This is the accepted version of the article: Espunyes, Johan; Lurgi Rivera, Miguel; Büntgen, Ulf; [et al.]. Different ef- fects of alpine woody plant expansion on domestic and wild ungulates. DOI 10.1111/gcb.14587 This version is avaible at https://ddd.uab.cat/record/203555 under the terms of the license This is the accepted version of the article: Espunyes, Johan; Lurgi Rivera, Miguel; Büntgen, Ulf; [et al.]. Different ef- fects of alpine woody plant expansion on domestic and wild ungulates. DOI 10.1111/gcb.14587 This version is avaible at https://ddd.uab.cat/record/203555 under the terms of the license This is the accepted version of the article: Espunyes, Johan; Lurgi Rivera, Miguel; Büntgen, Ulf; [et al.]. Different ef- fects of alpine woody plant expansion on domestic and wild ungulates. DOI 10.1111/gcb.14587 This version is avaible at https://ddd.uab.cat/record/203555 under the terms of the license This is the accepted version of the article: Espunyes, Johan; Lurgi Rivera, Miguel; Büntgen, Ulf; [et al.]. Different ef- fects of alpine woody plant expansion on domestic and wild ungulates. DOI 10.1111/gcb.14587 This version is avaible at https://ddd.uab.cat/record/203555 under the terms of the license 1 Title: Different effects of alpine woody plant expansion on domestic 2 and wild ungulates 3 Running head: “Effects of woody plant expansion on ungulates” 4 Johan Espunyes1, Miguel Lurgi2, Ulf Büntgen3,4,5,6, Jordi Bartolomé7, Juan Antonio Calleja8,9, 5 Arturo Gálvez-Cerón1,10, Josep Peñuelas9,11, Emmanuel Serrano1 6 7 1. Wildlife Ecology & Health group (WE&H) and Servei d’Ecopatologia de Fauna Salvatge 8 (SEFaS), Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat 9 Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain 10 2. Centre for Biodiversity Theory and Modelling. Theoretical and Experimental Ecology 11 Station, CNRS-Paul Sabatier University, 09200, Moulis, France. 12 3. Department of Geography, University of Cambridge, Cambridge, Downing Place, CB2 3EN, 13 UK 14 4. Swiss Federal Research Institute WSL, Zürcherstr 111, 8903, Birmensdorf, Switzerland 15 5. Global Change Research Centre (CzechGlobe), Bělidla 986/4a, 60300, Brno,Czech Republic 16 6. Department of Geography, Faculty of Science, Masaryk University, Kotlářská 2, 613 00, 17 Brno, Czech Republic 18 7. Grup de Recerca en Remugants, Departament de Ciència Animal i dels Aliments, Facultat 19 de Veterinària, Universitat Autònoma de Barcelon, 08193, Bellaterra, Barcelona, Spain 20 8. Unitat de botánica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, 21 Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain. 22 9. Centre de Recerca Ecològica i Aplicacions Forestals (CREAF), 08193, Cerdanyola del 23 Vallès, Spain. 24 10. Facultad de ciencias pecuarias, Universidad de Nariño, Pasto, Colombia 25 11. CSIC, Global Ecology Unit, CREAF-CSIC-Universitat Autònoma de Barcelona, 08193, 26 Bellaterra, Spain 1 27 28 Corresponding author: Johan Espunyes, (0034)935868190, johan.espunyes@gmail.com 29 30 Keywords: herbivory, diet preference, Pyrenean chamois, free-ranging livestock, habitat 31 change, shrubification, mountain ecosystems 32 Paper type: Primary Research Article 2 33 Abstract 34 Changes in land-use and climate affect the distribution and diversity of plant and animal 35 species at different spatiotemporal scales. The extent to which species-specific phenotypic 36 plasticity and biotic interactions mediate organismal adaptation to changing environments, 37 however, remains poorly understood. Woody plant expansion is threatening the extent of alpine 38 grasslands worldwide, and evaluating and predicting its effects on herbivores is of crucial 39 importance. Here, we explore the impact of shrubification on the feeding efficiency of Pyrenean 40 chamois (Rupicapra p. pyrenaica), as well as on the three most abundant coexisting domestic 41 ungulate species: cattle, sheep and horses. We use observational diet composition from May to 42 October and model different scenarios of vegetation availability where shrubland and woodland 43 proliferate at the expense of grassland. We then predicted if the four ungulate species could 44 efficiently utilise their food landscapes with their current dietary specificities measuring their 45 niche breath in each scenario. We observed that the wild counterpart, due to a higher trophic 46 plasticity, is less disturbed by shrubification compared to livestock, which rely primarily on 47 herbaceous plants and will be affected 3.6 times more. Our results suggest that mixed feeders, 48 such as chamois, could benefit from fallow landscapes, and that mountain farmers are at a 49 growing economic risk worldwide due to changing land-use practices and climate conditions. 50 51 Introduction 52 Environmental and climatic changes are affecting biological and ecological systems 53 across the globe at alarming rates (Steffen et al., 2005). These trends influence fauna and flora 54 in many ways, from habitat degradation to distributional range shifts, as well as phenological 55 mismatch (Parmesan & Yohe, 2003; Root et al., 2003; Pereira et al., 2010). In fact, global land- 3 56 use and climatic changes, through their influence on different aspects of the biology and ecology 57 of species, have caused numerous extinctions (Vitousek et al., 1997), with models predicting 58 an intensification of these trends over the next century (Loarie et al., 2009). Specialist species 59 are predicted to decline at a faster rate due to their limited adaptive potential within their narrow 60 environmental tolerances (Thuiller et al., 2005; Morrison et al., 2018). Understanding the 61 species-specific potential and limitation to cope with global change is thus a central aspect of 62 timely conservation studies (Nussey et al., 2005; Charmantier et al., 2008). 63 In this context, evaluating and predicting the impact of global change on wild herbivores 64 has become a conservation priority globally, since their protection and management has been 65 deemed crucial for the long-term conservation of ecosystems (e.g., Büntgen et al., 2014). 66 Indeed, herbivores fulfil key roles in the terrestrial trophic cascades and the maintenance of 67 ecosystem health by affecting nutrient cycles and maintaining the diversity and stability of 68 predators and primary producers (Bardgett & Wardle, 2010). They are also considered 69 “environmental engineers” due to their fundamental role in the structure, composition, and 70 functioning of ecosystems (Schmitz, 2008). Numerous questions regarding the effects of global 71 change must be answered including whether or not herbivores will be able to maintain their role 72 in a particular ecosystem subject to change. 73 European mountains are a paradigmatic representation of a changing ecosystem, host of 74 a wide variety of wild and domestic herbivores. As in other mountain ranges around the world, 75 they have undergone a biological shift since the mid-20th century due to profound agricultural 76 land-use and climatic changes (Sanz-Elorza et al., 2003; Mottet et al., 2006; Steinbauer et al., 77 2018). The dramatic decline in rural populations and agropastoral activities have led to a general 78 decline in livestock densities (Didier, 2001; Gartzia et al., 2016). Temperatures have 79 simultaneously increased (IPCC, 2007), which have affected these ecosystems, albeit to a lesser 80 degree, for example by stimulating shrub development or by upward shifting the tree line 4 81 (Peñuelas et al., 2007; Ameztegui et al., 2016). Consequently, plant succession at varying rates 82 leads to woody plant expansion (Prévosto et al., 2011; Mod & Luoto, 2016), resulting in a shift 83 in dominance from herbaceous to woody plants in one of the richest habitats in the world: alpine 84 grasslands (Wilson et al., 2012). However, this so-called shrubification (Martin et al., 2017) 85 generally decreases the diversity of plant species (Tasser & Tappeiner, 2002; Koch et al., 2015), 86 the productivity of the environment (Lett & Knapp, 2003) and the diversity and total abundance 87 of mammals (Stanton et al., 2018). Community composition and ecological interactions 88 between species, including herbivore-plant interactions, are being importantly restructured 89 (Lurgi et al., 2012). These trends will likely continue as models predict a continuous increase 90 in temperature (López-Moreno et al., 2008) and a decrease in rural agropastoral activities 91 (Verburg et al., 2010; Mann, 2013). 92 The Pyrenees, in southwest Europe, have experienced a major expansion and 93 densification of shrubland and forested areas over the last century. For this reason, they 94 constitute an ideal study case of the effects of land-use and climate change on natural 95 communities. Not only have tree line ecotones increased on average by 35m (Ameztegui et al., 96 2016), with forest cover in some areas expanding by at least two-thirds (Poyatos et al., 2003; 97 Lasanta-Martínez et al., 2005), but they have also experienced an increase in recent summer 98 temperatures occurring at an unprecedented rate (Büntgen et al., 2008, 2017). These locally 99 detected changes are consistent with a larger-scale trend across most (or even all) of the 100 European mountain systems (Fig. 1). 5 101 102 Figure 1. Schematic representation of the primary causes of woody plant 103 expansion in European alpine ecosystems. The decrease in mountain rural populations 104 causes a decline in agropastoral activities and therefore a reduction in livestock densities. At 105 the same time, temperatures increase due to climate change. The combination of these factors 106 leads to the expansion of woody plants in alpine grasslands (1. Gartzia et al., 2016; 2. Didier, 107 2001; 3. Metailié & Paegelow, 2005; 4. Lasanta-Martínez et al., 2005; 5. Collantes, 2006; 6. 108 Sturaro et al., 2005; 7. Büntgen et al., 2006; 8. Büntgen et al. 2017; 9. Cannone et al., 2007; 109 10. Roura-Pascual et al., 2005; 11. Bartolomé et al., 2005; 12. Kozak et al., 2007) 110 111 Through evolution, and in some cases further domestication, large herbivores present in 112 the Pyrenean grasslands display a wide range of body sizes, digestive systems and feeding 6 113 behaviours adapted to specific diets. They are consequently expected to respond differently to 114 habitat change (Somero, 2010) and are thus good models for the study of species-specific effects 115 of woody plant expansion. 116 The aim of this study is to explore the potential impact of the expansion of woody plant 117 coverage on four herbivore species in the eastern Spanish Pyrenees - the wild Pyrenean chamois 118 (Rupicapra p. pyrenaica), as well as seasonal domestic cattle, sheep and horses - that inhabit 119 the same alpine habitats. Traditional farming is based on livestock freely living and grazing on 120 alpine grasslands during the summer to reduce the economic costs involved in livestock 121 maintenance. Once the yield of these human-created grasslands decay, livestock is then moved 122 to the hay meadows in the lower parts of the valleys until the following spring (Montserrat & 123 Fillat, 1990). The Pyrenean chamois is a medium-sized wild caprinae well adapted to mountain 124 environments. They are considered mixed feeders, which consume a wide variety of resources 125 depending on the habitat and season, following the annual cycle of primary productivity 126 (Espunyes et al., 2019). Together, these four species represent a contrasting collection of 127 energetic requirements (large- vs medium-sized herbivores), feeding behaviours (grazers vs 128 mixed feeders), digestive physiologies (rumen vs hindgut fermenters), and origins (livestock vs 129 wildlife). 130 We devised various scenarios of shrubification based on the current vegetation cover, 131 where shrubland and woodland proliferate at the expense of grassland following models of 132 projected woody plant expansion. We then use these shrubification scenarios and the diet 133 composition of the ensemble of herbivores to predict how phenotypic plasticity and biotic 134 interactions mediate the effects of shrubification. Given the diverse dietary requirements of our 135 four species, we hypothesise that changes in land cover would affect each species differently, 136 with grazers being more severely and rapidly affected than mixed feeders. 7 137 138 Methods 139 Study area 140 The study was carried out in the Freser-Setcases National Game Reserve (FSNGR), in 141 the eastern part of the Spanish Pyrenees (42°22′N, 2°09′E). This area of 410 ha is known as 142 Costabona and ranges from 1500 to 2400 m.a.s.l. It belongs to the sub-humid subalpine and 143 alpine bioclimatic belts of the southern slopes of the Pyrenees, with a noticeable Mediterranean 144 climatic influence (Vigo, 2008). Mean annual temperature for 2009-2012 was 5.7 ºC (daily 145 min=-18.2, max=26.6), and mean yearly accumulated rainfall for the same period was 1042.4 146 mm (yearly min=762.6, max=1282.8). These data were obtained from the Nuria meteorological 147 station at 1971 m a.s.l. in the core of the FSNGR (Servei Meteorològic de Catalunya). 148 The vegetation cover of our study area was assessed in June 2011 following the line- 149 intercept method proposed by Cummings and Smith (2000). The cover of all plant species 150 present was recorded along six randomly selected transects of 10×0.1 m at different altitudes. 151 152 Collection and analysis of faeces 153 Fresh faecal samples from each of the four ungulates considered in this study (namely 154 Pyrenean chamois, cattle, horses and sheep) were collected monthly from June to October 2011 155 and 2012 (except in September 2012 when sampling was not possible due to adverse 156 meteorological conditions), coinciding with the presence of the four species in the area. Once 157 every month, two observers walked a transect of about 5 km, covering the entire altitudinal 158 range and main vegetation communities of the study area. Fresh faecal samples from at least 159 five individuals per species were collected and pooled together before being transported to the 8 160 laboratory and frozen at -20 ºC after every session. A total of nine pooled samples was obtained 161 per species. This sampling procedure was used to obtain a general overview of the variability 162 of feeding in the field during the three periods of plant phenology in our study area (namely: 163 green-up, plateau greenness and senescence periods; Villamuelas et al., 2016). 164 A faecal cuticle microhistological analysis was used to determine dietary composition, 165 adapted from a protocol described by Stewart (1967). The samples were thawed, washed and 166 ground to separate the epidermal fragments. Ten grams of sample were then placed in a test 167 tube containing 5 ml of 65% concentrated nitric acid, boiled in a water bath for 1 min, and 168 diluted with 200 ml of water. This suspension was passed through 1.00- and 0.25-mm filters. 169 The 0.25-1.00 mm fraction was spread on glass microscope slides in 50% glycerol, and cover- 170 slips were fixed with DPX microhistological varnish. Two slides were prepared from each 171 sample. The slides were microscopically examined by the same operator at magnifications of 172 100× and 400×, and 200 fragments of plant epidermis were identified per sample. An epidermis 173 collection of the 55 main plant species in the study area were collected and used for fragment 174 identification. Plant cuticles were identified to the species or genus level depending on the 175 difficulty of the task. 176 177 Simulation of woody plant expansion 178 Patterns of expansion of woody plants into grasslands have been studied worldwide 179 (Olsson et al., 2000; Bartolomé et al., 2005; Falcucci et al., 2007; Eldridge et al., 2011). This 180 plant succession can proceed at different speeds and with different numbers of stages depending 181 on land-use patterns, initial state, altitude, or topography (Tasser & Tappeiner, 2002; Vacquié 182 et al., 2016). This process can nevertheless be synthesised in a first successional stage when 183 herbaceous species are replaced by shrubs, followed by a second successional stage when 184 shrubs are replaced by trees (Gellrich et al., 2007; Tasser et al., 2007; Wallentin et al., 2008; 9 185 Améztegui et al., 2010). Succession can be fast; descriptive and predictive studies have 186 demonstrated that woody plant cover can increase by 0.5-5% per year (Barger et al., 2011; 187 Komac et al., 2013). 188 To simulate the effects of woody plant expansion, we devised eight hypothetical 189 scenarios where shrubland and woodland proliferate at the expense of grassland without any 190 agricultural practices or forestry management. Based on the original vegetation availability in 191 the study area, we designed shrubification scenarios where woody plants increased and 192 graminoids and forbs decreased proportionally. The initial state of the system (i.e. original 193 scenario) comprised a relative abundance of woody species of 21.4% (19.6% shrubs and 1.8% 194 trees). Total relative abundance of woody plants was then increased by intervals of 10% per 195 scenario until reaching 100% of woody plant cover (except the first scenario which increased 196 by 8.6% to achieve 30% woody plant cover). This procedure yielded nine scenarios of woody 197 plant cover (the original scenario plus eight hypothetical): 21.4%, 30%, 40%, 50%, 60%, 70%, 198 80%, 90% and 100%, respectively. The increase in woody plant cover was distributed 199 proportionally across the plant species included in this category according to their relative 200 abundance. For example, if woody plant cover increased by 20% and the plant Juniperus 201 communis represents 50% of the total woody plant cover then a 10% increase of that plant was 202 simulated. Conversely, the cover of forbs and graminoids was decreased by the same fraction 203 of woody plant increase. This decrease was also distributed proportionally between the species 204 of these categories (see Supplementary Table S1). 205 206 Landscape-use efficiency 207 The breadth of a resource niche can be used as a proxy for species performance 208 (Rotenberry & Wiens, 1980) or to quantify the extent to which organisms are able to exploit 10 209 their environment (Krebs, 1999). We used the measure of niche breadth proposed by Smith 210 (1982) as a proxy for the efficiency in the use of resources by the herbivores (i.e. landscape-use 211 efficiency, LUE). 212 LUE for each herbivore in each shrubification scenario was calculated as: 213 𝐿𝑈𝐸 = ∑ (√𝑃𝑗 × 𝐴𝑗 ) 214 where Pj is the proportion of plant j in the diet of the herbivore, and Aj is the proportion of plant 215 j available in the study area. This index ranges from nearly zero, for the narrowest possible 216 niche when a species is specialised in eating the rarest resources, to one, for the broadest 217 possible niche when a species uses resources in proportion to their availability. This index is 218 thus low when a species inefficiently uses the resources of its habitat and is high when a species 219 uses them efficiently (i.e. proportionally to the availability). 220 221 Statistical analysis 222 After describing the diets of our studied species by basic statistics, we performed 223 a non-parametric multivariate analysis of similarity (ANOSIM; Clarke, 1993) to check for 224 differences in diets between herbivores. The ANOSIM statistic R is based on the difference of 225 mean ranks between groups and within groups and a high value of R in this analysis indicates a 226 high dissimilarity between groups. A nonmetric multidimensional scaling (NMDS) plot based 227 on Bray–Curtis dissimilarity indices was created to visually identify the patterns in dietary 228 similarities between species. Stress, a measure of goodness of fit should be <0.2 in order to have 229 a good representation with no prospect of misinterpretation (Clarke & Warwick, 2001). Our 230 NMDS stress was 0.0985 so our representation was considered to be sufficiently well-described 11 231 in two dimensions. The ANOSIM and the NMDS plot were performed using the R vegan 232 package (version 2.4-5, Oksanen et al., 2017). 233 We then evaluated the impact of woody plant expansion (i.e. woody plant abundance in 234 the scenarios) on the LUE of each species by a linear model (LM). LUE of each species was 235 the response variable in our LM whereas the interaction between animal species and degree of 236 woody plant expansion were our fixed explanatory factors. Interspecific differences of LUE 237 values were analysed with a pairwise Mann-Whitney U test using the FSA package (version 238 0.8.17, Ogle, 2017). 239 Normality of residuals and homogeneity of variance assumptions were checked previous 240 to the performance of any analysis. All statistical analyses were performed using R version 3.4.3 241 (R Core Team, 2018). 242 243 244 Results 245 Initial state of the system and herbivore diets 246 We recorded 65 plant species in our study area. Graminoids represented half of the 247 vegetation cover (51.6%), where Festuca spp. was clearly dominant (32.3%), followed by 248 Carex cariophyllea (12.4%). Forbs covered almost one-third of the area and were dominated 249 by Trifolium alpinum (7.5%), followed by Trifolium repens (1.5%) and Hippocrepis comosa 250 (1.4%). The other plants were woody species (shrubs and trees, 21.2%), where dwarf shrubs 251 (Calluna vulgaris and Juniperus communis) and legumes Cytisus spp. were the most common 252 (See Table S1). 253 The ANOSIM indicated that the differences in dietary composition were higher between 254 Pyrenean chamois and the livestock than amongst the livestock species (Table 1). Among the 12 255 livestock diets, horse diet differed the most from the rest, while cattle and sheep showed more 256 similar dietary compositions. The NMDS plot supported these interspecific dietary differences 257 (Fig. 2). Cattle Horses Sheep R=0.506 R=0.692 R=0.569 Chamois p= 0.002 p=0.001 p=0.001 R=0.246 R=0.427 Sheep p=0.013 p=0.001 R=0.386 Horses p=0.002 258 Table 1. Differences in dietary composition between alpine ungulates in the Pyrenees. 259 Summary of the pairwise ANOSIM of the differences in dietary composition between 260 Pyrenean chamois, cattle, horses, and sheep from June to October 2011 and 2012 in the 261 eastern Spanish Pyrenees. A high value of R in this analysis indicates a high dissimilarity 262 between groups. 13 263 264 Figure 2 Diet dissimilarity among alpine ungulates in the Pyrenees. Nonmetric 265 multidimensional scaling (NMDS) plot representing dietary dissimilarity for seasonal 266 livestock (cattle, horses, and sheep) and Pyrenean chamois from June to October 2011 and 267 2012 in eastern Spanish Pyrenees. When stress, a measure of goodness of fit, is <0.2, NMDS 268 reproduces an adequate depiction of the groups. 269 270 Analysis of diet composition for livestock during summer and autumn showed a larger 271 overall content of graminoid and forb fragments compared to woody plants. Horses were the 272 most extreme livestock species with the highest consumption of graminoids (63.7%) and the 273 lowest consumption of woody plants (5.9%; see Table 2). On the contrary, Pyrenean chamois 274 faeces had the highest content of woody plant fragments (48.6%) and the lowest content of 275 graminoids (25.8%) and forbs (25.3%). Cattle and sheep had similar diets (R=0.246, see table 14 276 1), but cattle showed a higher content of graminoids (cattle: 49.8%; sheep: 45.9%) and woody 277 plants (cattle: 16.2%; sheep: 12.4%) and a lower content of forbs (Cattle: 34.1%; sheep: 41.6%). 278 Chamois Cattle Horses Sheep Woody plants Calluna vulgaris 24.6 10.9 2.2 2.1 (0.0-6.0) (1.0-22.5) (0.0-12.0) (0.0-29.5) 17.3 0.1 0.0 0.3 Cytisus spp. (3.5-51.5) (0.0-0.5) (0.0-0.0) (0.0-6.0) 7.1 5.2 3.7 10.1 Other woody plants (0.0-17.5) (2.0-8) (0.5-9.5) (3.0-17.0) Total 48.6 16.2 5.9 12.4 Graminoids 22.0 40.4 52.4 40.4 Festuca spp. (8.5-50.0) (26.5-52.0) (43.0-63.5) (27.5-52.0) 1.8 3.2 1.8 2.1 Avenula pratensis (0.0-6.5) (0.0-6.5) (0.0-6.0) (0.0-4.5) 2.1 6.2 9.5 3.4 Other graminoids (0.0-6.5) (3.0-11.5) (5.0-13.0) (2.0-7.0) Total 25.8 49.8 63.7 45.9 Forbs 1.3 2.2 1.5 4.1 Anthyllis vulneraria (0.0-5.0) (0.0-7.0) (0.0-4.5) (2.0-6.5) 3.6 3.5 3.9 5.2 Plantago monosperma (0.0-9.0) (2.0-5.0) (2.0-8.0) (3.0-10.0) 2.5 3.9 2.2 4.7 Potentilla spp. (0.0-6.0) (2.0-6.0) (0.5-4.5) (1.5-8.0) 11.2 13.4 13.8 14.2 Trifolium spp. (2.0-17.5) (5.5-21.0) (7.0-20.0) (6.0-21.5) 6.7 10.9 9.1 13.5 Other forbs (2.5-10.5) (4.0-17.0) (5.0-19.0) (8.5-20.0) Total 25.3 34.1 30.4 41.6 279 280 Table 2. Dietary composition of Pyrenean chamois and seasonal cattle, horses, and sheep 281 in the Pyrenees. Data from June to October 2011 and 2012 in the Freser-Setcases National 282 Game Reserve (eastern Spanish Pyrenees). Values represent mean percentages of fragment 283 frequency (min-max). 284 285 Simulation of woody plant expansion and LUE 286 Changes in LUE through the hypothetical scenarios of woody plant expansion suggested 287 that horses would be most affected by the disappearance of grasslands in the Pyrenees. Pyrenean 288 chamois, on the other hand, could even benefit during the early stages of expansion (Fig. 3). 15 289 Current LUE is lower for chamois (median=0.72, min=0.57, max=0.75) than livestock (cattle: 290 median=0.76, min=0.70, max=0.79; horses: median=0.74, min= 0.71, max=0.79; sheep: 291 median=0.73, min=0.69, max=0.77; significantly different only between cattle and chamois, 292 w=73, p=0.0028). Our LM revealed that 76.8% of the observed LUE variability was explained 293 by the interaction between woody plant expansion and ungulate species (F3,316 = 149.2, 294 p<0.001). 295 296 16 297 Figure 3. Evolution of the landscape-use efficiencies (LUE) of Pyrenean chamois and 298 seasonal livestock along different scenarios of woody plant expansion. Slopes of the linear 299 regression are also reported for each species. 300 301 The LUE of all the herbivores studied decreased to some degree from the initial to the 302 final scenario, (Fig. 3). However, this decrease was sharper in livestock species. In fact, the 303 LUE of chamois remained practically stable until woody plant cover reached 90%. It even 304 increased slightly in scenarios of initial shrubification and then began to decrease moderately 305 from the fourth hypothetical scenario (60% woody plant abundance) onwards, acquiring at the 306 same time the highest values relative to the other species. Livestock, however, lost their 307 foraging resilience at very early degrees of shrubification (Fig. 3). The LUE of cattle (β = - 308 0.005, SE = 0.0005, p < 0.001), sheep (β = -0.004, SE = 0.0005, p < 0.001) and horses (β = - 309 0.005, SE = 0.0005, p < 0.001), steadily decreased from the first scenario of shrubification. The 310 LUE of sheep and horses were significantly smaller than chamois from the third hypothetical 311 scenario onwards (50% woody plant cover; w= 74, p<0.005 and w==75, p<0.005, respectively) 312 and cattle differed from the fourth scenario onwards (60% woody plant cover; w=14, p<0.05). 313 The lowest LUE values were detected in our final scenario of shrubification, where 314 woody plants occupied the entire area, notably decreasing livestock LUE (cattle: median=0.29, 315 min=0.14, max=0.41; sheep: median=0.17, min=0.06, max=0.29; horses: median=0.09, 316 min=0.14, max=0.29). LUE was significantly higher for chamois (median=0.56, min=0.21, 317 max=0.70) than cattle (w=13, p=0.014), sheep (w=76, p<0.0001), and horses (w=79, p<0.001) 318 due to its moderate decrease in niche breadth along the scenarios. Livestock LUEs differed 319 significantly only between cattle and horses (w=71, p=0.006). 320 17 321 Discussion 322 Our results suggest that woody plant expansion in an unmanaged environment will 323 affect herbivores in alpine grasslands during summer and autumn but that the magnitudes and 324 direction of these effects will vary between species. Animals with a preference for herbaceous 325 plants will have difficulties to follow the same diet and they will need to acclimatise to a higher 326 consumption of woody plants in order to remain in these areas. At the same time, competition 327 for the most consumed plants, such as Festuca spp. or Trifolium spp., would lead to overgrazing, 328 one of the main causes of rangeland degradation worldwide (Du Toit et al., 2010; Hilker et al., 329 2014). 330 Changes in woody plant cover importantly restructure the wild herbivore assemblage as 331 grazer densities decrease when woody cover increases (Smit & Prins, 2015). The increasing 332 woody plant expansion in alpine environments will render grazers less efficient users of their 333 landscape, as our predictions suggested, therefore decreasing their density in response to food 334 availability. Livestock are highly dependent on the availability of montane grasslands, and the 335 number of livestock grazers will have to decrease to prevent a reduction in productivity. In fact, 336 increases in woody plant cover are already having repercussions on livestock production and 337 reproduction (Anadon et al., 2014). The need to maintain sustainable levels of production will 338 force farmers to move livestock to more suitable areas. Habitat diversity will consequently 339 decline even faster in alpine areas, because plant species richness is maintained by grazing in 340 these human-created herbaceous communities (Bakker, 1998; Boulanger et al., 2018). 341 Horses feed mostly on graminoids and, to a lesser extent, on forbs and thus would be 342 more quickly and broadly affected by woody plant expansion. This strong dependence on 343 herbaceous plants has been widely described in feral and free-ranging horses (Olsen & Hansen, 344 1977; Salter & Hudson, 1979; Patrick, 1992; Celaya et al., 2011). The consumption of grasses 18 345 (50% of total consumption) can be lower in some extreme environments, such as the 346 Chihuahuan Desert (Hansen, 1976), but animal growth is usually restricted when high-quality 347 pastures are not readily available (Dawson et al., 1945; Andreyev, 1971; Celaya et al., 2011). 348 The production of horse meat in the Pyrenees, as in other parts of Europe, is exclusively free- 349 range. Animals make use of grasslands at different altitudes according to the season and, as a 350 consequence, depend highly on montane pastures to subsist (Martin-Rosset & Trillaud-Geyl, 351 2015). Woody plant expansion is therefore a real threat to horse meat production. 352 Our results support those of several studies that found that free-ranging cattle generally 353 consume higher proportions of forbs and woody plants than horses (Krysl et al., 1984; Menard 354 et al., 2002; Celaya et al., 2011; Scasta et al., 2016). Woody plant expansion would thus affect 355 cattle less than horses. Diets can be more variable and contain more woody species in free- 356 ranging cattle than horses, although diets of cattle can also be high in graminoids and forbs 357 (Aldezabal et al., 2002; Scasta et al., 2016). The high content of plant secondary metabolites in 358 shrubs, such as tannins, can affect intake, digestion, and metabolism in herbivores and can be 359 toxic if consumed in large amounts (Hanley et al., 1992; Burrit & Provenza, 2000). Cattle can 360 consume a relatively high proportion of woody plants in specific habitats and conditions, but 361 this rusticity and adaptability are only observed in some local breeds (Guevara, 1996; Bartolomé 362 et al., 2011). However, local breeds have been gradually abandoned in recent decades for the 363 benefit of highly productive commercial breeds (Taberlet et al., 2008) and consequently, many 364 locally adapted breeds have already become extinct (Scherf, 2000). At the same time, the use 365 of these breeds is impaired by important inbreeding situations and small effective population 366 sizes (Taberlet et al., 2008), highlighting future challenges of livestock farming in areas were 367 local adaptations will be needed. 368 Medium-sized herbivores, such as sheep, tend to have a proportionally higher 369 maintenance cost per body weight unit (Kleiber, 1961). They therefore need to forage on plants 19 370 higher in nutritional value compared to larger herbivores, such as cattle or horses. Sheep can 371 select preferred components in fine-scale mixtures due to their smaller size, which determines 372 gape size, and can therefore feed on the more nutritional parts of plants (Gordon & Illius, 1988). 373 Studies on the composition of diets have reported higher contents of forbs and woody plants by 374 sheep than by large herbivores (La Morgia & Bassano, 2009; Karmiris & Nastis, 2010). Still, 375 the consumption of graminoids and forbs by sheep and cattle in our study was similar, probably 376 due to the high availability of these resources in our study area, generating a strong overlap in 377 the use of resources. 378 The societal demand for livestock products is increasing the development of research 379 programmes focusing on animal behaviour and genetics for developing animals able to use 380 shrubs more efficiently (Estell et al., 2012). The productivity of these breeds, however, is 381 currently relatively low (Verrier et al., 2005), and animals in mountainous areas have 382 adaptations and functional traits of interest for the montane farming system but a lower 383 production of muscle or milk than commercial breeds (Verrier et al., 2005). The use of shrub- 384 dominated areas for meat or milk production does not presently meet animal requirements 385 (Casasús et al., 2005). These practices appear unsustainable due to the necessity of management 386 intervention (e.g. thinning and spraying) and intensive supplementary feeding (Gutman et al., 387 2000; Brosh et al., 2006). As a consequence, livestock farming in areas suffering from woody 388 plant expansion will be at high economic risk due to the impossibility of maintaining sustained 389 economic incomes. 390 Livestock farming in mountainous areas is important to local economies and cultural 391 heritages and is often essential for the livelihood of rural populations worldwide (Mann, 2013). 392 In fact, 32% of Kenyans inhabiting mountains mainly depend on livestock farming to subsist 393 and in Nepalese mountains, where 59% of the population lives below the poverty line, livestock 394 contribute to 21.2% of total household incomes (Abington, 1992; Golicha et al., 2012). Besides 20 395 a direct nutritional income through meat or milk, livestock also play a vital role in supporting 396 farming systems providing wool, manure, working traction, transportation, cash income and 397 risk diversification (Sherman, 2005). In rural areas where subsistence agriculture is prevalent, 398 the loss of pasture land would have a dramatic socio-economic impact, regardless of the causes 399 of the local shrubification. 400 We found that Pyrenean chamois during summer and autumn would be favoured by a 401 moderate to high expansion of woody plants due to their ability to balance their feeding 402 behaviour between grazing and browsing. In fact, chamois can have extremely diverse dietary 403 preferences depending on the habitat and season (Herrero et al., 1996; La Morgia & Bassano, 404 2009) and can even be exclusively dependent on woody species (Yockney & Hickling, 2000). 405 This high phenotypical plasticity is due to their capacity to alternate between ruminal and 406 hindgut fermentation depending on forage quantity and quality (Hofmann, 1989). Because 407 chamois evolved during thousands of years in unmanaged environments (Masini & Lovari, 408 1988) it is not surprising that the reversion to unmanaged conditions could favour them. The 409 chamois diet in our study area differed from the diets of all the livestock species and showed an 410 evenly distributed consumption of herbaceous and woody plants, despite a lower availability of 411 the latter. This behaviour will allow the chamois to have a higher theoretical LUE than the other 412 herbivores in scenarios of future woody plant expansion. Furthermore, mixed feeders may even 413 be able to slow shrub expansion (Olofsson et al., 2009; Schulze et al., 2018), highlighting the 414 importance of the conservation of these herbivores to maintain open habitats. Our results 415 suggest that the LUE of chamois would be impaired in scenarios of extreme shrubification, 416 despite their adaptation to a high consumption of woody plants. However, the phenotypic 417 capacity of chamois could allow them to consume more woody plants than observed in our 418 study area but further studies would be necessary to assess the effect of these dietary adaptations 419 on the performance of this species. At the same time, this study focussed on summer and autumn 21 420 diets of chamois because there are key for the reproduction and survival of the species (Garel 421 et al., 2011; Scornavacca et al., 2016). Food availability during winter may also determine 422 chamois survival, but there is no information on that process. Hence, further investigations will 423 be required to determine the impact of shrubification on the survival of chamois during winter. 424 Our data also suggest that extensive land management and human intervention (e.g. 425 manual shrub clearance), will be necessary for maintaining semi-natural grasslands and free- 426 range farming systems. Keeping in mind that half of the European network of Natura 2000 sites 427 are associated with farming, agricultural land abandonment may have important impacts on 428 landscape and biodiversity in Europe (MacDonald et al., 2000). Moreover, in a kind of vicious 429 circle, the capital investment needed and the time and effort of the demanding work to reverse 430 woody plant expansion on agricultural land is leading to an intensification of the abandonment 431 of farmland and rural areas, accelerating shrubification (MacDonald et al., 2000). Being a driver 432 of woody plant expansion, temperature increase in mountainous environments will also impair 433 biodiversity and efforts to limit global warming will be capital for the future of these 434 environments (Steffen et al., 2018). 435 Finally, the consequences of our results are not only representative of a Pyrenean 436 scenario or a European montane ecosystem. Considering that shrubification is a global issue 437 affecting other habitats and ecosystems worldwide, from the African savannah to the arctic 438 tundra (Tape et al., 2006; Naito & Cairns, 2011), lessons learned from this paradigmatic case 439 example can be extrapolated to a global scale. 440 441 Acknowledgements 442 We would like to thank the National Game Reserve of Freser-Setcases personnel for 443 their authorization to carry out this study. We also thank Marco Apollonio and an anonymous 22 444 reviewer for valuable feedback on the early draft of this manuscript. JE acknowledges a pre- 445 doctoral grant from the Government of Andorra, ATC015 - AND - 2015/2016, 2016/2017 and 446 2017/2018. ML is supported by the French ANR through LabEx TULIP (ANR-10-LABX-41; 447 ANR-11-IDEX-002-02), by the Midi-Pyrénées region (grant number CNRS 121090), and by 448 the FRAGCLIM Consolidator Grant, funded by the European Research Council under the 449 European Union’s Horizon 2020 research and innovation programme (grant agreement number 450 726176). ES was funded by the Spanish Ministerio de Economia y Competitividad (MINECO) 451 through a Ramon y Cajal agreement (RYC-2016-21120). 452 453 454 References 455 Abington JB (ed.) (1992) Sustainable Livestock Production in the Mountain Agro-ecosystem 456 of Nepal. Food and Agriculture Organization of the United States, Rome, Italy. 457 Aldezabal A, García-González R, Gómez D, Fillat F (2002) El papel de los herbívoros en la 458 conservación de los pastos. 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