# Nitrogen deposition and climate change has increased vascular plant species richness and altered the composition of grazed subalpine grasslands

Marion Boutin, Emmanuel Corcket, Didier Alard, Luis Villar, Juan-José Jiménez, Cian Blaix, Cedric Lemaire, Gilles Corriol, Thierry Lamaze, André Pornon

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## ► To cite this version:

Marion Boutin, Emmanuel Corcket, Didier Alard, Luis Villar, Juan-José Jiménez, et al.. Nitrogen deposition and climate change has increased vascular plant species richness and altered the composition of grazed subalpine grasslands. Journal of Ecology, 2017, 105 (5), pp.1199-1209. 10.1111/1365-2745.12743 . hal-01608704

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Distributed under a Creative Commons Attribution 4.0 International License
*Journal of Ecology* 2017, **105**, 1199–1209

doi: 10.1111/1365-2745.12743

# Nitrogen deposition and climate change have increased vascular plant species richness and altered the composition of grazed subalpine grasslands

Marion Boutin¹,², Emmanuel Corcket³,⁴, Didier Alard³,⁴, Luis Villar⁵, Juan-José Jiménez⁵, Cian Blaix⁶, Cédric Lemaire³,⁴, Gilles Corriol⁷, Thierry Lamaze² and André Pornon*¹

¹CNRS, ENFA, UMR5174 Laboratoire Evolution & Diversité Biologique, Université Toulouse 3 Paul Sabatier, 118 route de Narbonne, F-31062 Toulouse, France; ²UMR 5126, Centre d'Etudes Spatiales de la Biosphère, Université Toulouse 3 Paul Sabatier, 18 av. Edouard Belin, F-31401 Toulouse, France; ³UMR1202 BIOGECO, Université de Bordeaux, F-33615 Pessac, France; ⁴UMR1202 BIOGECO, INRA, F-33610 Cestas, France; ⁵Instituto Pirenaico de Ecología, IPE-CSIC, Avda. Nuestra Señora de la Victoria 12, Jaca, E-22700 Huesca, Spain; ⁶Institute of Life Sciences, Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, I-56127 Pisa, Italy; and ⁷Conservatoire Botanique National Pyrénées Midi-Pyrénées, Vallon de Salut, BP 70315, F-65203 Bagnères-de-Bigorre, France

## Summary

1. Atmospheric nitrogen (N) deposition and climate warming are two major components of global change that drive species richness and composition in plant communities. However, their combined effects have been insufficiently investigated across large spatial and temporal scales particularly in high-elevation, nutrient-limited ecosystems.

2. We examine whether and how N deposition and climate warming have altered the plant richness and the composition of subalpine semi-natural, extensively grazed grasslands of the Pyrenees, using two complementary approaches: (i) analysis of 553 relevés to explore vegetation changes across large ecological gradients including temperature and N deposition (spatial approach) and (ii) a re-sampling of a subset of 40 sites among the 553 sites to assess temporal changes over the past decades (temporal approach).

3. Both approaches showed that the vascular plant species richness increased when temperature and cumulative N deposition increase, shifting the species composition towards more thermophilic and eutrophic communities.

4. Synthesis. We hypothesize that the release from abiotic constraints (milder temperature and higher nitrogen availability) due to global changes and long-standing extensive grazing counteracting the negative effects of nitrogen deposition have been responsible for the diversity and compositional changes of plant communities over the last decades in the Pyrenees. Thus, in contrast with other grasslands, high-elevation grazed grasslands may increase in species diversity with nitrogen deposition under climate warming.

**Key-words:** abiotic constraints, global warming, grazing, plant biodiversity and composition, Pyrenees, soil acidification, soil eutrophication, spatial pattern, subalpine grasslands, temporal trend

## Introduction

In Europe, decadal average temperature has increased by c. 1·3 °C (±0·11 °C) since pre-industrial times. Meanwhile, atmospheric nitrogen (N) deposition has increased considerably during the last two centuries (on a global basis, from 34 Tg N year⁻¹ in 1860 to 100 Tg N year⁻¹ in 1995) and is

expected to continue increasing world-wide (Galloway *et al.* 2008). The hot spots of biodiversity of mountains may be particularly threatened by global warming and atmospheric N deposition because of (i) the role of low temperature and N availability in regulating cold ecosystems (Körner 1999; Sala *et al.* 2000) and (ii) the greater temperature increase at high altitude (Füssel *et al.* 2012).

Some studies have revealed an increase in diversity (Pauli *et al.* 2012) or plant growth (Jarrad *et al.* 2009) with temperature increase in high altitude habitats. The changes in

<footnote>*Correspondence author. E-mail: andre.pronon@univ-tlse3.fr</footnote>
diversity were attributed either to an upward migration of species (Lenoir *et al.* 2008; Gottfried *et al.* 2012) or to the expansion of species already present at a site (Cannone & Pignatti 2014). However, others have reported no (Price & Waser 2000; Spasojevic *et al.* 2013; Windmaißer & Reisch 2013), weak (Vittoz *et al.* 2009) or negative effects (Sebastià *et al.* 2008a; Pauli *et al.* 2012) of climate warming on plant biodiversity. Impacts of local factors such as grazing or habitat heterogeneity and changes in precipitation amounts (Sebastià *et al.* 2008b; Spasojevic *et al.* 2013) have been proposed to explain the divergent responses of these habitats to climate warming. For instance, grazing tends to promote plant biodiversity (de Bello, Lepš & Sebastià 2006; Sebastià *et al.* 2008b; Komac, Domenech & Fanlo 2014) except in xeric areas (Deléglise, Loucougaray & Alard 2011), overgrazed areas (Yan & Lu 2015) or extremely high elevation grasslands (Speed, Austrheim & Mysterud 2013). By limiting the expansion of woody plants and litter accumulation (Dirnböck, Dullinger & Grabherr 2003; Klein, Harte & Zhao 2004), both of which increase with temperature, grazing can provide refuge for alpine plant species and dampen species loss due to climate warming.

Atmospheric N deposition has negative effects on plant diversity in low-altitude extensively managed grasslands. This occurs mainly as a consequence of soil acidification and/or soil eutrophication exacerbating competition for light between species (Stevens *et al.* 2004; Duprè *et al.* 2010; Maskell *et al.* 2010; van den Berg *et al.* 2011; Gaudnik *et al.* 2011).

Although more distant from the main N emission sources than many low-elevation habitats, most European mountain ranges still receive rates of N deposition several times higher than presumed pre-industrial levels (Hiltbunner, Schwikowski & Körner 2005), likely exceeding the N critical loads (500–1000 mg N m⁻² year; Bobbink *et al.* 2010; Phoenix *et al.* 2012) in subalpine grasslands (Kirchner *et al.* 2014; Boutin *et al.* 2015). Due to their low N availability and limited pH buffer capacities (Körner 1999) high-elevation ecosystems are likely to be among the most sensitive systems to eutrophication, acidification and base cation depletion following N depositions (Horswill *et al.* 2008). However, no studies have really investigated the effects of atmospheric N deposition across large spatial and/or temporal gradients in high-elevation above tree line grasslands.

Most of the small-scale experimental studies conducted in mountain habitats indicate an increase in biomass and changes in the proportion of species in response to N addition (Bowman *et al.* 1993; Onipchenko *et al.* 2012; Bassin, Volk & Fuhrer 2013) and a decrease in plant species richness (see Humbert *et al.* 2016 for a review). Positive effects of N addition on species richness have sometimes been observed (Olofsson & Shams 2007) or predicted for particularly unproductive habitats (Grime 1977; Huston & DeAngelis 1994). Grazing can attenuate the short-term impacts of temperature and N availability increases on ecosystem function (Gill 2014) and promote community stability in grasslands exposed to N addition through a reduction in competitive exclusion (Pasari, Hernandez & Zavaleta 2014; Beck *et al.*

2015). However, these findings may be transient as the chronic impact of long-term N accumulation from low level N deposition (Phoenix *et al.* 2012) and long-term effects of grazing have not been explored.

The extent to which climate warming may interact with N deposition is still unclear. In lowland acidic grasslands, Gaudnik *et al.* (2011) highlighted that N deposition was related to significant community composition change at the regional scale, whereas climate warming was the predominant driver at a larger scale. Experimental studies revealed additive effects of N deposition and climate warming on vegetation productivity (Shaw *et al.* 2002; Hutchison & Henry 2010), interactive effects on plant growth (Parsons *et al.* 1994) or opposite effects on species diversity in grasslands (Zavaleta *et al.* 2003). To our knowledge, no studies have investigated the effects on subalpine grasslands of both climate warming and N deposition. Thus, there is a considerable uncertainty about their combined impacts on the vegetation of high-altitude ecosystems.

Here, we examine whether and how N deposition and climate warming have impacted the species richness and composition of the subalpine semi-natural extensively grazed grasslands of the Pyrenees (southwest Europe; Fig. S1, Supporting Information) using two complementary approaches. First, we analysed 553 relevés covering the entire Pyrenees massif (Fig. S1 and Table S1). This approach (hereafter named spatial approach), already successfully used on northern European acidic grasslands (Duprè *et al.* 2010), aimed to explore the relationships between ecological factors (including temperature and cumulative N deposition) and plant community richness and composition across large ecological gradients. However, this approach cannot always separate temporal and spatial causes of vegetation change. Therefore, in a second approach (hereafter named temporal approach), we compared historical and recently re-sampled vegetation relevés at 40 sites selected among the 553 sites (Fig. S1 and Table S2) to detect temporal changes in species richness and composition over the past six decades (Gaudnik *et al.* 2011).

Using this design we addressed three major questions: (i) How are plant richness and composition of subalpine grasslands related to the spatial gradients of cumulative N deposition and temperature across the Pyrenees? (ii) Has plant species richness and composition of these grasslands changed over the past decades?, and (iii) Can these changes be related to cumulative N deposition and/or temperature increase since N availability and temperature are the main constraints on ecosystem functions in high altitude ecosystems (Körner 1999)? According to these three questions we hypothesized that: (i) The composition of the grasslands would shift towards more thermophilic and eutrophic plant communities both along spatial gradients of increasing cumulative N deposition and temperature (spatial approach) as well as through time (temporal approach); (ii) If temperature was the most influential factor across the Pyrenees we would observe in the temporal approach a general species richness increase as a result of climate warming and of species suited to warmer temperatures becoming established as is expected in relatively humid
habitats (de Bello, Lepš & Sebastià 2006); and (iii) If the N deposition was the most influential factor in the Pyrenees, we would expect in the temporal approach a decrease in species richness with cumulative N deposition all across the entire massif as observed in low-altitude grasslands (Stevens et al. 2004; Duprè et al. 2010; Maskell et al. 2010; Gaudnik et al. 2011). However, the grazing might have dampened the impacts of N (Klein, Harte & Zhao 2004), promoted the establishment of new species (Olofsson & Shams 2007; Deléglise, Loucougaray & Alard 2011) and thus contributed to maintaining plant diversity in the Pyrenees. Lastly, the temporal changes in species richness would have varied across the Pyrenees, either increasing or decreasing where climate warming or N deposition has been the most influential factor.

# Materials and methods

## FLORISTIC AND ENVIRONMENTAL DATA

### Floristic data

We selected 553 among 839 compiled relevés from the *Nardion strictae* Br.-Bl. 1926 sensu lato community type, mainly from the *Festucetalia spadiceae* Barbero 1970 (see de Foucault 2012) covering the 1948–2010 period, and present in most of the subalpine Pyrenees area (Table S1, Fig. S1; see Supplementary methods in Appendix S1 for the selection of relevés and species nomenclature). These extensively grazed, acidic, oligotrophic grasslands, mostly dominated by *Nardus stricta*, *Festuca eskia*, *Festuca nigrescens* and *Trifolium alpinum* share edaphic and floristic attributes with the low-elevation *Violion caninae* Schwick 1944 alliance on which the effects of N deposition have been well studied in Europe (Stevens et al. 2004; Duprè et al. 2010; Gaudnik et al. 2011). Because many relevés lacked information about bryophytes and lichens, only vascular species were accounted for in this study.

### Cumulative N deposition

We calculated cumulative N deposition (CumN, the sum of NOₓ and NH₃ depositions in mg N m⁻²) from 1940 to the sampling year for each site from modelled estimations of N deposition from the CHI-MERE chemistry-transport model (Menut et al. 2013) and trends in N pollutant emissions over the years from the CITEPA (French Interprofessional Technical Center for the Study of Atmospheric Pollution) and Erisman et al. (2007). Nitrogen deposition data were spatially interpolated at the coordinates of our sites by the nearest neighbour method (see Appendix S1 for more details about CumN calculations).

### Climatic data

Temperature and precipitation data were compiled from the French SAFRAN dataset (grid of 8 × 8 km, 1958–2013; Quintana-Seguí et al. 2008) and the Spanish SPAIN02 v2.1-March 2012 dataset (grid of 20 × 20 km, 1950–2007; Herrera et al. 2012). Despite a larger resolution, SPAIN02 predictions (especially for precipitation) have been shown to correlate well with observations (Herrera et al. 2012) (Appendix S1 for details).

As N deposition data, climatic data were spatially interpolated at the coordinates of our sites by the nearest neighbour method. To

account for the difference of elevation between the sites of the relevés and the corresponding grid point from the models, temperature was corrected according to the adiabatic gradient of -0.56 °C by 100 m of elevation.

## Other environmental variables

Latitude (Lat), longitude (Lon) coordinates (recorded by the relevés' authors or completed from location information provided) and values for elevation (Elev, in meters), slope (Slope, in degrees) and aspect (see Appendix S1) were attributed to each relevé. Community indices were used as surrogates for moisture (F<sub>ci</sub>), pH (R<sub>ci</sub>), fertility (N<sub>ci</sub>) of soils and community thermophily (T<sub>ci</sub>) and heliophily (L<sub>ci</sub>) as no values were provided for these parameters in the relevés. These indices were calculated as the mean (not cover-weighted) of species F, R, N, T and L indicator values derived from Ellenberg et al. (1992) but adapted to the French context and completed by Julve (1998).

## SPATIAL APPROACH

This first approach using the dataset of 553 relevés aimed at identifying variations in species richness and composition along large ecological gradients, particularly temperature and cumulative N deposition. The effects of environmental variables on species richness were explored through a Generalized Additive Model (GAM) accounting for nonlinear responses (package mgcv, R Development Core Team 2008; Wood 2014; see Appendix S1). To avoid issues of multicollinearity, the geographical (Lat, Lon, Elev) and temporal (Year) variables behind these gradients were not included in the models since they were correlated with Tmean, Prcp and CumN. Explanatory environmental variables included in the analyses were thus Slope, NSasp, WEasp, Tmean, Prcp and CumN and communities' indices R<sub>ci</sub>, N<sub>ci</sub> and F<sub>ci</sub>. Because we selected plots in a relatively homogeneous community type, the use of community indices in regression with species richness should not be subject to important bias (Zelený & Schaffers 2012). Backward selection of the variables was applied according to their significance. Plot size was not accounted for in the analyses. Indeed, it was mentioned for only 430 relevés (among the 553) and where it was mentioned, we did not find any positive correlation between plot size and species richness (Spearman correlation test, *P* = 0.203). This was to be expected because, in practice, phytosociologists usually tended to adjust the plot area to the minimal area of the community and expand the area in species-poor communities (Chytrý 2001).

The effects of environmental drivers on species composition were explored through a redundancy analysis (RDA, a constrained ordination method) using the package vegan (Oksanen et al. 2013) on Helinger transformed species data. The constraining variables were Slope, NSasp, WEasp, Tmean, Prcp and CumN since Lat, Long, Elev and Years were projected a posteriori, as non-constraining variables. The significance of both types of variable was tested by a permutation test (999 permutations). Because mean ecological community indices always inherit information on compositional similarity (Zelený & Schaffers 2012) F<sub>ci</sub>, R<sub>ci</sub>, N<sub>ci</sub>, T<sub>ci</sub> and L<sub>ci</sub> indices were also projected a posteriori using a modified permutation test (999 permutations).

## TEMPORAL APPROACH

### Site selection

The protocol for the selection of sites and the resampling of relevés was adapted from Gaudnik et al. (2011). We selected 40 of the 553
sites (Fig. S1 and Table S2) according to the date of the historical relevés (before 1987), detailed information about location and topography, and the presence in both historical and recent relevés of four species from the following list: *N. stricta*, *F. eskia*, *F. nigrescens* agg., *Jasione* sp., *Plantago alpina*, *Calluna vulgaris*, *Luzula pediformis*, *Potentilla erecta*, *Campanula scheuchzeri* agg., *Agrostis capillaris*, *Avenella flexuosa*, *Polygala* sp. and *Galium saxatile*.

## Resampling protocol

In the summers of 2013 and 2014, in each of the 40 sites we randomly delimited eight 2-m² quadrats in a topographically and floristically homogeneous area, apart from boggy or woody areas, tracks/ paths or particular microtopographic elements (rock contours, holes...). We measured the average vegetation height and cover, and we recorded grazing clues (presence of herds in the vicinity, animal droppings, grazed vegetation) and attributed one out of three levels of grazing intensity (0 = none, 1 = moderate, 2 = intense). On each quadrat we identified all vascular plant species and estimated their cover from 1 to 10 according to the Domin scale (Rich *et al.* 2005). These cover values were later lined up with the phytosociological scale (Braun-Blanquet 1932) for comparison with the historical data.

At the centre of the five quadrats randomly selected among the eight, we took a soil sample at 0–15 cm and pooled the five samples of the site. Soil samples were then air-dried and sieved at 2 mm. Sub-samples were used for the measurement of pH in water and the analysis of C and N contents (CHONS Thermo Electron FlashEA1112) after fine milling (Retsch MM200).

## Data analyses

Some species commonly known to be difficult to distinguish (especially as they often lacked flowers at the time of the surveys) were grouped together both in the historical and recent relevés, (see Appendix S1). Comparisons of means between the historical and recent groups of relevés were performed for explanatory variables (*Tmean*, *Prcp* and *CumN*), community indices (*F*<sub>ci</sub>, *R*<sub>ci</sub>, *N*<sub>ci</sub>, *T*<sub>ci</sub> and *L*<sub>ci</sub>) and species richness. The one-tailed paired Student test was used

for *S*, *Tmean*, *N*<sub>ci</sub> (after square-root transformation), *T*<sub>ci</sub> and *L*<sub>ci</sub> and a two-tailed paired Student test was used for *R*<sub>ci</sub> after checking for normality and equality of the variances. Nonparametric, one-tailed paired Wilcoxon rank sum tests were used for variables that departed from these assumptions even after transformations (*Prcp*, *CumN* and *F*<sub>ci</sub>). Correspondence analysis (vegan package; Oksanen *et al.* 2013), with unconstrained ordination, was used to explore the species compositional temporal trajectories of the communities between the two sampling dates, after downweighting of the species present in less than 5% of the sites. Two-tailed paired Wilcoxon tests were used to assess the significance of the shifts in relevés' coordinates between historical and recent samplings along the first two axes. Spearman correlation tests were used to measure the correlations of environmental variables with each of the first two axes. The significance of the projection of the community indices in the first plane was tested by the modified permutations test (999 permutations; Zelený & Schaffers 2012).

Finally, we calculated the change in frequency of species occurrence (Δ freq.) between the historical and the recent pools of relevés. We recorded ecological values and altitudinal ranks (Gottfried *et al.* 2012) only for the species with a Δ freq. strictly exceeding 0.1 in absolute value (i.e. if the difference of occurrence was of more than four sites), thus limiting the risk of overinterpretation.

## Results

### SPECIES RICHNESS AND COMPOSITION ALONG ECOLOGICAL GRADIENTS (SPATIAL APPROACH)

The GAM (explaining 48.1% of the deviance in species richness (*S*) with a $R^2_{adj}$ of 0.46) showed a positive relationship of species richness with soil pH ($R_{ci}$, *F* = 13.13, *P* < 0.001), *Tmean* (mean temperature, *F* = 0.54, *P* = 0.028) and *CumN* (cumulative N deposition; *F* = 1.11, *P* < 0.001; Fig. 1a,c,e). The negative relationship of richness with *NSasp* (Aspect; *F* = 0.78, *P* = 0.008; Fig. 1d) indicated that higher plant richness was found in north- rather than on south-facing aspects, although it must be recalled here that aspect is a

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Relationship between species richness and each significant explanatory variable of the Generalized Additive Model (holding all the other explanatory variables constant). *S response* represents the deviation from mean species richness. The dashed line indicates a *S* response of zero. Grey zones are 95% confidence intervals. Ticks on the *x*-axes show jittered values actually taken by the variables in the dataset. *Tmean* = mean temperature; *Prcp* = precipitation; *CumN* = cumulative N deposition; *NSasp* = North–South aspect; *R*<sub>ci</sub>, *N*<sub>ci</sub> and *F*<sub>ci</sub> = ecological community indices for soil pH, fertility and moisture respectively.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Relationship between species richness and both mean temperature and cumulative N deposition (holding all the other explanatory variables constant) as predicted by the Generalized Additive Model. [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

complex integrator of several ecological factors including not only temperature but also moisture, wind exposure, freeze-thaw cycles (Winkler *et al.* 2016). The model highlights an additive effect of *CumN* and *Tmean* on species richness: the impact of both *CumN* and *Tmean* corresponded approximately to the sum of the impacts of each individual factor (Fig. 2). The species richness had a curved relationship with indices of soil fertility (*N*<sub>ci</sub>, *F* = 2.43, *P* < 0.001), soil moisture (*F*<sub>ci</sub>, *F* = 3.37, *P* < 0.001) and *Slope* (*F* = 2.39, *P* < 0.001) with the highest richness at intermediate values of these variables (Fig. 1f–h). Species richness had a significant nonlinear relationship with *Prcp* (mean precipitation; *F* = 1.69, *P* = 0.002) with a peak for annual precipitation around 1100 mm (Fig. 1b).

The first two axes of the RDA were significant (*P* = 0.005, permutation test) and explained, respectively, 60.6% and 18.2% of the constrained variance (which represented 9.9% of the total variance) of the floristic composition. Climatic variables (*Tmean* and *Prcp*) and *CumN* explained community composition mostly along these two axes. Permutation test of the *a posteriori* projections of the variables *Years*, *Lat*, *Lon* and *Elev* on the first two axes showed their significance (*P* = 0.001 for all variables). Their projection in the first plane (Fig. 3a) highlights their collinearity, which prevents the separation and interpretation of temporal and spatial gradients in this analysis. Modified permutation tests of the *a posteriori* projections of the community indices showed that the community heliophily (*L*<sub>ci</sub>, *P* = 0.032), and thermophily (*T*<sub>ci</sub>, *P* = 0.001), the soil pH (*R*<sub>ci</sub>, *P* = 0.003) and fertility (*N*<sub>ci</sub>, *P* = 0.031) but not soil moisture (*F*<sub>ci</sub>, *P* = 0.065) were significantly correlated with the first plane of the RDA. The horizontal axis was a gradient of temperature (*Tmean*), and to a lesser extent, of *CumN* both increasing towards an extreme to

the left (Fig. 3a). The species on the left side (for instance *Festuca nigrescens* agg., *A. capillaris*, *Galium verum*, *Carex caryophyllea*, *Lotus corniculatus*, *Achillea millefolium* and *Pilosella lactucella*) were associated with higher temperatures, increasing soil fertility, pH and cumulative N deposition (see Fig. 1 for the range of variation in these variables), while the species on the right (for instance *T. alpinum*, *Ranunculus pyrenaeus*, *F. eskia*, *Alopecurus gerardi*, *Carex pyrenaica* and *Scorzoneroides pyrenaica*) were more characteristic of colder, more oligotrophic and acidic, but sunnier, conditions. The second axis was mainly negatively related to *CumN* and positively to *Prcp* (Fig. 3a) and opposed species associated with sites receiving higher levels of cumulative N deposition and lower levels of precipitation (*P. lactucella*, *Thymus pulegioides*, *C. caryophyllea*, *Epikeros pyrenaeus* and *Alchemilla flabellate*) to species associated with sites receiving lower levels of cumulative N deposition and higher levels of precipitation (*P. alpina*, *P. erecta*, *Conopodium majus*, *Jasione laevis* and *Colchicum montanum*; Fig. 3b).

## CHANGES IN SPECIES RICHNESS AND COMPOSITION OVER THE PAST 60 YEARS (TEMPORAL APPROACH)

For the past 60 years, mean cumulative N deposition on re-sampled sites has increased by 20 162 mg N m⁻² (i.e. about

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Redundancy analysis ordination bi-plots on the first two axes. (a) displays ordination of sites, constraining variables (in black) and *a posteriori* projected years and geographic variables (in grey). (b) displays ordination of species (selected among the most abundant species and to allow reading of all the labels displayed) and *a posteriori* projected mean ecological indices (in grey). See Table S2 for a list of species acronyms. On each graph, only the significant *a posteriori* projected variables are shown. *Tmean* = mean temperature; *Prcp* = precipitation; *CumN* = cumulative N deposition; *NSasp* = North–South aspect; *WEasp* = West–East aspect; *L*<sub>ci</sub>, *T*<sub>ci</sub>, *R*<sub>ci</sub> and *N*<sub>ci</sub> = community indices for heliophily, thermophily, soil pH and fertility respectively.</figcaption></figure>
200 kg N ha⁻¹), mean temperature increased by 1.1 °C on average and annual precipitation has slightly diminished by about 100 mm on average (Table 1). The higher values of community thermophily (T<sub>ci</sub>) and soil fertility (N<sub>ci</sub>) and lower values of soil moisture (F<sub>ci</sub>) for the recent relevés compared to the historical ones indicate that plant communities have consistently responded to these environmental changes (Table 1). In contrast, no significant changes in community indices for heliophily (L<sub>ci</sub>) or soil pH (R<sub>ci</sub>) were observed (Table 1).

Over the past decades, species richness has increased by 13.8 species on average (Table 1). This could not result from a sampling plot size effect since (i) there was no correlation between plot size and species richness in this dataset (see Materials and methods section for an explanation) and (ii) the area used for the recent relevés (16 m²) was on average smaller than the area used for the historical ones (63 ± 40 m², n = 28). Thus, we assume that some recent relevés have, in fact, underestimated the increase in species richness.

Correspondence analysis (15.9% of the total variance in species composition explained by the first two axes) showed a significant shift in species composition between the historical and recent relevés on both the first and second axes towards their negative extremities (*P* < 0.001 and *P* = 0.008 respectively; Fig. 4). This shift was correlated with an increase in *CumN* (along the first axis only), *Tmean*, the community indices *T*<sub>ci</sub>, *R*<sub>ci</sub>, *N*<sub>ci</sub> and with a decrease in *L*<sub>ci</sub> (Table 2). Thus, species most frequent and abundant in the recent group of relevés (see Fig. S2) were mainly associated with high *CumN*, *Tmean*, *T*<sub>ci</sub>, *R*<sub>ci</sub> and *N*<sub>ci</sub>. In contrast, species most frequent and abundant in the historical group were typical of low *CumN*, *Tmean*, *T*<sub>ci</sub>, *R*<sub>ci</sub> and *N*<sub>ci</sub>. Both axes were also positively correlated with elevation suggesting the arrival

<table><caption>**Table 1.** Mean (±SD) of the environmental and vegetation characteristics and ecological community indices for the historical and recent groups of relevés, and the significance of the shift between the two periods. Parametric paired Student tests were used for *S* (species richness), *Tmean* (mean temperature), *L*<sub>ci</sub>, *T*<sub>ci</sub>, *R*<sub>ci</sub> and *N*<sub>ci</sub> (community indices for heliophily, thermophily, soil pH and fertility respectively). Nonparametric paired Wilcoxon rank sum tests were used for *Prcp* (precipitation), *CumN* (cumulative N deposition) and *F*<sub>ci</sub> (community index for soil moisture). A permutation test was used for *D* (Simpson dissimilarity). Bold indicates significant values</caption><thead><tr><th>Variable</th><th>Historical</th><th>Recent</th><th>P-value</th></tr></thead><tbody><tr><td><strong>Environmental factors</strong></td><td></td><td></td><td></td></tr><tr><td><em>Tmean</em> (°C)</td><td>4.5 ± 1.1</td><td>5.6 ± 1.2</td><td><strong>&lt;0.001</strong></td></tr><tr><td><em>Prcp</em> (mm)</td><td>1409 ± 471</td><td>1303 ± 445</td><td><strong>&lt;0.001</strong></td></tr><tr><td><em>CumN</em> (mg m<sup>-2</sup>)</td><td>11 263 ± 5416</td><td>31 425 ± 7081</td><td><strong>&lt;0.001</strong></td></tr><tr><td><strong>Vegetation structure</strong></td><td></td><td></td><td></td></tr><tr><td><em>S</em></td><td>21.4 ± 6.9</td><td>35.2 ± 9.5</td><td><strong>&lt;0.001</strong></td></tr><tr><td><em>D</em></td><td>0.7 ± 0.2</td><td>0.6 ± 0.1</td><td><strong>&lt;0.001</strong></td></tr><tr><td><strong>Vegetation ecology</strong></td><td></td><td></td><td></td></tr><tr><td><em>L</em><sub>ci</sub></td><td>7.5 ± 0.3</td><td>7.4 ± 0.3</td><td>0.261</td></tr><tr><td><em>T</em><sub>ci</sub></td><td>3.7 ± 0.5</td><td>3.9 ± 0.5</td><td><strong>0.007</strong></td></tr><tr><td><em>F</em><sub>ci</sub></td><td>5.0 ± 0.4</td><td>4.9 ± 0.2</td><td><strong>0.002</strong></td></tr><tr><td><em>R</em><sub>ci</sub></td><td>4.0 ± 0.6</td><td>4.1 ± 0.7</td><td>0.813</td></tr><tr><td><em>N</em><sub>ci</sub></td><td>3.0 ± 0.5</td><td>3.2 ± 0.5</td><td><strong>0.034</strong></td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Correspondence analysis ordination of pairs (linked by a grey line) of historical (circles) and recent (triangles) relevés in the 1–2 axes factorial plan. The black arrow indicates the shift from the centroid of the historical group to the centroid of the recent group. *L*<sub>ci</sub>, *T*<sub>ci</sub>, *R*<sub>ci</sub> and *N*<sub>ci</sub>: a posteriori projected mean ecological indices community for heliophily, thermophily, soil pH and fertility respectively.</figcaption></figure>

<table><caption>**Table 2.** Relationships between ecological variables and the first two axes of the Correspondence analysis. The coefficients and significance of the correlation with each axis (Spearman correlations tests) are indicated for all variables except community indices. For community indices, the scores on each axis and the significance of their projection on the 1–2 axes plane (permutation test modified) are indicated. Bold indicates significant values</caption><thead><tr><th></th><th>Axis1</th><th>Axis2</th></tr></thead><tbody><tr><td><strong>Geography</strong></td><td></td><td></td></tr><tr><td><em>Elev</em></td><td><strong>0.45</strong>***</td><td><strong>0.48</strong>***</td></tr><tr><td><em>Lon</em></td><td>-0.22</td><td>0.04</td></tr><tr><td><em>Lat</em></td><td>0.21</td><td><strong>-0.44</strong>***</td></tr><tr><td><strong>Topography</strong></td><td></td><td></td></tr><tr><td><em>Slope</em></td><td>-0.20</td><td><strong>0.28</strong>*</td></tr><tr><td><em>NSasp</em></td><td>-0.03</td><td><strong>-0.42</strong>***</td></tr><tr><td><em>WEasp</em></td><td><strong>0.38</strong>***</td><td>0.04</td></tr><tr><td><strong>N deposition</strong></td><td></td><td></td></tr><tr><td><em>CumN</em></td><td><strong>-0.47</strong>***</td><td>-0.14</td></tr><tr><td><strong>Climate</strong></td><td></td><td></td></tr><tr><td><em>Tmean</em></td><td><strong>-0.54</strong>***</td><td><strong>-0.35</strong>**</td></tr><tr><td><em>Prcp</em></td><td>0.16</td><td>-0.16</td></tr><tr><td><em>Grazing intensity</em></td><td><strong>-0.46</strong>*</td><td>0.24</td></tr><tr><td><strong>Soil</strong></td><td></td><td></td></tr><tr><td><em>% N</em></td><td>0.15</td><td>-0.16</td></tr><tr><td><em>% C</em></td><td>0.22</td><td>-0.19</td></tr><tr><td><em>pH</em></td><td><strong>-0.60</strong>***</td><td>0.17</td></tr><tr><td><em>C:N</em></td><td><strong>0.35</strong>*</td><td>-0.16</td></tr><tr><td><strong>Vegetation</strong></td><td></td><td></td></tr><tr><td><em>Cover</em></td><td>0.02</td><td>0.00</td></tr><tr><td><em>Height</em></td><td>0.14</td><td><strong>-0.37</strong>*</td></tr><tr><td><strong>Community indexes</strong></td><td></td><td></td></tr><tr><td><em>L</em><sub>ci</sub></td><td><strong>0.20</strong>*</td><td><strong>0.98</strong></td></tr><tr><td><em>T</em><sub>ci</sub></td><td><strong>-0.58</strong>***</td><td><strong>-0.81</strong></td></tr><tr><td><em>F</em><sub>ci</sub></td><td>0.92</td><td>-0.38</td></tr><tr><td><em>R</em><sub>ci</sub></td><td><strong>-0.99</strong>***</td><td><strong>-0.06</strong></td></tr><tr><td><em>N</em><sub>ci</sub></td><td><strong>-0.78</strong>*</td><td><strong>-0.63</strong></td></tr></tbody></table>

Levels of significance: *P* < 0.05; **P* < 0.01; ***P* < 0.001.
of species typical of conditions at lower sites during the past decades. Although only recent relevés held information about these variables (except soil pH also mentioned in five historical relevés), grazing intensity and soil pH were significantly negatively correlated with the first axis. In contrast, C : N ratio in soil was significantly positively correlated with the first axis. Vegetation height was negatively correlated with the second axis (Table 2).

Changes in species frequencies of occurrence between the historical and recent groups revealed that far more species increased in occurrence (214, of which 123 were new for this pool of sites) than the species which decreased (80, of which 56 were no longer present in the recent group) or remained stable (27). The species which most largely increased in occurrence have higher mean *T*, *N* and *R* ecological values than the species which decreased in occurrence, although the small number of species in this last category did not allow us to test the significance of these differences (Table 3). Among the species which increased in occurrence, 37% have a very large distribution area (either European, Eurasian or cosmopolitan; Table 3).

Discussion

POSITIVE EFFECTS OF CUMULATIVE N DEPOSITION
AND TEMPERATURE INCREASE ON PLANT RICHNESS

The increase in cumulative N deposition and mean tempera-
ture, and the reduction in precipitation observed across the
Pyrenees were consistent with trends reported in other studies
(IPCC 2007; Galloway et al. 2008; Gaudnik et al. 2011;
Miquel 2012). The higher values of community thermophily
(*T*<sub>ci</sub>) and soil fertility (*N*<sub>ci</sub>) and lower values of soil moisture
(*F*<sub>ci</sub>) for the recent relevés compared with historical ones indi-
cated that plant communities have consistently responded to
these environmental changes.

In both the spatial and temporal approaches we observed a
significant increase in plant richness and a shift in species com-
position towards more thermophilic and less oligotrophic com-
munities with increasing cumulative N deposition and
temperature. These convergent results strongly suggest that
these factors have amply contributed to profound changes in
plant composition (according to our first hypothesis) and an
increase in local plant richness in the subalpine Pyrenees grass-
lands. The positive impacts of temperature and cumulative N
deposition (Fig. 2) were evidenced by a shift in plant composi-
tion from typically high-elevation specialist species relatively
well adapted to low temperatures and/or low nutrient availabil-
ity, to generalist species having lower altitudinal range optima
and a very large distribution area. (Fig. 3, Fig. S2, Table 3).
For example, among those species, *T. alpinum* and *P. alpina*
were also reported as disadvantaged by N inputs in Alpine
communities (Bassin, Volk & Fuhrer 2013), whereas *Ceras-
tium fontanum* was, consistent with our findings, associated
with high levels of N deposition in lowland acidic grasslands
(Stevens et al. 2011).

Overall, our results agree well with our second hypothesis and previous studies that have shown a positive effect of climate warming on species diversity in higher elevation belts mainly as a result of upward migration of species (Lenoir *et al.* 2008; Gottfried *et al.* 2012). In contrast, they are not consistent with our third hypothesis and most survey studies concluding to detrimental impact (Stevens *et al.* 2004; Duprè *et al.* 2010; Maskell *et al.* 2010; van den Berg *et al.* 2011; Gaudnik *et al.* 2011) or absence of impact (McLauchlan *et al.* 2014) of N deposition on plant diversity. Yet, none of these studies concerned grazed subalpine grasslands, which suggests that the originality of our results might be linked to the specificities of these habitats. Furthermore, plant richness and eutrophic or mesotrophic species may increase simultaneously in mountains, suggesting that enhancing nutrient availability may not always have negative consequences on plant richness in such habitats (Bühler & Roth 2011). Finally, it is worth noticing that the increase in plant species richness at the site scale might have come with a homogenization of these communities across sites. Indeed, the increasing species tended to be found in a great number of sites (e.g. *C. caryophyllea*, *A. capillaris* and *C. scheuchzeri* agg. rose from occurrences of 20–40% to occurrences of 80–90%), whereas the declining species were species already present in just a small number of sites (e.g. *Leontodon pyrenaicus*, *Agrostis rupestris* and *Phyteuma hemisphaericum* decreased from occurrences of 20–30% to occurrences of 5–20%). An increase in common and generalist species and a decrease in some orophyte species leading to homogenization has already been reported in Switzerland (Bühler & Roth 2011) and in the Highlands of Scotland, UK (Ross *et al.* 2012).

OTHER FACTORS POTENTIALLY INVOLVED IN
VEGETATION CHANGES

Grasslands of the Pyrenees have experienced long-standing and extensive grazing and therefore, changes in grazing management could have deeply driven the community dynamics. We lacked accurately localized historical data, which prevented the inclusion of this variable in our analyses. However, several elements suggest that if changes in grazing have occurred they are not likely to have been responsible for the pattern and the very consistent richness increase observed across all our sites. Indeed, extensive grazing in the Pyrenees usually promotes or, at least, maintains species richness (de Bello, Lepš & Sebastià 2006; Sebastià *et al.* 2008a; Barrantes, Reiné & Ferrer 2013; Komac, Domenech & Fanlo 2014). However, since 1970 livestock units have declined across subalpine grasslands in the French Pyrenees by about 18% on average (Bozzolo 2012), which might have led to either no change or a decline in floristic diversity (Galop *et al.* 2011). Moreover, the grazing decline has not been homogeneous across the massif, both (i) at the regional scale, with a marked drop in the Eastern Pyrenees, a lower one in the Central Pyrenees and almost no change in the Western Pyrenees (Galop *et al.* 2011) where the grazing intensity is currently the
<table><caption>**Table 3.** Change in species occurrence (Δ freq.) between historical (hist.) and recent (rec.) relevés. Only species for which the occurrence changed by more than four sites (i.e. Δ freq. >0.1) are shown. For each species, its ecological values for heliophily (L), thermophily (T), soil moisture (F), pH (R) and fertility (N), altitudinal rank (according to Gottfried *et al.* 2012; lowest values indicate higher elevations) and chorology (according to Julve 1998) are indicated. In bold, species absent from the historical relevés. The averages are calculated for the decreasing and increasing species groups. See Table S2 for a list of the species acronyms</caption><thead><tr><th>Species</th><th>Δ freq. (hist.; rec.)</th><th>L</th><th>T</th><th>F</th><th>R</th><th>N</th><th>Alt. rank</th><th>Chorology</th></tr></thead><tbody><tr><td colspan="9"><strong>Decreasing species</strong></td></tr><tr><td>Scorzoneroides pyrenaica</td><td>-0.17 (0.25; 0.08)</td><td>8</td><td>3</td><td>5</td><td>3</td><td>2</td><td>4</td><td>Orophyte</td></tr><tr><td>Agrostis rupestris</td><td>-0.14 (0.32; 0.18)</td><td>8</td><td>2</td><td>4</td><td>2</td><td>1</td><td>3</td><td>Orophyte</td></tr><tr><td>Phyteuma hemisphaericum</td><td>-0.13 (0.18; 0.05)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>3</td><td>Orophyte</td></tr><tr><td>Averages</td><td></td><td>8</td><td>2.5</td><td>4.5</td><td>2.5</td><td>1.5</td><td>3.3</td><td></td></tr><tr><td colspan="9"><strong>Increasing species</strong></td></tr><tr><td>Carex caryophyllea</td><td>0.58 (0.2; 0.78)</td><td>8</td><td>5</td><td>4</td><td>3</td><td>2</td><td>NA</td><td>Eurasian</td></tr><tr><td>Agrostis capillaris</td><td>0.54 (0.38; 0.92)</td><td>7</td><td>5</td><td>5</td><td>4</td><td>4</td><td>6</td><td>Holarctic</td></tr><tr><td>Galium sp.</td><td>0.42 (0.4; 0.82)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Campanula scheuchzeri</td><td>0.35 (0.45; 0.8)</td><td>8</td><td>2</td><td>5</td><td>4</td><td>3</td><td>4/6</td><td>Oroph./euras.</td></tr><tr><td>Viola sp.</td><td>0.35 (0.15; 0.5)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Pilosella officinarum</td><td>0.33 (0.15; 0.48)</td><td>7</td><td>5</td><td>4</td><td>4</td><td>2</td><td>5</td><td>Eurasian</td></tr><tr><td>Veronica chamaedrys</td><td>0.3 (0.12; 0.42)</td><td>6</td><td>5</td><td>5</td><td>5</td><td>5</td><td>NA</td><td>Eurasian</td></tr><tr><td><strong>Thymus praecox</strong></td><td>0.28 (0; 0.28)</td><td>8</td><td>4</td><td>3</td><td>6</td><td>2</td><td>4</td><td>Orophyte</td></tr><tr><td>Veronica officinalis</td><td>0.27 (0.15; 0.42)</td><td>5</td><td>5</td><td>5</td><td>3</td><td>3</td><td>6</td><td>Circumboreal</td></tr><tr><td>Trifolium repens</td><td>0.27 (0.25; 0.52)</td><td>7</td><td>5</td><td>5</td><td>5</td><td>6</td><td>6</td><td>Eurasian</td></tr><tr><td>Crocus nudiflorus</td><td>0.27 (0.08; 0.35)</td><td>8</td><td>6</td><td>5</td><td>3</td><td>3</td><td>6</td><td>Basque</td></tr><tr><td>Polygala serpyllifolia</td><td>0.25 (0.15; 0.4)</td><td>8</td><td>4</td><td>5</td><td>2</td><td>2</td><td>NA</td><td>Atlantic</td></tr><tr><td>Alchemilla flabellata</td><td>0.25 (0.2; 0.45)</td><td>8</td><td>1</td><td>5</td><td>2</td><td>1</td><td>5</td><td>Orophyte</td></tr><tr><td>Veronica serpyllifolia</td><td>0.24 (0.08; 0.32)</td><td>7</td><td>5</td><td>5</td><td>5</td><td>5</td><td>NA</td><td>Circumboreal</td></tr><tr><td>Euphrasia sp.</td><td>0.24 (0.18; 0.42)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Gentianella campestris</td><td>0.23 (0.02; 0.25)</td><td>8</td><td>5</td><td>5</td><td>4</td><td>2</td><td>NA</td><td>European</td></tr><tr><td>Cerastium fontanum</td><td>0.22 (0.18; 0.4)</td><td>7</td><td>5</td><td>5</td><td>5</td><td>6</td><td>4</td><td>Cosmopolitan</td></tr><tr><td>Trifolium pratense</td><td>0.22 (0.35; 0.57)</td><td>7</td><td>5</td><td>5</td><td>5</td><td>6</td><td>5</td><td>Eurasian</td></tr><tr><td>Poa alpina</td><td>0.2 (0.08; 0.28)</td><td>8</td><td>2</td><td>4</td><td>7</td><td>2</td><td>5</td><td>Orophyte</td></tr><tr><td>Taraxacum sp.</td><td>0.2 (0.1; 0.3)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Vaccinium myrtillus</td><td>0.2 (0.22; 0.42)</td><td>5</td><td>4</td><td>5</td><td>2</td><td>3</td><td>6</td><td>Eurasian</td></tr><tr><td>Galium verum</td><td>0.2 (0.28; 0.48)</td><td>7</td><td>5</td><td>3</td><td>7</td><td>3</td><td>NA</td><td>Holarctic</td></tr><tr><td><strong>Carduus defloratus</strong></td><td>0.18 (0; 0.18)</td><td>7</td><td>2</td><td>3</td><td>8</td><td>4</td><td>5</td><td>Orophyte</td></tr><tr><td>Lotus corniculatus</td><td>0.18 (0.52; 0.7)</td><td>7</td><td>5</td><td>4</td><td>7</td><td>3</td><td>4/5</td><td>Euras./oroph.</td></tr><tr><td>Festuca nigrescens agg.</td><td>0.18 (0.8; 0.98)</td><td>7</td><td>5</td><td>5</td><td>3</td><td>2</td><td>5</td><td>Holarctic</td></tr><tr><td>Luzula campestris</td><td>0.17 (0.05; 0.22)</td><td>7</td><td>5</td><td>4</td><td>3</td><td>3</td><td>NA</td><td>Cosmopolitan</td></tr><tr><td>Achillea millefolium</td><td>0.17 (0.18; 0.35)</td><td>8</td><td>5</td><td>5</td><td>5</td><td>4</td><td>6</td><td>Eurasian</td></tr><tr><td>Cruciata glabra</td><td>0.17 (0.18; 0.35)</td><td>7</td><td>3</td><td>5</td><td>5</td><td>5</td><td>6</td><td>European</td></tr><tr><td>Antennaria dioica</td><td>0.16 (0.12; 0.28)</td><td>8</td><td>4</td><td>4</td><td>3</td><td>2</td><td>5</td><td>Circumboreal</td></tr><tr><td>Poa chaixii</td><td>0.16 (0.02; 0.18)</td><td>6</td><td>5</td><td>5</td><td>3</td><td>4</td><td>NA</td><td>European</td></tr><tr><td>Poa supina</td><td>0.15 (0.05; 0.2)</td><td>8</td><td>2</td><td>5</td><td>6</td><td>6</td><td>NA</td><td>Orophyte</td></tr><tr><td>Epikeros pyrenaeus</td><td>0.15 (0.35; 0.5)</td><td>8</td><td>3</td><td>8</td><td>2</td><td>2</td><td>NA</td><td>Orophyte</td></tr><tr><td><strong>Ajuga reptans</strong></td><td>0.15 (0; 0.15)</td><td>5</td><td>5</td><td>5</td><td>5</td><td>6</td><td>NA</td><td>Eurasian</td></tr><tr><td><strong>Erigeron uniflorus</strong></td><td>0.15 (0; 0.15)</td><td>9</td><td>1</td><td>5</td><td>6</td><td>2</td><td>2</td><td>Arctico-alpine</td></tr><tr><td>Ranunculus gouanii</td><td>0.15 (0.1; 0.25)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Orophyte</td></tr><tr><td>Pilosella lactucella</td><td>0.15 (0.42; 0.57)</td><td>8</td><td>3</td><td>5</td><td>4</td><td>2</td><td>5</td><td>European</td></tr><tr><td>Luzula pediformis</td><td>0.14 (0.08; 0.22)</td><td>7</td><td>2</td><td>5</td><td>2</td><td>2</td><td>NA</td><td>Orophyte</td></tr><tr><td>Cerastium sp.</td><td>0.13 (0.02; 0.15)</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Rumex acetosa</td><td>0.13 (0.02; 0.15)</td><td>8</td><td>5</td><td>5</td><td>5</td><td>6</td><td>6</td><td>Holarctic</td></tr><tr><td><strong>Helictotrichon sedenense</strong></td><td>0.12 (0; 0.12)</td><td>9</td><td>2</td><td>4</td><td>9</td><td>2</td><td>4</td><td>Orophyte</td></tr><tr><td><strong>Thesium pyrenaicum</strong></td><td>0.12 (0; 0.12)</td><td>8</td><td>3</td><td>4</td><td>4</td><td>2</td><td>NA</td><td>Eurasian</td></tr><tr><td><strong>Ranunculus repens</strong></td><td>0.12 (0; 0.12)</td><td>5</td><td>5</td><td>7</td><td>5</td><td>7</td><td>NA</td><td>Holarctic</td></tr><tr><td>Prunella vulgaris</td><td>0.12 (0.1; 0.22)</td><td>5</td><td>5</td><td>5</td><td>6</td><td>6</td><td>NA</td><td>Cosmopolitan</td></tr><tr><td>Averages</td><td></td><td>7.2</td><td>4.0</td><td>4.8</td><td>4.5</td><td>3.5</td><td>5.0</td><td></td></tr></tbody></table>

highest (ACP-Pyrénées 2010; Bozzolo 2012) and (ii) at the local scale, where, grazing has persisted in all of the 40 sites studied but at different intensities.

Precipitation decreases could also have altered species richness (Sebastià *et al.* 2008b). Indeed, the soil moisture index, $F_{ci}$, has decreased significantly but in a range that has induced

no significant changes in plant richness (Table 1, Fig. 1g). Moreover, according to the complex relationship between precipitation and species richness (Fig. 1b) the latter would have either increased or decreased depending on whether historical precipitation ranged between 1500 and 1100 mm or was just below 1100 mm. Finally, $Prcp$ and $F_{ci}$ were not significantly
involved in the temporal floristic change in the re-sampled
communities (Fig. 4). Overall, these observations do not sup-
port a driving role of precipitation changes in the global
floristic shift and the consistent richness increase observed
across the Pyrenees.

MECHANISMS INVOLVED IN VEGETATION CHANGES

Nitrogen addition in a tightly N-limited ecosystem may have
two opposed and potentially independent effects on plant
communities: (i) increasing plant richness by favouring the
establishment of more nitrophilous species (Huston &
DeAngelis 1994; Grime 2001; Olofsson & Shams 2007), (ii)
decreasing plant richness by inducing competitive exclusion
of stress-tolerant oligotrophic species through an increase in
biomass of eutrophic species leading to light limitation (Hau-
tier, Niklaus & Hector 2009) or/and soil acidification (Maskell
et al. 2010). Therefore, the local plant diversity should reflect
the net influence of these two opposed effects and how both
are influenced by the local environmental context.

The increase in mean temperature and the addition of exogenous N (mainly NO₃⁻ and NH₄⁺) readily available for plants could have alleviated the low temperature and nutrient availability constraints (Körner 1999), thus reducing the strength of the abiotic filter on plant communities. This could have allowed less stress-tolerant and more generalist species (Grime 1977), physiologically adapted to warmer temperatures and richer soils, to become established. This hypothesis is supported by the curved relationship between plant richness and soil fertility (Fig. 1f), which perfectly fits with the hump-backed model of Grime (2001). The model states that few stress-tolerant plant species can survive on particularly poor soils. Species diversity increases with fertility (Huston & DeAngelis 1994; Olofsson & Shams 2007) until attaining a peak from which it decreases as a few species highly competitive in the use of resources out-compete other species. In our communities the richness peak was around 3·5 N₉ᵢ and more than 90% of our sites had lower values. The temporal trajectory of community composition along the elevation gradient in the temporal analysis provides evidence that both temperature and N increases have favoured the upward migration of species and were chiefly responsible for the observed richness and compositional shifts.

Nitrogen supply commonly drives plant richness losses of inferior competitors for light (Hautier, Niklaus & Hector 2009; Borer et al. 2014). Experiments have, nevertheless, shown that trampling and grazing can alleviate competitive exclusion through biomass removal (Kohler et al. 2004; Bonanomi, Caporaso & Allegrezza 2009; Lanta et al. 2009) offsetting light limitation and competitive exclusion (Borer et al. 2014). Thus, while changes in management practices are not likely to have been the main drivers of the community shift observed across the Pyrenees, grazing persistence could have limited the competitive exclusion of local species. Actually, the subalpine grasslands studied here have low vegetation height (10·5 cm on average on our sites) as a result of extensive grazing. Furthermore, no significant change in

community heliophily (Lci) has been observed over the past
decades contrary to expectations if typical heliophilous spe-
cies were lost following an increase in the competition for
light. The hypothesis of a positive effect of grazing is sup-
ported by the finding of Pasari, Hernandez & Zavaleta
(2014), who observed that native species richness declined in
fertilized plots removed from grazing, whereas it increased in
grazed, fertilized plots. Actually, grazing can stabilize ecosys-
tem function against the impacts of global change through N
and biomass exports (Gill 2014). Grazing could also have
mitigated soil eutrophication through N exportation by live-
stock (Gill 2014) and facilitated seedling establishment by
creating microsites suitable for plant recruitment (Olofsson &
Shams 2007) or as a vector of seed dispersal.

Soil acidification enhancing the availability of toxic ions is another main cause of species loss following N addition (Malkell et al. 2010). In this study, we did not observe signs of a decrease in soil pH (R<sub>ci</sub>, Table 1) over the past decades (temporal approach). Moreover, the species associated with higher temperature and N deposition were also associated with higher pH (Table 2) and R<sub>ci</sub> values in the multivariate analyses. Finally, in the spatial approach we observed a strong positive relationship between pH and plant richness as shown by other studies (Grime 1977; Pärtel et al. 2004; Stevens et al. 2004). These results suggest that cumulative N deposition has not led to detectable soil acidification in these habitats.

Conclusions

This is, to our knowledge, the first study showing that cumu-
lative N deposition has had, with climate warming, a positive
impact on local vascular plant species richness of grazed sub-
alpine grasslands over the past decades. We propose that this
original finding results from: (i) the establishment of less
stress-tolerant species following the decrease in temperature
and nutrient constraints and (ii) the long-standing persistence
of grazing that has prevented competition for light and
allowed the survival in the communities of species typical of
stressful habitats. These processes have outweighed the
decrease and the extirpation of some plant species. Thus, in
contrast with other grasslands, high-elevation grazed grass-
lands may shift towards enhanced species diversity with N
deposition under climate warming.

Authors’ contributions

M.B., E.C., D.A. and A.P. designed the study; M.B., E.C., D.A., L.V., J.J.J., C.B., C.L., G.C and A.P. performed field work; M.B., C.B., C.L. and T.L. worked data; M.B. and A.P. wrote the manuscript with contributions from E.C., D.A. and G.C. and corrections from all authors. All authors approved this manuscript.

Acknowledgments

We are grateful to the CBNPMP, SOPHY and SIVIM databases, and to the
authors of the historical vegetation relevés whose past work made this study
possible. We thank François Lamarque for improving the English. We thank
the referees for helpful comments on the manuscript. This work was funded by
the French Environment and Energy Management Agency (ADEME;
ANEMONE project No. 1262C0013), the Communauté de Travail des Pyrénées through the Region Midi-Pyrénées (ANEMONE project No. 11051284) and Aragón (ANEMONE project No. CTPP13/11) and the Observatoire Homme Milieux Pyrénées (OHM) du Haut Vicdessos. This work was supported by the French Laboratory of Excellence project 'TULIP' (ANR-10-LABX-41; ANR-11-IDEX-0002-02).

## Data accessibility

The historic vegetation-plot data are archived by the Conservatoire Botanique National Pyrénées Midi-Pyrénées and are listed in Table S1 of the Supporting Information.

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Received 9 May 2016; accepted 23 December 2016

Handling Editor: Meghan Avolio

## Supporting Information

Details of electronic Supporting Information are provided below.

**Fig. S1.** Location of the sites over the Pyrenean massif.

**Fig. S2.** Correspondence analysis displaying the ordination of the plant species.

**Table S1.** References of the vegetation-plot data collected for this study.

**Table S2.** Detailed information about the 40 sites revisited.

**Table S3.** List of the species acronyms.

**Appendix S1.** Supplementary materials and methods.