ·
Department of Ecology, University of Innsbruck, Sternwartestraße 15, Innsbruck 6020, Austria
e-mail: lucia.laorden-camacho@uibk.ac.at

K. Grigulis · B. Lyonnard · M.-P. Colace · C. Gallet · S. Lavorel ·
Laboratoire d'Écologie Alpine, UMR 5553, Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, 38000 Grenoble, France
et al. 2011; Naito & Cairns 2011; Stevens et al. 2017; Van Auken 2009; Wookey et al. 2009). Increased shrub cover has been linked to multiple drivers, including climate warming in arctic and subarctic tundra (Cannone and Malfasi 2024; Elmendorf et al. 2012; Myers-Smith and Hik 2018), increased rainfall in savanna (García Criado et al. 2020; Venter et al. 2018), or recovery from disturbances like fires or anthropogenic pressures (D’Odorico et al. 2012; Eldridge et al. 2011). In European mountains, increasing temperature and land use change (i.e. grazing cessation) since the 1950s have facilitated the colonisation by shrub species of grass-dominated ecosystems (Ameztegui et al. 2016; Anthelme et al. 2007), including the Pyrenees (Gartzia et al. 2014) and the Alps (Cannone et al. 2007). Subalpine grasslands are key providers of multiple ecosystem services such as fodder production, and regulation of climate, water quality or soil fertility (Lavorel et al. 2017), even far beyond mountain boundaries (Schirpke et al. 2019). Shrub encroachment is expected to alter the biogeochemical functioning of these grasslands through indirect effects from plant communities to soil properties, and thereby to impact the provision of important ecosystem services.

Recent studies have increased our understanding of the functional impacts of shrub encroachment. Increased shrub density modifies plant community structure (Illa et al. 2017), plant-soil stoichiometry (Urbina et al. 2020), soil properties like soil bulk density, soil compressive strength and soil shear strength (Li et al. 2016; Liu et al. 2023) and water balance (Liu et al. 2022). These changes in plant and soil properties have also been associated with changed soil microbial communities (Broadbent et al. 2022; Grau et al. 2019; Li et al. 2017; Urbina et al. 2020). However, the complexity, site-specificity and the multivariate nature of these multiple processes makes it difficult to generate comprehensive understanding. For example, while some studies have shown that shrub encroachment decreases nutrient availability (Urbina et al. 2020), others have found increased soil fertility with leguminous shrub presence (Yang et al. 2024b) or in semi-arid savanna (Ward et al. 2018). Eldridge et al. (2011) showed that in arid ecosystems shrub height and shape could be linked to microclimate and soil fertility, also consistent with expectations of shrub effects on microclimate in tundra ecosystems (Myers-Smith et al. 2019). Moreover, few

studies have investigated the underlying mechanisms of aboveground-belowground linkages in response to shrub expansion (Broadbent et al. 2022; Grau et al. 2019; Urbina et al. 2020). These studies have shown that shrub expansion results in stoichiometric changes in aboveground plant tissues which are coupled with changes in soil nutrients, possibly linked to nutrient allocation (e.g. nutrient accumulation in shrub species due to higher biomass) and shifts in soil microbial communities (e.g. increase of ericoid mycorrhiza fungi fostering nitrogen immobilization).

Trait-based ecology has made it possible to mechanistically link vegetation changes to ecosystem services by quantifying links between community-level plant traits and ecosystem functions that are generalizable beyond specific taxonomic composition (Lavorel 2013). The plant economics framework links shifts from acquisitive plants, with traits such as high tissue nitrogen content and associated fast growth living in environments with high resource availability, to conservative plants with high tissue density and associated slow growth in resource-poor environments (Díaz et al. 2016; Reich 2014). Because these traits are also those determining carbon and nutrient cycling processes (primary production, litter decomposition and mineralisation), this gradient in turn translates to shifts from fast to slow carbon and nutrient turnover (Lavorel and Garnier 2002). Studies of subalpine grasslands have confirmed these global patterns (Grigulis et al. 2013; Laliberté and Tyliankis 2012; Moretti et al. 2013), however its relevance to shrub ecosystems has received little attention (de Bello et al. 2010). Specifically, while subalpine shrub encroachment has established effects on soil carbon and nutrient pools (Seeber et al. 2022; Tasser et al. 2005; Urbina et al. 2020) these effects have not been linked to community-level shifts in plant traits. The relevance of the plant economics framework to indirect effects from shrub encroachment to nutrient cycling thus remains as a critical knowledge gap.

Shrub encroachment is considered a ‘Plant Functional Type Transformation’ with cascading effects on carbon and nutrient cycling processes (Wookey et al. 2009). Because woody plants induce a qualitative, hysteretic change in the tissue chemistry of plant communities, and hence decomposability (Hattenschwiler and Vitousek 2000), their increase in ecosystems is expected to cause nonlinear changes in carbon and nutrient cycling. Yet, research to date in
subalpine grasslands has not tested whether increas-
ing shrub density results in such nonlinear changes,
as opposed to gradually slower cycling and hence
nutrient accumulation as expected from the plant eco-
nomics spectrum. The primary effect of shrub growth
is increasing biomass, which directly increases lignin
amounts in ecosystems. Lignin is a structural and
highly polymerised compound whose concentra-
tion, and abundance relative to highly decomposable
nitrogen, has been shown to modify decomposition
of leaf and stem litter (Cornwell et al. 2008; Yang
et al. 2024a). According to the biomass ratio hypoth-
esis (Grime 1998), shrub species traits could then
be expected to have the greatest effect on ecosystem
functions. Furthermore, given the qualitative and
quantitative differences in lignin between stems and
leaves (Lourenço and Pereira 2018), effects of shrub
encroachment on soil processes requires considering
traits of entire plants, rather than just leaf traits as
done previously (Myers-Smith et al. 2019; Zuo et al.
2018). Given that litter decomposition is influenced
by the distribution of biomass across woody plant
organs (Guo et al. 2022a) the consideration of bio-
mass allocation along with traits of leaves and stems
thus appears warranted in community-level trait cal-
culations (Garnier et al. 2004).
This paper investigates the trait-based mechanisms
through which shrub expansion modifies grassland
functioning, with a focus on nutrient cycling. Using
plant trait and soil data collected along gradients
of shrub encroachment at two subalpine grassland
sites in the European Alps, we tested indirect effects
of shrub encroachment on ecosystem functions by
quantifying direct and indirect (i.e. biotic mediated
(Díaz et al. 2007)) effects of increased shrub bio-
mass on plant traits and soil properties. The aim of
our research was to test: (1) whether shrub encroach-
ment in subalpine grasslands affects soil properties
through functional changes in plant communities, and
(2) whether these indirect effects are linearly linked
to the plant economics spectrum as opposed to a
nonlinear functional shift as woody tissues become
prevalent. Most studies have analysed the functional
effect of woody species based on their presence or
absence, but to our knowledge, quantitative stud-
ies along gradients of shrub density are rare (Blaser
et al. 2014; Pornaro et al. 2017; Zhou et al. 2017), or
in some cases limited to the inclusion of one or two
intermediate successional stages from grassland to

This paper investigates the trait-based mechanisms
through which shrub expansion modifies grassland
functioning, with a focus on nutrient cycling. Using
plant trait and soil data collected along gradients
of shrub encroachment at two subalpine grassland
sites in the European Alps, we tested indirect effects
of shrub encroachment on ecosystem functions by
quantifying direct and indirect (i.e. biotic mediated
(Díaz et al. 2007)) effects of increased shrub bio-
mass on plant traits and soil properties. The aim of
our research was to test: (1) whether shrub encroach-
ment in subalpine grasslands affects soil properties
through functional changes in plant communities, and
(2) whether these indirect effects are linearly linked
to the plant economics spectrum as opposed to a
nonlinear functional shift as woody tissues become
prevalent. Most studies have analysed the functional
effect of woody species based on their presence or
absence, but to our knowledge, quantitative stud-
ies along gradients of shrub density are rare (Blaser
et al. 2014; Pornaro et al. 2017; Zhou et al. 2017), or
in some cases limited to the inclusion of one or two
intermediate successional stages from grassland to

shrubland (Crofts et al. 2018; Ding et al. 2019; Liu
et al. 2023; Urbina et al. 2020). Here, we analysed
changes in soil properties along a continuous gradient
of increasing shrub density. Our trait-based approach
targeted plant economics (leaf and stem) traits, scaled
according to plant allometries. Our hypothesized data
model (Fig. 1) articulates increasing shrub biomass
in a grassland to direct effects on plant trait commu-
nity weighted means (CWM) and to subsequent indi-
rect effects on soil properties. The model comprises
the following links. (1) Encroached grasslands store
higher amounts of aboveground phytomass compared
to managed grasslands (Seeber et al. 2022), which
results in higher aboveground stocks of carbon (C),
nitrogen (N) and phosphorus (P) (Urbina et al. 2020).
(2) Combined with the conservative traits of shrub
species and their different biochemical composition,
this increases community-level dry matter content
(DMC) and lignin concentration. (3) These changes
in community-level traits slow down nutrient cycling,
reducing soil nutrient availability. (4) Lignin-rich lit-
ter forms recalcitrant compounds, which slows down
the decomposition of soil organic matter (SOM) (Hat-
tenschwiler and Vitousek 2000; Kraus et al. 2003).
(5) Decreased decomposability in turn reduces soil
pH and (6) increases stocks of SOM of changed stoi-
chiometric quality (C:N ratio). While these mecha-
nisms have been studied individually, we tested them
as a causal chain, using structural equation modelling,
a hypothesis confirmatory approach (Shipley 2018)
(Fig. 1). We hypothesized that: 1) Shrub encroach-
ment results in a shift towards more conservative,
nutrient- poor plant communities; 2) These commu-
nity-level plant functional changes feed forward to
soil properties characteristic of slow nutrient cycling
ecosystems; 3) Linear changes in plant community
traits along the shrub density gradient result in non-
linear changes in soil properties.

**Materials and methods**

Study sites

We conducted our study at two locations in the Euro-
pean Alps: Lautaret (France) and Stubai Valley (Aus-
tria) where changes in management over the past dec-
ades have resulted in a mosaic of plant communities
(Lavorel et al. 2017).
<figure><img src="image_2.png" /></figure>

**Fig. 1** Hypothesized direct effects of shrub density on plant communities (green arrows) and indirect effects on soil properties (orange arrows). (1) Shrub biomass is expected to directly affect the nutritional quality of plant communities. (2) Shrub encroachment is expected to directly affect the community-level resource strategy and its tissue chemical composition, including recalcitrant secondary compounds. (3) Shrub encroachment is expected to have an indirect effect on soil nutritional quality. (4) The recalcitrant tissues of shrubs are

expected to affect litter quality and decomposability. Shrub encroachment is expected to have an indirect effect on (5) soil pH and (6) soil organic matter quantity and quality mediated by changes in litter quality and decomposability. In brackets we indicate measured (a) plant traits: N (nitrogen), P (phosphorus), DMC (dry matter content), Lignin; (b) soil properties: NH₄⁺ (ammonium), NO₃⁻ (nitrate), PO₄·3⁻ (phosphate), pH, SOM (soil organic matter), CN (C:N ratio)

The Lautaret site is located in the Central French Alps on the southern slopes of the Guisane valley (45.03°N, 6.24°E), at an average altitude of 1900 m above sea level (m.a.s.l.) on flysch sediment. The soils are characterized by loamy to clay-loam textures (Fig. S1a). The climate is sub-alpine with a pronounced continental influence and summer drought. Mean annual precipitation is 956 mm and mean monthly temperatures range between -7.4 °C in February and 19 °C in July (1991–2020). Significant shrub encroachment has taken place due to the extension of livestock production, with summer grazing by cattle since conversion from mowing to grazing in the 1970s (Lavorel et al. 2017). The site is divided into two grassland areas with homogenous management histories: one dominated by the grass species *Bro mus erectus* and the other by *Patzkea paniculata*. *B. erectus* grassland is colonised by the evergreen shrub *Juniperus communis* and the deciduous *Rosa spinosa*.

*P. paniculata* grassland is colonised by *J. communis* or by the deciduous *Vaccinium myrtillus* and *V. uliginosum* (Fig. 2). Both grasslands are currently lightly grazed by cattle in summer (approx. 2.5 cattle/ha for one week).

The Stubai site is located in Austria on the southern slopes of the Stubai valley (47.11°N, 11.30°E) at approximately 2000 m.a.s.l. on a siliceous substrate. The soils are characterized by loamy textures (Fig. S1b). The climate is humid continental. Mean annual precipitation is 1097 mm and mean monthly temperatures range between -9 °C in January and 12 °C in August (1991–2020). The grassland abandoned 25 years ago (Lavorel et al. 2017) is divided into two areas with homogenous management histories (i.e. mowed and grazed by cattle): one dominated by the grass species *Nardus stricta*, and other dominated by *Festuca rubra* and *Sesleria albicans*. *N. stricta* grassland is colonised by shrub deciduous
<figure><img src="image_3.png" /><figcaption>**Fig. 2** Study design at the Lautaret (L) and Stubai (S) sites. The location map indicates site locations within the European Alps. Different combinations of shrub and grass communities result in 4 types of plots at the Lautaret site and 2 types of plots at the Stubai site. Coloured square boxes indicate the main grassland communities in the plot. Lautaret has 2 types of grassland communities (green boxes) dominated by *Bromus erectus* (light green) or *Patzkea paniculata* (dark green). Stubai has 2 types of grassland communities dominated by *Nar-*</figcaption></figure>

dus stricta (gray-blue) or *Festuca rubra* and *Sesleria albicans* (bright-blue). Small icons represent the main shrub species for Lautaret and Stubai: *Juniperus communis*, *Rosa spinosa*, *Vaccinium uliginosum*, *Vaccinium myrtillus*, *Rhododendron ferrugineum*, *Calluna vulgaris*, *Vaccinium vitis-idaea*. The gradient of shrub density is represented by an inverted triangle from low (light grey) to high density (dark grey). The numbers on the inverted triangles and squares represent the number of plots

species, *V. myrtillus* and *V. uliginosum*. *F. rubra*—*S. albicans* grassland is colonised by evergreen species, *Rhododendron ferrugineum*, *Calluna vulgaris* and *V. vitis-idaea*. In both grasslands, shrubs have a diffuse distribution within the herbaceous matrix (Fig. 2).

Sampling design

We used a classic gradient approach to assess the
functional effects of increasing shrub density on plant
community traits and soil functions (Ter Braak and
Prentice 1988). Gradient designs have been shown to
outperform replicated designs to detect complex eco-
logical responses to environmental variation (Krey-
ling et al. 2018). Here, in order to account for a priori
effects of herbaceous communities and associated

colonising shrub species on ecosystem functioning,
we stratified shrub density gradients within each of
the two grassland communities at each site (Fig. 2).
At each site, we selected homogenous and relatively
flat (slope <10°) 10 × 10 m plots with increasing
shrub cover. We collected data on plant traits and soil
properties from a total of 54 plots, each with a size of
10 m× 10 m.

The Lautaret site comprised 26 plots. Within
each grassland community we sampled a gradient
of increasing shrub cover on plots encroached by *J.
communis* (from 0–68%—7 plots within each grass-
land type), by *R. spinosa* (from 0–45%—7 plots) and
by *Vaccinium* species (from 0–90%—5 plots). The
Stubai site comprised a total of 28 plots. Within each
grassland community we sampled along a gradient of
increasing shrub cover on plots encroached by *Vaccinium* species (from 9–76%—15 plots), by *R. ferrugineum* (from 39–86%—9 plots) and *C.vulgaris* (from 7–41%—4 plots). In total, the 54 plots covered a continuous gradient of increasing shrub cover from 0 to 90% (Fig. S2).

All soil samples and biomass estimates were taken at peak biomass during summer 2021. At Lautaret, herbaceous plant traits were measured during summer 2021 for *P. paniculata* plots and during summer 2022 for *B. erectus* plots, considering them as functionally distinct plant communities (Quétier et al. 2007). In Stubai, herbaceous trait measurements were spread distributed across the two grasslands in 2021 (13 plots) and 2022 (15 plots). Interannual variation was not significant at the Stubai site (Table S1).

## Biomass estimation

Estimates were made of shrub species and herbaceous standing biomass in each of the 10 by 10 m experimental plots. We used allometric techniques to estimate biomass, which are often used as a more rapid and non-destructive method opposed to the more labour-intensive harvesting ones (Lavorel et al. 2008).

Shrub cover was estimated using the point method (Levy and Madden 1933). One hundred points were recorded every meter along ten transects laid out at one-meter intervals. At each point the presence or otherwise of shrub species or herbaceous vegetation was noted in a 10 cm diameter circle around the measurement point. The percentage cover of each component of the vegetation was expressed as the number of points at which it was present over the 100 points.

Table height (height of vegetation canopy closure) of each shrub species (if any) and/or total herbaceous vegetation was also measured at each of the measured 100 points in each plot. Plot mean canopy height was then calculated for each shrub species and the herbaceous vegetation component. These measurements then allowed us to estimate the volume occupied by each shrub species in each plot (total plot area in m² multiplied by the proportion area covered by each shrub, multiplied by the mean canopy height of that shrub in the plot). Standing biomass of leaves and stems of each shrub species was then estimated using allometric equations developed for each shrub species relating shrub volume or cover to shrub standing

biomass of leaves and stems. For the herbaceous component, total standing biomass was estimated using allometric equations relating the mean plot herbaceous canopy height to biomass in tonnes/hectare (then converted to kg based on the plot area of 100 m²). Methods used to develop these equations and their fit and precision are presented in supplementary materials.

## Plant functional traits

### Shrub traits

For each shrub species we measured leaf and stem dry matter content (LDMC, StDMC), leaf and stem carbon concentration (LCC, StCC), leaf and stem N concentration (LNC, StNC), leaf and stem P concentration (LPC, StPC), and stem lignin concentration. Leaf and stem lignin concentration in herbaceous species was considered negligible in comparison to shrubs, and approximated to zero for further calculations. For each species, 10 samples of a single leaf and adjoining stem were taken from ten well developed individuals from the immediate area of the plots. *C. vulgaris* was taken as a combined sample due to unfeasibility of leaf-stem separation. Methods used for the measurement of DMC (at full hydration), C, N and P concentrations and lignin content followed standard trait measurement protocols (Cornelissen et al. 2003). C and N concentrations were measured using a standard elemental analyser. P concentrations were quantified after acid extraction using an inductively coupled plasma—optical emission spectrometer following European standard ISO 11885. For lignin analyses, 1 g of ground (1 mm) plant material was subjected to a dissolution in 100 ml acidic detergent, followed by a second attack by $H_2SO_4$ 72%. After washing until neutralisation, the dry organic residue (ADL-lignin) was determined as the difference between the dry residue (oven drying at 103 °C) and the residue after mineralisation (oven at 550 °C) (Soest 1963).

### Herbaceous traits

Herbaceous traits were measured at plot level using a variation of the taxonomic-independent trait transect method (Lavorel et al. 2008). This method seeks to directly measure plant traits on a representative sample of herbaceous community biomass
sampled directly in a plot, rather than by estimating species relative abundances and using this to weight species-specific trait values. Samples were taken at 50 cm intervals along the two 14.4 m diagonals of each plot (56 points per plot). At each point, a sample of one well developed mature leaf was taken for each well-grown herbaceous species within a 10 cm diameter cylinder. The sampled leaves from each point were transported to the laboratory in a cool box (ca. 4 °C), pooled and measured using standard trait methods (Cornelissen et al. 2003) for LDMC (at full hydration), LCC, LNC and LPC concentrations. Leaves from each point were considered as an aggregate sample for LDMC, then averaged across the 56 points to calculate a plot value. Dry leaves were pooled to three aggregate samples per plot for

analyses of C, N and P concentrations then averaged to calculate a plot value.

Calculation of community weighted plant traits

Estimates of the standing biomass of leaves and stems of each shrub species and of the herbaceous vegetation component in each plot were used to calculate the proportional representation of total plot community biomass represented by leaves and stems of each shrub species and by herbaceous vegetation (Fig. 3). CWM of each plant trait for each plot was then estimated by extending the biomass weighted mean calculation (Garnier et al. 2004) to integrate allometric distribution of plant material across leaves and stems. For this, trait values estimated for leaves and stems of each shrub species and mean plot-based values for

<figure><img src="image_4.png" /><figcaption>Fig. 3 Calculation of community weighted mean (CWM) for each plant trait and plot, integrating values for traits of three biomass components: herbaceous leaves, shrub leaves and shrub stems. Allometric equations were used to estimate total aboveground biomass. Herbaceous leaf traits were calculated</figcaption></figure>

as a mean per plot from leaf samples collected using the trait-transect method. Shrub leaf and stem traits were mean values for each shrub species. Superscripts (a, b, c) denote: a) Mean value at plot level; b) Mean value per shrub species; c) Value per plot
herbaceous vegetation were weighted by their relative biomass representation in the plot. Such CWM values were calculated for each plot for DMC, C, N, P concentrations and lignin content as follows (Eq. 1):

$$CWMtrait = \sum_{Sp=i}^{n} \%Biom_i \times (\%Leaf_i \times Leaftrait_i + \%Stem_i \times Stemtrait_i) \quad (1)$$

where %Biom_i is the relative biomass of species i to the total biomass in the community; %Leaf_i and %Stem_i are the relative leaf and stem abundances, respectively, of species i to the its total biomass; Leaftrait_i and Stemtrait_i is the trait value for the species i and n the total number of species.

## Soil physical-chemical properties

Soil sampling and processing methods followed Grigulis et al. (2013). Nine soil cores of 5 cm diameter and 15 cm depth were taken from each experimental plot, pooled to a composite soil sample and taken back to the lab in cool boxes (ca. 4 °C) for the measurement of soil physical-chemical properties. Sampling was stratified based on the proportion of shrub cover in plots (i.e. in plots with > 80% shrub cover all cores were taken under shrubs, in plots with 40–60% shrub cover 2–3 cores were taken under shrubs, and in plots with 0–20% shrub cover 0 to 1 cores were taken under shrubs, with the balance of cores being taken from under herbaceous vegetation). Composite soil samples were sieved at 2-mm and then analysed for pH (with CaCl₂), C and N concentration (colorimetric analysis after KCl extraction in Lautaret; elemental analyser in Stubai), P Olsen and SOM (loss on ignition).

## Plant available N and P

Plant available N and P ions were measured using cation- and anion-exchange resin membranes that adsorb ammonium ($NH_4^+$), and nitrate ($NO_3^-$) and phosphate ($PO_4^{3-}$) ions, respectively, from the soil solution following Hovenden et al. (2017). These sticks simulate plant roots and when left in the soil for four weeks provide a way of estimating the concentrations of ions to which a plant root would be exposed

in situ, providing an integrated view of inorganic ion availability across this time window.

Briefly, during the period of peak biomass production, ten charged (using a 0.5 M $NHCO_3$ solution) 5 cm by 1 cm anion resin strips (AXM-100S Anion Exchange Membrane Sheet, Membranes International Incorporated—https://membranesinternational.com/) and five charged (using a 0.5 M NaCl solution) 5 cm by 1 cm cation resin strips (CXM-200S Cation Exchange Membrane Sheet, Membranes International Incorporated) were placed vertically into the soil in each plot. The incubated resins were removed from the soil after four weeks, rinsed clean with distilled water and ions were extracted. Extractant solutions were then analysed for $NH_4^+$, $NO_3^-$ and $PO_4^{3-}$ using colorimetric analysis. The ion concentrations measured from the five resins per plot destined for the measurement of each ion were averaged to provide mean plot values of available $NH_4^+$, $NO_3^-$ and $PO_4^{3-}$ and standardized per time in the field and resin size (units: µg L$^{-1}$ week$^{-1}$ cm$^{-2}$).

## Statistical analysis

We used principal component analysis (PCA) to visualize general patterns of variation. CWM N and Ns were removed from the PCA analysis due to high correlation with other variables (i.e. Pearson's correlation coefficient > 0.6) and to avoid redundancy (e.g. CWM N is included in CWM Lignin:N and CWM C:N ratios). We added individual PCAs for plots encroached by ericaceous species to confirm the similarity of Vaccinium communities dominated by common species at both sites at a 95% confidence interval (Fig. S3). Hence, all Vaccinium deciduous dominated plots from Stubai and Lautaret site were grouped into the same shrub type ericaceous deciduous ("Ericaceous D"). For our analyses we then classified the plots into four categories: "Ericaceous E" (ericaceous evergreen dominated plots), "Ericaceous D" (ericaceous deciduous dominated plots), "J. communis" (Juniperus communis dominated plots), "Rosa sp." (Rosa spinosa dominated plots) (Table 1). We used R packages FactoMineR (Le et al. 2008) and factoextra (Kassambara and Mundt 2020) for the PCA analysis.

We used graphical representation to analyse the linearity of changes along shrub encroachment gradients using the ggplot2 package in R. Simple linear models compared using the Akaike Information
<table><caption>**Table 1** Categories for grouping shrub species in data analysis: Ericaceous deciduous (Ericaceous D), Ericaceous evergreen (Ericaceous E), *J. communis* and *Rosa* sp. Site: Stubai (S) or Lautaret (L)</caption><thead><tr><th>Category</th><th>Dominant encroaching shrub species</th><th>Site</th><th>Grass dominant species</th></tr></thead><tbody><tr><td rowspan="2">Ericaceous D</td><td rowspan="2">Vaccinium myrtillus, Vaccinium uliginosum</td><td>S</td><td>Festuca rubra and Sesleria albicans</td></tr><tr><td>L</td><td>Patzkea paniculata</td></tr><tr><td rowspan="2">Ericaceous E</td><td>Rhododendron ferrugineum</td><td rowspan="2">S</td><td rowspan="2">Nardus stricta</td></tr><tr><td>Calluna vulgaris, Vaccinium vitis-idaea</td></tr><tr><td>J. communis</td><td>Juniperus communis</td><td>L</td><td>Patzkea paniculata or Bromus erectus</td></tr><tr><td>Rosa sp.</td><td>Rosa spinosa</td><td>L</td><td>Bromus erectus</td></tr></tbody></table>

Criterion (AIC) were used to test the effects of shrub encroachment on measured CWM traits and soil properties using the *stats* R package. All assumptions for normality, independence and homoscedasticity were checked. We performed a joint analysis for plots at Stubai and Lautaret as exploratory analysis showed no site differences.

To test our hypothesis of indirect effects of shrub encroachment, we used piecewise structural equation models (SEM), which allowed us to test for causal chains of mechanistic links (Shipley 2016). Following the trait-based approach, we expected that those traits relevant for the processes of interest and which we included in the initial SEM would capture differences across shrub species. However, a preliminary analysis indicated that while this was correct for most species, there was a qualitative difference in relationships between plant traits and soil properties in plots colonised by *J. communis*, indicating an identity effect (Díaz et al. 2007) (Fig. S4). Accordingly, we included *J. communis* presence/absence as a random effect for links between community plant traits and soil properties by using mixed effect linear models (lme) in the SEM. We used simple linear models (lm) for links between shrub encroachment and CWM of plant traits. All assumptions for the linear and mixed effects models were checked. Variables were transformed when necessary to meet model assumptions (i.e. log($PO_4^{3-}$)). We tested two hypothetical models: a first model including all significant and non-significant links (model 1 hereafter), and a second model with only significant links (model 2 hereafter). All SEM analyses were performed using R packages *piecewiseSEM* (Lefcheck 2016) and *nlme* (Pinheiro and Bater 2023). All statistical and graphical analyses were conducted using the open-source R Statistical Software v. 4.2.2 (RStudio Team 2022).

## Results

### General patterns

PCA analysis suggested that plots varied along two main independent dimensions (Fig. 4). The first axis (Dim 1) explained 45.1% of the variance, while the second axis (Dim 2) explained 19.1%. The variables that contributed most to Dim 1 were CWM Lignin:N (14% contribution), CWM DMC (13% contribution), CWM CN (11% contribution) (Fig. S5a). Higher values of these traits were associated with higher shrub biomass. Soil properties and nutrients had a lower contribution to Dim 1 compared to CWM traits. Amongst soil properties, SOM and soil C contributed most, with their higher values being associated with higher values of CWM CN and CWM DMC at higher shrub biomass.

The second axis (Dim 2) differentiated plots by nutrients in plant tissues and in the soil. Soil $NO_3^-$ and CWM P were the two most contributing variables to Dim 2 (23% and 20% respectively), followed by pH (10%) (Fig. S5b). Ericaceous colonised plots had higher CWM P, lower pH and $NO_3^-$ compared to *J. communis* colonised plots. These differences were more pronounced at higher shrub biomass.

Linearity of changes along the gradient of increasing shrub biomass

Herbaceous plant traits differed across the two sites (Fig. S6) reflecting different composition and especially dominant species within each grassland type. However, herbaceous plant traits responded similarly to increases of shrub biomass both between sites and within sites (Fig. S7). Furthermore, their variation
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Principal component analysis based on plant traits and soil properties. Points represent sampled plots (n = 54). Green arrows represent measured plant trait variables: community weighted mean lignin/nitrogen ratio (CWM Lignin:N), community weighted mean dry matter content (CWM DMC), community weighted mean carbon concentration (CWM C), community weighted mean carbon/nitrogen ratio (CWM CN), community weighted mean phosphorus concentration (CWM P), community weighted mean nitrogen/phosphorus ratio NP (CWM NP). Brown arrows represent measured soil property variables: soil organic matter concentration (SOM), soil carbon concentration (Cs), soil carbon/nitrogen ratio (CNs), soil</figcaption></figure>

phosphorus concentration (Ps), available soil phosphorus or PO₄³⁻(Phosphate), available soil nitrate or NO₃⁻ (Nitrate), available soil ammonium or NH₄⁺ (Ammonium). The length of the arrow represents the contribution of each variable to the PCA. Symbols represent the main shrub species encroaching each plot: *Juniperus communis* (*J. communis*), *Rosa spinosa* (*Rosa* sp.), Ericaceous deciduous (Ericaceous D) and Ericaceous evergreen (Ericaceous E). Each point is colored according to the encroachment gradient (% shrub biomass in each plot) ranging from low % of shrub biomass (yellow) to high % of shrub biomass (red)

along the encroachment gradient (from 0.9% increase for carbon to 21% increase for nitrogen), whether for the entire data set or within individual sites, were of small magnitude as compared to differences between mean herbaceous and shrub traits (e.g. 44% carbon in herbaceous species vs 48% of carbon shrubs; 1.9% nitrogen in herbaceous species vs. 1.2% nitrogen for shrub species). Simple linear models of CWM traits with increasing shrub biomass were significant for all measured community-level plant traits (Fig. S4a-i, Table S2). By contrast, we found fewer significant regressions for simple linear models of soil properties along the gradient (Fig. S4j-t, Table S2). Additionally, the results from the model comparison using AIC showed that linear models were a better fit compared to most of the logarithmic models (Table S3). Although some of the logarithmic and quadratic

models showed a lower AIC compared to the linear models, these differences were either small (AIC difference <10 points) or mainly driven by the presence of *J. communis* (Fig. S4, Table S3). For instance, although the quadratic model for CWM CP with increasing biomass is a better fit than the linear one (AIC difference of -28 points, Table S3), this nonlinear relationship is driven by higher values of CWM CP in *J. communis* plots rather than by shrub density (Fig. S4e).

CWM C (R² = 0.87), CWM DMC (R² = 0.78) and CWM Lignin:N ratio (R² = 0.89) increased significantly with increasing shrub biomass (Table S2). CWM DMC increased by 60% from low to high density plots (Fig. 5a), reflecting increasing prevalence of dense shrub tissue and especially stems. Traits related to nutritional quality of plant communities such as
CWM N and CWM P decreased significantly with increasing shrub biomass (Table S2) and, while also following linear trends, showed a higher variability at lower shrub biomass (Fig. 5b-c).

Plant-soil parameter relationships also suggested linear changes in soil properties and nutrients as a response to changes in plant traits (Fig. 5d-i). pH decreased with increasing CWM DMC, while SOM

and soil CN increased with increasing CWM DMC (Fig. 5d-f). Available soil P (PO₄³⁻) increased significantly with increasing CWM P (Fig. 5g), while available soil N nutrients (NO₃⁻ and NH₄⁺) showed no significant changes with increasing CWM N (Fig. 5h-i). Plant-soil relationships were influenced by shrub type (Fig. 5d-f), reflecting the presence of *J. communis*. We also identified this species effect in linear

<figure><img src="image_6.png" /><figcaption>**Fig. 5** Simple linear regressions. (a-c) Responses of community weighted means (CWM) of plant traits to increasing shrub biomass%. (d-f) Responses of soil properties (pH, soil organic matter concentration (SOM), soil carbon/nitrogen ratio (C:N soil) to increasing community weighted mean dry matter content (CWM DMC)). (g-i) Responses of soil available nutrients: phosphate (PO₄³⁻) to increasing community weighted mean</figcaption></figure>

phosphorus concentration (CWM Phosphorus); nitrate (NO₃⁻) and ammonium (NH₄⁺) to increasing community weighted mean nitrogen concentration (CWM Nitrogen). The main shrub type in each plot is indicated as Ericaceous D (yellow), Ericaceous E (turquoise), *Rosa sp.* (pink), *J. communis* (purple). Significant regressions (p < 0.05; solid lines) and non-significant regressions (p ≥ 0.05; dashed lines) are indicated
regressions of soil properties with shrub biomass (Fig. S4j-t). The shrub identity effect was particularly evident for pH, with decreasing values from 7 to 5 with increasing CWM DMC for Ericaceous E, Ericaceous D and Rosa sp plots, in contrast to a constant value for *J. communis* plots (Fig. 5d).

## Indirect effects of shrub encroachment on soil properties

The SEM analyses showed that increasing shrub biomass had direct effects on plant community traits which lead to indirect effects on soil properties. To illustrate these effects, we present two piecewise SEMs.

Model 1 (Fig. 6a) including all causal relationships hypothesized in our conceptual model (Fig. 1) differed significantly from observed data structure ($p < 0.05$) and thus cannot be supported as a reasonable representation of the data. However, all links in this hypothetical model were significant, other than those between CWM N and available N ions ($NH_4^+$ and $NO_3^-$) (Supplementary Material).

Model 2 (Fig. 6b), which was a sub-set of Model 1 where we removed the non-significant links between CWM N and available $NO_3^-$ and $NH_4^+$, did not differ significantly from observed data structure (p-value > 0.05). Model 2 shows direct effects of shrub biomass on CWM of plant traits and indirect effects on soil properties. First, increasing shrub biomass led to significant decreases in CWM N and CWM P, as well as a significant increase in CWM DMC, with shrub biomass explaining 87% of the variance of the DMC of the plant community (Fig. 6b). These effects were followed by significant effects of plant community traits on soil properties. Increasing CWM DMC was significantly related to increasing CNs, increasing SOM, and decreasing soil pH. Thus, increasing shrub biomass led to more acidic soils, increased SOM, which then led to increases in soil CNs. Decreasing tissue P concentration (CWM P) with increasing shrub biomass led to decreasing soil available P ($PO_4^{3-}$). In contrast, increasing shrub biomass led to decreasing N in plant tissues (CWM N) but with no significant effects on soil N availability.

Including *J. communis* as a random effect in the analysis of plant-soil linkages increased the proportion of variance explained by the model (Table S4). This was evidenced by a higher conditional $R^2$

(including random effects) vs. marginal $R^2$ (excluding random effects) for all dependent variables, with the exception of $PO_4^{3-}$ which remained constant. In particular, the amount of explained variance in soil pH increased from 8 to 39% when accounting for the presence of *J. communis*.

## Discussion

This study shows that shrub encroachment in subalpine grasslands has indirect effects on soil properties via plant community traits (Fig. 6b). Specifically, we found that the succession toward shrubland leads to plant communities with tougher and nutrient-poor tissues (Fig. 7). These in turn increased SOM and soil CN, and decreased pH and P availability (Fig. 7), confirming our hypothesis that shrub encroachment has indirect effects on soil properties via changed plant traits. These indirect effects did however not apply to soil N availability. Contrary to our expectations, linear changes in plant community traits resulted in gradual rather than nonlinear changes in soil properties along the shrub density gradient. Lastly, our results suggest that aboveground community weighted mean DMC, an easily measurable trait, can be used as an indicator of tissue quality and decomposability in shrub encroached subalpine grasslands. Our results support general hypotheses on changes in community-level plant strategies in shrub encroached ecosystems and their implications for carbon and nutrient cycling (Myers-Smith et al. 2019; Wookey et al. 2009), thereby complementing evidence for these understudied ecosystems in different biomes (Ambrosino et al. 2023; Crofts et al. 2018; Garnier et al. 2004; Guo et al. 2022b; Kemppinen et al. 2021; Nunes et al. 2019; Vogel et al. 2022).

## Responses of plant community traits to shrub encroachment

Most of the variation in plant traits in response to increasing shrub biomass (axis 1 of the PCA in Fig. 3) was associated with a gradient of plant resource economics. Communities became more conservative along succession towards shrubland, with lower N and P concentration and greater investment in tougher tissues (higher community weighted mean CN and community weighted mean DMC), reflecting
(a) Hypothesized model

<figure><img src="image_7.png" /></figure>

(b) Selected model

<figure><img src="image_8.png" /></figure>

**Fig. 6** Structural equation models (SEM) exploring the direct effects of increasing shrub biomass (dark green box) on plant community traits (light green boxes) and indirect effects on soil properties (brown boxes). (a) Model 1. Hypothesized model showing the relationships between increasing shrub biomass and plant community weighted mean phosphorus concentration (CWM P), community weighted mean dry matter content (CWM DMC) and community weighted mean nitrogen concentration (CWM N), and onwards indirect effects on soil properties (brown boxes) of soil organic matter concentration (SOM), soil carbon/nitrogen ratio (CNs), soil pH and soil available phosphate (PO₄³⁻), nitrate (NO₃⁻) and ammonium

(NH₄⁺). Arrows indicate the direction of hypothesized positive (light red) and negative (grey) relationship. (b) Model 2. Selected model including only significant relationships. Arrows indicate unidirectional causal relationships between variables. Black arrows indicate positive relationships and red arrows negative relationships. Arrows for non-significant paths (*p* < 0.05) are dashed. The thickness of each arrow indicates the magnitude of the standardized regression coefficient, given on top of the arrow. Adjusted R² are displayed for each relationship (grey boxes). Fischer's C statistic, p-value and degrees of freedom (Df) are given for each model (n = 54)
<figure><img src="image_9.png" /><figcaption>**Fig. 7** Effects of shrub encroachment on plant communities and indirect effects on soil properties. Plant communities at high shrub density (right) have harder and more nutrient-poor tissues compared to low encroached communities (left). Increasing shrub density leads to poorly decomposable litter due to the high amount of recalcitrant molecules, which results in the accumulation of soil organic matter, low soil pH, high soil carbon/nitrogen ratio (CN) and low soil available phosphorus (P). Black-dashed boxes indicate measured parameters, grey-dashed boxes indicate parameters that were not measured.</figcaption></figure>

a mass effect of higher DMC, higher C concentra-
tion and lower N concentration in shrub stems com-
pared with herbaceous leaves. Indeed, the response
of herbaceous traits themselves along the succession
to shrubland was considerably lower than that of the

The horizontal arrow indicates the gradient of shrub density from low (orange) to high (red) encroachment. Triangular bars at the bottom indicate the linear increase or decrease of measured plant traits (green) and soil properties (brown) along the shrub encroachment gradient: community weighted mean dry matter content (CWM DMC), community weighted mean phosphorus concentration (CWM P), community weighted mean nitrogen concentration (CWM N), soil organic matter concentration (SOM), soil carbon/nitrogen ratio (CNs), soil pH and soil available phosphate (PO4 3-)

whole plant community, confirming the primary role
of shrub traits (Fig. S8). Our results support previous
evidence of shrub encroachment in subalpine grass-
lands in European mountains resulting in plant com-
munities with tougher, more carbon-rich tissues and

<figure><img src="image_10.png" /></figure>
lower nutrient content (Grau et al. 2019; Seeber et al. 2022; Urbina et al. 2020). They also match patterns of secondary succession elsewhere, including Mediterranean shrublands (Navas et al. 2010).

In addition, Urbina et al. (2020) showed that changes in plant C, N and P with increasing shrub density were combined with an increase in their aboveground stocks, given that high shrub biomass per se overrides the dilution of nutrients in their aboveground biomass. Accordingly, despite the decrease of nutrient concentrations in the aboveground biomass, we found an increase of total aboveground stocks of N and P with increasing shrub biomass (Fig. S9c-f). This increase in stocks was mainly driven by the stem biomass of shrub species, which emphasizes the importance of considering biomass allocation to understand nutrient cycling and storage in these ecosystems.

## Linear changes of plant traits and soil properties in shrub encroached subalpine grasslands

Contrary to our expectations, we found no indication of nonlinear trends in soil processes with increasing shrub density. All our linear models for plant traits were significant with a high R², with the exception of P concentration which, while still significant, showed a higher variance. These results confirm a gradual functional response of plant communities to increasing shrub biomass along gradients of encroachment, at least for low shrubs such as those considered here in subalpine grasslands of the Alps. Similarly, although soil properties showed a higher variance, we did not detect abrupt changes along the shrub density gradient. Soil C, soil CN and SOM increased significantly and linearly with increasing shrub biomass. However, we measured concentrations of total soil SOM and soil C, as opposed to specific carbon fractions or phenolic compounds. While some studies have found nonlinear changes of soil organic C and lignin in shrub encroached temperate grasslands (Zhou et al. 2017), they included a narrower range of density (0 to 30%) and different functional groups (i.e. leguminous shrub *C. microphylla*) compared with our study. Instead, our results are consistent with linear increases of soil C found in subalpine grasslands with increasing cover of *R. ferrugineum* in the Eastern Italian Alps (Pornaro et al. 2017).

## Indirect effects of shrub encroachment on soil properties via plant community traits

Our SEM showed that the increase of woody material, which has high CN, high DMC and high lignin (and Lignin:N), led to increased SOM and soil CN and to decreased pH. These results are consistent with well-known trait effects on decomposition (Cornwell et al. 2008; Rosenfield et al. 2020; Zuo et al. 2018) and hence on soil carbon accumulation (De Deyn et al. 2008; Huys et al. 2022; Sokol et al. 2022). Recalcitrance of woody tissues results from their high fibre density (Bani et al. 2018) and their high concentration in polyphenols (i.e. tannins, lignin) (Kahl et al. 2017), compared to leaves. Accordingly, leaf and wood litter traits (i.e. lignin) have been shown to be independent within woody tree species, resulting in different effects on decomposition (Kobayashi et al. 2024). Lignin can impact decomposition and nutrient cycling in multiple ways. First, it can have direct effects on decomposer communities such as saprotrophic fungi or soil macrofauna (Sanghaw et al. 2023; Yang et al. 2024a; Zuo et al. 2014). Second, lignin can interact with soil minerals and be protected from microbial access, therefore influencing decomposition (Liao et al. 2022). Our model also showed decreasing soil pH with shrub encroachment, which was also reported in other studies (Collins et al. 2020; Eldridge et al. 2011), and might be linked to the leaching of organic acids into the soil (Swift et al. 1979; Whitford 1992).

Shrub encroachment decreased soil P availability as a result of the lower P concentration in plant communities, and despite the increase in total aboveground P stocks (Fig. S9e-f) and belowground P stocks (Fig. S4 l). These results highlight the importance of quality, and not only quantity, for nutrient release in the case of P, thus confirming reported effects of plant functional traits on nutrient cycling in shrub encroached grasslands (Yang et al. 2022; Zhang et al. 2008a). Moreover, P cycling depends on other mechanisms such as the ability of fungal communities to transform organic P into forms of P available for plants (Ferrol et al. 2019; Wang et al. 2023), which can be inhibited by soil low pH (Zhang et al. 2008b). We suspect that the immobilization of P in aboveground and belowground compartments, together with decreased pH, caused a decline in available P in shrub encroached plant communities.
Surprisingly, we found that decreases of N in plant communities did not lead to decreased soil N availability. We expected that the increase of woody biomass with high concentrations of lignin and tannins, which are known to inhibit N release (Rahman et al. 2013), would result in decreasing soil available N. Broadbent et al. (2022) observed decreased soil N availability in Austrian subalpine grasslands encroached by ericaceous species. However, seasonality, soil biota or microclimate might offset this effect. For instance, Crofts et al. (2018) found no differences in N availability in tundra ecosystems with increasing shrub density, potentially linked to a lower pulse of nutrients in summer (vs. spring), when the sampling was done. The symbiosis of shrubs with ericaceous mycorrhizae and ectomycorrhiza may also impact N cycling by immobilizing soil nutrients (Broadbent et al. 2022). Lastly, microclimatic factors such soil moisture or temperature due to rainfall interception or shading by shrubs can affect N cycling (Aguirre et al. 2021). It is likely that the multifactorial determinism of N cycling, including microbial communities, dominant functional shrub species, and abiotic factors such as precipitation regimes and soil water content, made it difficult to observe decreasing trends of available N in this study.

At our sites, land use history with varying duration since mowing (Lautaret) and more intensive grazing (Stubai), along with idiosyncratic colonisation into initially homogenous grasslands (Lavorel et al. 2017) have resulted in varying shrub density. Our results based on a directional structural equation model show that this shrub density gradient then influences soil properties via plant community traits, potentially affecting plant-soil feedbacks (Bennett et al. 2017; Wardle et al. 2004). In fact, multiple studies have shown the effect of aboveground traits on belowground processes (Kahl et al. 2017; Sokol et al. 2022), reflecting coordination with relevant belowground traits (de la Riva et al. 2016; Freschet et al. 2010; Pérez-Ramos et al. 2012). However, we cannot exclude that changed soil conditions (greater amounts of more recalcitrant organic matter, lower pH and P availability) may result in a feedforward loop further favouring shrub colonisation through changes in microclimate and/or soil microbial communities (Myers-Smith et al. 2019; Wookey et al. 2009). For

example, the presence of ericoid mycorrhiza fungi in ericaceous species can outcompete the ubiquitous arbuscular mycorrhiza fungi in the obtention of nutrients from recalcitrant litter (Wurzburger and Hendrick 2009) and limit N availability to non-ericoid mycorrhizal plants, including the herbaceous matrix (Northup et al. 1998; Ward et al. 2021). Uncovering such feedback effects, along with detailed testing of causal mechanisms requires experimental approaches including long-term shrub removal experiments (Aguirre et al. 2024; Broadbent et al. 2021; Fanin et al. 2022; Urcelay et al. 2009).

Leaf dry matter content as an indicator of tissue quality and decomposability

While some previous studies have used CN as a proxy for tissue quality, we found that community weighted mean DMC was a more significant indicator of the amount of recalcitrant compounds (especially lignin) in shrub encroached grassland communities, and thus of decomposability (Bumb et al. 2018; Freschet et al. 2012; Pietsch et al. 2014) and integration into soil organic matter (Cheng et al. 2023). In the PCA, community weighted mean DMC was positively correlated with both community weighted mean CN and community weighted mean Lignin:N. Importantly, alternative SEM models incorporating community weighted mean Lignin:N had a lower level of support compared with models including community weighted mean DMC as an indicator of tissue quality (Fig. S10).

Our integration of leaf and stem DMC into community weighted mean DMC progresses on previous research that focused on leaf traits only (Grau et al. 2019; Seeber et al. 2022; Urbina et al. 2020). We demonstrate the relevance of integrating stem traits (Kobayashi et al. 2024; Zuo et al. 2018) by incorporating biomass allocation to leaves vs. stems in woody plants (Guo et al. 2022a). Thereby we confirm that considering allocation is important in community trait-based studies (Garnier et al. 2004) in which not only species dominance but also structural components are relevant to understand ecosystem functioning.
Effects of shrub species identity

The functional type of the dominant species has been
found to be one of the main drivers of the functional
structure of alpine subshrub communities (Illa et al.
2017), and of functional effects of shrub encroach-
ment globally (Eldridge et al. 2017). The effect of
ericaceous species on soil properties has been well
documented (Hattenschwiler and Vitousek 2000),
and their negative effect on N availability shown in
various ecosystems, including boreal forests (Fanin
et al. 2022) and subalpine grassland (Broadbent et al.
2022). In this study, we found consistent changes in
responses of soil properties and nutrient availabil-
ity to shrub density for ericaceous species, whether
deciduous or evergreen, across the two sites. In con-
trast, our second SEM incorporating *J. communis*
as a source of variation confirmed that the effects of
shrub encroachment on soil properties were species
dependent (Fig. 4). Plots with high abundance of *J.
communis* had soils with higher pH, lower concentra-
tions of PO₄³⁻ and NH₄⁺ and higher concentrations
of NO₃⁻ compared to plots dominated by ericaceous
species or *R. spinosa*, while grassland plots did not
significantly differ across corresponding communi-
ties. Responses to shrub density also differed in *J.
communis* encroached communities, with limited
changes in pH, SOM or soil CN. The higher litter
persistence of *J. communis* (Illa et al. 2017) might
explain the lower incorporation of plant material into
SOM and corresponding stability in C:N stoichiom-
etry. Grau et al. (2019) found similar results in sub-
alpine grasslands encroached by *Juniperus sabina* in
south facing slopes of the Pyrenees, with no change
in pH between young and mature shrublands. Con-
sistent with these results, in our study *J. communis*
dominated communities had lower P concentrations
in plant tissues than other, and especially ericaceous
dominated communities, reflecting lower P concentra-
tion in *J. communis* than in grassland or other shrub
species. In the Pyrenees, this resulted in decreases in
total aboveground and belowground P stocks in plots
encroached by *J. communis* or *J. sabina* (Urbina et al.
2020). Sequestration of nutrients in the abundant bio-
mass of shrubs and especially *Juniper* species (Illa
et al. 2017) might explain the observed decrease of
available P. Despite the non-significant change of N
availability with increasing shrub density, we found
higher concentrations of NO₃⁻ in plots encroached

by *J. communis* compared with plots encroached by
ericaceous species. The later also showed lower val-
ues of pH at high shrub densities (Fig. S4q), which
can have negative impacts on N availability (Wang
and Kuzyakov 2024), and thus explain the lower
NO₃⁻ concentrations compared with plots encroached
by *J. communis*.

Implications for the quantification of ecosystem
services in shrub-encroached grasslands

Shrub encroachment is expected to significantly
modify societal values of grasslands through changes
in ecosystem services (Bardgett et al. 2021). Quan-
tification of these changes with trait-based models
is required to assess their magnitude and project the
effects of future climate and land use change sce-
narios (Barros et al. 2017; Kambach et al. 2024).
Previous ecosystem service models were developed
for herbaceous communities (Grigulis et al. 2013;
Lavorel et al. 2011), and have been applied to project
their responses to subalpine grassland management
scenarios (Lamarque et al. 2014; Lavorel 2018; Schir-
pke et al. 2017). These models based on the plant
economics spectrum and especially on traits reflect-
ing plant size (height) and aboveground tissue com-
position (dry matter content, nutrient concentrations)
could however not be applied to shrub-encroached
communities until demonstrating the validity of these
traits beyond grasslands. With this study, we show
that this extrapolation is now warranted.

Conclusion

Grazing extensification or abandonment and climate
warming have fostered shrub encroachment in Euro-
pean subalpine grasslands. The succession from
grassland to shrubland is expected to have strong
impacts on ecosystem functions, but their specific
mechanisms are not fully understood. In this study
at two sites in the Alps, we tested the hypothesis that
increasing shrub biomass has indirect effects on soil
properties via community-level plant functional traits.
First, our investigation of community-level traits and
soil properties along a unique continuous gradient of
encroachment showed that, based on a shift to preva-
lence of woody tissues, their changes were linear
with shrub density. Secondly, our structural equation
model identified the direct and indirect effects of increasing shrub biomass on soil properties via the continuous shift to more conservative communities with lower nutrient concentrations and tougher tissues. The production of these more recalcitrant tissues led to soil organic matter accumulation, more acid and phosphorus-poor soils. These effects were moderated by shrub identity, where colonisation by *Juniperus communis* qualitatively modified community traits (especially community weighted mean phosphorus concentration) and soils (especially pH). Our results also suggest that weighted means of a single trait like tissue density (dry matter content), assessed at whole community level with contributions of leaf and stem biomass, could be a good indicator for indirect impacts of shrub encroachment on soil properties. Further research will need to explore associated mechanisms via litter decomposition, changed soil carbon fractions, changes in root traits and impacts on soil microbial communities and their activities. Lastly, our confirmation that the plant resource economics spectrum is applicable to shrubland succession indicates that models of ecosystem services developed for grasslands are also applicable to communities dominated by low shrubs. This will enable scenario-based projections of ecosystem services of subalpine landscapes under land use and climate change scenarios.

**Acknowledgements** We acknowledge Elia Barnier, Amanda Casanova, Philippine Magnin and Morgane Storey for field help at Lautaret, and Jardin du Lautaret (UAR 3370, Université Grenoble Alpes, CNRS) for logistic support. Erich Tasser, Anna-Lena Neunteufel, Ramona Leineweber, Julian Maindok, Katrin Müller, Carmen Hassler, Vanessa Meier, Hannah Chessell, Martin Tasser, Emma Egger, Joachim Hochrainer for field help at Stubai.

**Author contributions** Sandra Lavorel, Karl Grigulis and Georg Leitinger conceptualized and designed this study. The data collection was done by Lucía Laorden-Camacho, Elena Tello-García, Marie-Pascale Colace and Blandine Lyonnard. Lucía Laorden-Camacho and Karl Grigulis performed the data analysis. The first draft of the manuscript and data visualization was done by Lucía Laorden-Camacho, in close consultation with Karl Grigulis, Elena Tello-García and Georg Leitinger. Sandra Lavorel made substantial contribution to the final draft. All authors contributed to the review and editing of the manuscript.

**Funding** Open access funding provided by University of Innsbruck and Medical University of Innsbruck. This study was conducted as part of LUCSES project, funded by the L'Agence

Nationale de la Recherche (ANR) and the Austrian Science Fund (FWF) (ANR-20-CE91-0009 and FWF, I4969-B). It contributes to Zone Atelier Alpes and the Lautaret eLTER platform. The Stubai site is part of the LTSER platform Tyrolean Alps, belonging to the national and international long-term ecological research network (LTER-Austria, LTER Europe and ILTER). This research was funded in part by the Austrian Science Fund (FWF) 10.55776/14969. For open access purposes, the author has applied a CC BY public copyright licence to any author accepted manuscript version arising from this submission.

**Data availability** The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

**Declarations**

**Competing interests** The authors have no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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