Integrative Zoology 2022; 17: 78–92 doi: 10.1111/1749-4877.12572 ORIGINAL ARTICLE Positive effect of spring advance on the diet quality of an alpine herbivore Johan ESPUNYES,1,2,3 Emmanuel SERRANO,1 Sara CHAVES,1,4 Jordi BARTOLOMÉ,4 Pierre MENAUT,5 Elena ALBANELL,4 Pascal MARCHAND,6 Kévin FOULCHÉ5 and Mathieu GAREL6 1 Wildlife Ecology and Health group (WE&H) i Servei d’Ecopatologia de la Fauna Salvatge (SEFaS), Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain, 2 Wildlife Conservation Medicine Research Group (WildCoM), Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain, 3 Research and Conservation Department, Zoo de Barcelona, Barcelona, Spain, 4 Group of Ruminant Research (G2R), Department of Animal and Food Science, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 French Agency for Biodiversity, Direction Régionale Occitanie, Service d’Appui aux Acteurs et Mobilisation du Territoire, Villeneuve de Rivière, France and 6 French Agency for Biodiversity, Direction de la Recherche et Appui Scientifique, Unité Ongulés Sauvages, Gières, France Abstract Changes in vegetation phenology related to global warming are having alarming effects on the life history traits of many herbivore species. Such changes are particularly critical in alpine ecosystems, where strong climate lim- itations on plant growth make seasonal synchronization imperative for the growth, reproduction and survival of herbivores. However, despite the pivotal role of resource-use strategies on the performances of such species, few studies have explicitly assessed the mechanistic impact of climate change on their diets. We aimed to fill this gap by studying the effect of spring onset on the dietary composition and quality of a medium-size alpine herbivore while considering density-dependent processes and age- and sex-specific differences in foraging behavior. Using an exceptional, long-term (24 years) direct individual-based dietary monitoring of a Pyrenean chamois population (Rupicapra pyrenaica pyrenaica), we showed that ongoing earlier onsets of spring are leading to an earlier access to high-quality forage and therefore a higher diet quality at a fixed date, without apparent changes in diet compo- sition. We also showed that at high densities, intraspecific competition reduced diet quality by driving animals to feed more on woody plants and less on nutritious forbs and graminoids. By assessing the mechanistic effects of global warming on the dietary patterns of species at the center of trophic networks, this study is an essential step for predictive models aiming at understanding the ongoing ecosystem consequences of the global climatic crisis. Key words: cuticle microhistological analysis, diet composition, near-infrared spectroscopy, Rupicapra pyre- naica Correspondance: Johan Espunyes, Wildlife Conservation Medicine Research Group (WildCoM), Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Spain. Email: johan.espunyes@uab.cat 78 © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial- NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Spring advance and herbivore diet INTRODUCTION The timing of resource availability and quality are assumed to be central in the mechanisms of population Ongoing anthropogenic climate change is having dra- regulation of large herbivores (Mysterud et al. 2003). matic effects on ecological systems and processes on all These latter are adapted to give birth during the vegetation continents and across oceans (IPCC 2014), resulting in green-up period in order to maximize the access to nutri- changes in the phenology, distribution and behavior of or- tious forage during high energetically demanding periods ganisms (Parmesan 2006; Cleland et al. 2007). Yet, the of late gestation and lactation (Rutberg 1987; Clutton- direction and magnitude of these responses can differ and Brock et al. 1989). The access to high-quality resources many underlying mechanisms are still unclear (Moritz & after winter scarcity is key for reproductive success, re- Agudo 2013; Stopher et al. 2014). It is therefore urgent to building body reserves, juvenile growth and survival and understand the mechanisms by which climate change is finally population dynamics (Pettorelli et al. 2005b, 2007; affecting the life history traits of organisms if we want to Herfindal et al. 2006). In alpine ungulates, the protein monitor, predict and mitigate future biodiversity changes content of the diet correlates with plant phenology during (Sala et al. 2000). the growing season, typically rising with the increasing Climate triggers numerous periodic events in the life access to high-quality vegetation following snow melt and of organisms (Mysterud et al. 2003) and the global in- plant emergence (Hamel et al. 2009; Gálvez-Cerón et al. crease in temperature is greatly affecting the seasonality 2013). However, the ongoing increase in spring tempera- of those events (Cleland et al. 2007). In temperate ecosys- tures is advancing the timing of plant growth in temperate tems, researchers are observing advancements of spring regions and therefore are also time-shifting the access to events such as the flowering of plants and the unfolding high-quality vegetation (Pettorelli et al. 2005b; Cleland of leaves (Menzel 2000; Franks et al. 2007) and even the et al. 2007). Because plant growing season is a key time of bird reproduction and migration (Charmantier & period for the growth, reproduction and survival of alpine Gienapp 2014; Tomotani et al. 2018). However, the herbivores, any factor affecting these seasonal patterns scarcity of long-term monitoring data and the difficulty to of forage quality and availability may have long-lasting test multiple confounding factors hampers the assessment effects on their life-history traits (Gaillard et al. 1996; of these phenological shifts caused by climate change Solberg et al. 2008). In arctic ecosystems it has been (Kharouba & Wolkovich 2020). already shown that climate change disrupts population Alpine species are robust indicators of the ongoing dynamics and trophic interactions (Post & Forchhammer changes that can be used to forecast their effects on 2008; Hansen et al. 2013). Thus, studying the dietary the life-history traits of many organisms (Inouye & consequences of environmental change is fundamental to Wielgolaski 2013; Mason et al. 2014). Indeed, alpine understand the evolutionary and demographic processes ecosystems are among areas where climate warming is in ungulate populations, especially in the context of the particularly intense, making alpine species as sentinels ongoing climate change (Nielsen et al. 2012). that may allow gaining critical insights on forthcom- Climatic variability is not the only process affecting ing effects expected elsewhere (Beniston 2005; EEA the diet of large herbivores, and ultimately population 2012). In addition, because mountain species face strong dynamics. Population density has also a clear impact limiting environmental conditions for which they have on resource availability due to intraspecific competition developed unique adaptations, they are expected to be (Bonenfant et al. 2009). High densities alter the habitat particularly vulnerable to climate change (IPCC 2014; use (Mobaek et al. 2009), shape the diet composition White et al. 2018). For instance, the expected upward (Kausrud et al. 2006) and therefore affect the diet quality shift of trees due to climate warming constitutes a ma- (Blanchard et al. 2003). Furthermore, density-dependent jor threat to range-restricted high-altitude species, by processes often interact with climatic variations in a shrinking the area of their suitable habitat (Dirnböck complex interplay (Bonenfant et al. 2009). Usually, et al. 2011). By imposing intense seasonal changes in intraspecific competition is stronger when climatic condi- resource availability and quality (Post & Stenseth 1999; tions (and therefore resource abundance) are unfavorable Inouye 2020), the strong climate limitation of plants and harsh climatic conditions impose sharper constraints growth in alpine ecosystems is also expected to have in populations experiencing high densities (Coulson et al. indirect effects on primary consumers, whose central 2001; Fattorini et al. 2018a). place in trophic networks may broadcast the cascading While climatic and demographic fluctuations should ecological consequences of climate changes on the whole influence the diet of large herbivores through re- ecosystem. source availability and quality, individual traits should © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, 79 Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. J. Espunyes et al. Figure 1 Trends in (a) the GDD in April and (b) the abundance of Pyrenean chamois in the National Game and Wildlife Reserve of Orlu from 1992 to 2015. The red lines and surrounding colored areas show the linear regression and their confidence intervals at 95%. GDD, accumulated growing degree day during April. furthermore influence resource selection processes tics. We benefited from an exceptional long-term dietary through, for instance, age- and sex-specific foraging monitoring (24 years) of a Pyrenean chamois popula- behavior and habitat selectivity (Marchand et al. 2015). tion (Rupicapra pyrenaica pyrenaica Bonaparte, 1845), a Increases in foraging efficiency with age and experience mountain ungulate highly selective for high-quality plants have been documented in several vertebrates, especially (Duparc et al. 2020), experiencing large variation in pop- birds (Daunt et al. 2007). Although much less commonly ulation density (Beaunée et al. 2015) and facing an earlier reported, a new body of evidence reveals patterns of onset of spring (see Results and Fig. 1a). Working with a increased experience and intraspecific aggression over direct individually-based measure of diet quality (fecal ni- life in ungulates (Fattorini et al. 2018b; Froy et al. 2018), trogen; Gálvez-Cerón et al. 2013), we monitored >250 in- potentially resulting in higher protein intake at old ages. dividuals and were provided with a unique opportunity to Likewise, female large herbivores are constrained to assess consistent mechanistic links between environmen- compensate for the increased energetic requirements tal changes and dietary consequences, while accounting linked to pregnancy and lactation and should have a diet for age- and sex-specific effects on foraging behavior. higher in protein than males during the growing season More specifically, we first studied the relationship (Miranda et al. 2012). between diet quality and diet composition in order to So far, the effect of density-dependent and density- understand which type of plants enhance the dietary independent factors on the use of resources by large her- quality of this highly selective herbivore. We then hy- bivores have most often been studied by combining rough pothesized that an earlier onset of spring will advance the proxies of food availability and quality, such as habitat access to more nutritious and digestible plants, increasing categories, vegetation indexes and meteorological vari- their consumption (Menzel & Fabian 1999) and the ables (Pettorelli et al. 2005a; Nielsen et al. 2012; but see diet quality (as indexed by fecal nitrogen) of chamois. Duparc et al. 2020). This is often due to logistical con- Conversely, high population abundance should force indi- straints preventing the collection of dietary information viduals to feed on resources with lower nutritional quality on individuals. Accordingly, very few studies have been due to intra-specific competition (Stewart et al. 2011). able to link climate change, population density, individual This effect could interact with variation in spring on- characteristics and diet quality, through individual forag- set, increasing in strength in years with later springs ing choices in term of diet composition. We aimed at fill- (Bonenfant et al. 2009). We also expected these patterns ing this gap by studying the effect of global warming on of diet composition and quality to be influenced by selec- spring onset and its consequences on the dietary composi- tion processes related to individual characteristics, with tion and quality of a medium-size herbivore, considering females expected to select for higher quality food items density-dependent processes and individual characteris- than males due to their increased energetic requirements 80 © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. Spring advance and herbivore diet during spring (e.g. Miranda et al. 2012). Similarly, old in- Concurrently, a long-term capture-mark-recapture dividuals might benefit from their experience in selecting monitoring has been performed since 1985 (Loison et al. and securing the best food resources in term of quality 1999). The number of animals trapped each year varied (Fattorini et al. 2018b; Froy et al. 2018). from 2 to 66, using different methods such as corral nets, snares, drive nets or darting. The sex and age of ani- mals were recorded since 1985 and fecal samples were collected on captured animals since 1992 (except during MATERIALS AND METHODS 1994 and from 1996 to 1999). It should be noted that fe- cal pellets are mainly representative of the 24 h previous Study area and chamois data collection diet (Espunyes et al. 2019b). The age was determined by The study was carried out in the National Game and counting horn growth annuli (Corlatti et al. 2015) and fe- Wildlife Reserve of Orlu (NGWR-O), Eastern Pyrenees, cal droppings were collected and stored frozen at −20°C France (42.66°N, 1.97°E). The reserve is an area of until laboratory analysis. The captures and sampling of 4243 ha ranging from 950 to 2765 m.a.s.l. extending from Pyrenean chamois were performed by the Office Français montane to nival ecosystems. The area is characterized de la Biodiversité, under the authority of the Ministry of by a mountainous oceanic climate with annual mean tem- Agriculture, Agrifood, and Forestry and the Ministry for peratures of 9.2°C and mean yearly-accumulated rainfall the Ecological Transition. of 1260 mm. The mountain stage is mainly composed of Other herbivores such as red deer (Cervus elaphus beech forests (Fagus sylvatica) with a substrate of genus Linnaeus, 1758) and roe deer [Capreolus capreolus Vaccinium, Galium and Luzula among others. The sub- (Linnaeus, 1758)] are present at low densities in the study alpine stage is mainly composed of heathlands dominated area. A herd of 1600 domestic sheep (Ovis aries)—whose by genus Rhododendrum and Vaccinium, grasslands of number has not significantly changed during our study Festuca spp. and Nardus stricta and scarce mountain pine period— is also present during the summer months. This (Pinus uncinata) forests. Finally, grasslands, rocks and herd has been present since several decades before the cliffs are the prevailing habitats in the highest slopes. start of our sampling. A negligible effect of inter-specific The abundance of Pyrenean chamois in the NGWR- competition for food resources is thus expected for O has been monitored since 1984. Abundance was ini- chamois during spring period (April-June; see below). tially monitored by annual single block counts in late spring from 1984 to 2008 and was progressively replaced Plant phenology in 2005 by helicopter surveys replicated 5 to 8 times each year to determine uncertainty around abundance es- We used the accumulated growing degree day (GDD) timates. Aerial counts were numerically scaled to block during April to monitor the annual onset of spring and counts (see Gilot-Fromont et al. 2018). Although these therefore assess the changes in spring temperatures since approaches systematically underestimate the true popula- 1992 (Moyes et al. 2011). GDD was estimated as the cu- tion size to a variable extent (Morellet et al. 2007), they mulative sum of the daily mean temperatures above 5°C provide a relatively fair overview of the long-term trend over the month of April (Studer et al. 2005) and was cal- in population abundance due to the very contrasted de- culated as follows: n    mographic periods encountered (Beaunée et al. 2015).  Tmax − Tmin Indeed, since the early 1990s, this population has been GDD = −5 repeatedly infected by Border Disease virus, a pestivirus i=1 2 that has caused dramatic decreases, and in some cases col- lapses, of Pyrenean chamois populations during the last where Tmax and Tmin are respectively, the maximum and 30 years along the Pyrenees (Marco et al. 2015; Lambert minimum daily air temperature (in°C) and n are the days et al. 2018). As a consequence, the abundance of chamois in April. Growing degree days are summed only if Ti in the NGWR-O has continuously decreased since the first is higher than the threshold temperature of 5°C. High contact with the virus and until the early 2010s when the values of GDD were attributed to early springs while virus disappeared from the population without interven- lower values were attributed to latter springs (Moyes et al. tion (Gilot-Fromont et al. 2018). In this population, most 2011). Daily temperatures in our study area were obtained births are highly synchronized and occur between mid- at the Météo France meteorological station of Ascou- May and mid-June, with a peak during the first week of Pailhères (42.72°N, 1.89°E, 1120 m a.s.l.; 10 km away June (Loison 1995). from NGWR-O). © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, 81 Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. J. Espunyes et al. Diet quality counting data of each plant genus were converted to per- centages. Epidermal fragments of floral parts, stems, peti- All fecal samples collected on captured animals in oles, and midribs were not registered due to the difficul- April-June from 1992 to 2015 were analyzed (n = 277, ties in species identification. Recorded plants were pooled median date of capture: April 28). Frozen feces were into 5 functional groups (see Espunyes et al. 2019a, b) thawed and oven-dried at 60°C for 24 h and then ground for a similar approach), namely leguminous woody plants with a 1-mm pitch laboratory mill (Cyclotec 1093, FOSS (hereafter LW), non-leguminous woody plants (NLW), Tecator, Höganäs, Sweden). graminoid plants (Gr), leguminous forb plants (LF) and The fecal nitrogen (fN), acid-detergent fiber (fADF) non-leguminous forb plants (NLF). and lignin (fL) estimations were carried out using Near-Infrared Spectroscopy (NIRS), a method already calibrated for fecal samples of Pyrenean chamois Statistical analyses (Villamuelas et al. 2017). The fN is a reliable indi- We first analyzed the relationship between diet com- cator of diet quality widely used in studies of herbivores position and diet quality by building predictive models diet (Robbins 1983; Hamel et al. 2009) while fL, as a using a partial least squares (PLS) multivariate regression digestive retardant, is a complementary indicator of low approach (Carrascal et al. 2009). The PLS approach digestibility and low nutritive content of forage (Hatfield copes better with collinearity than linear models and & Fukushima 2005). The fADF was used to correct fN can be used to explore relationships between a block values following the recommendations of Gálvez-Cerón of explanatory and correlated covariates and a block of et al. (2015). Ground fecal samples were scanned over response variables. The fN content was corrected by the a wavelength from 1100 to 2500 nm using a NIRSys- fADF content (obtaining fNc), following Gálvez-Cerón tems 5000 scanning monochromator (FOSS, Hillerød, et al. (2015). The proportions of plant functional groups Denmark). Reflectance was recorded at 2 nm intervals (namely: LW, NLW, Gr, LF and NLF) were used as the and each sample was scanned in duplicate. Finally, Win- predictor variables and fNc and fL concentrations were ISI III software (v. 1.6) was used to analyze the spectra used as the response variables. We then investigated the and predict fN, fADF and fL values. influence of environmental and biological variables on the diet composition and diet quality of chamois. For diet composition, we used beta-regressions in which the Diet composition responses variables were composed of the proportions We performed a fecal cuticle microhistological anal- (ranging from 0 to 0.9) of woody plants, graminoids and ysis on a subset (n = 155) of the previous samples and forbs in the diet of chamois. For diet quality, we used obtained a representative summary of the different plant linear normal regressions in which fN was the response taxa ingested by each animal (Storr 1961). Up to 6 sam- variable. The GDD during April, the abundance of ples (when available) were randomly selected each month. chamois in the reserve, the sex and the age were included Between 2 to 3 g of each fecal sample was mixed with as additive predictor variables. The day of sampling was 3 mL of 65% concentrated HNO3 . These test-tubes were included to control for sampling design and for the rapid placed for 1 min in a bath of boiling water and diluted change in fN values during growing season (Fig. S1, with 200 mL of water. This suspension was then passed Supporting Information). fADF was also included in through 1.00 mm and 0.25 mm filters. Later, 50% aque- diet quality models as predictor to control for the acid- ous solution of glycerine was dispersed on a microscope detergent fiber content and for making fN a better proxy slide. The fraction between 0.25 to 1.00 mm was then of diet quality (Gálvez-Cerón et al. 2015). The interaction spread on these glass slides trying to avoid any fragment between the abundance of chamois in the reserve and overlap. Finally, cover-slips (24 × 60 mm) were fixed to the GDD during April was also tested (Bonenfant et al. the slides with DPX microhistological varnish and left to 2009). We standardized all predictor variables (X̄ = 0, dry overnight. For each sample, 3 slides were prepared SD = 1) but sex (the only 2 levels factor), so that co- and examined by the same observer (SC) under an opti- efficient estimates could be interpreted as the relative cal microscope at 100× and 400× magnification by view- contribution of each variable to the model (Gelman & ing 2-mm- wide and 60-mm- long traverses, with 3 mm Hill 2006). between traverses. Up to 200 fragments per sample were We tested all combinations of predictor variables. identified to the genus level based on the shape of epider- The parameter estimates for each variable included in mal cells, trichomes and stomata surrounding cells. The a confidence set of models with model weight ωi > 82 © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. Spring advance and herbivore diet 0.05 were obtained using conditional model-averaging of the variables. The package “betareg” (version 3.1-3; in the AICc framework (Burnham & Anderson 2002). Cribari-Neto & Zeileis 2010) was used to perform the Inferences based on model-averaging are recommended beta regressions and the package “visreg” (version 2.5- over model selection because the averaged estimators 1; Breheny & Burchett 2017) was used to represent the present a better precision and reduced bias when no effect of a predictor variable of interest on the response single model has a clearly superior weight to the others variable after correcting for the effect of the other model in the set (Burnham & Anderson 2002). We checked for predictor variables (partial residual plots). multi-collinearity of the predictors by using the variance inflation factors (VIF). Following (Zuur et al. 2010), if a predictor showed a VIF > 3, it would have been re- RESULTS moved from a given model due to collinearity with other predictors. In preliminary analyses, we used Generalized Chamois diet Additive Models (GAM) to explore potential non-linear In all the studied period (1992–2015; no data in relationships between our predictors and fN. A non-linear 1994 and 1996–1999), 39 plant genera were detected in relationship would have justified the incorporation of a Pyrenean chamois feces samples collected during spring, superior polynomial term but this was not the case here, but none of these genera were detected in all the sam- excepted for the day of sampling (Fig. S1, Supporting ples. Overall, the most consumed group was graminoids Information). However, for this covariate, the relationship (Gr; 40.5%, CI95 = 36.9–44.2), mainly composed of only detracted from a linear pattern in the very last days Festuca spp. (26.1%, CI95 = 23.3–28.9) and Carex spp. of June and including a quadratic relationship in the best (7.2%, CI95 = 6.2–8.3), followed by the non-leguminous model selected (see Table S3, Supporting Information) woody plant group (NLW; 25.3%, CI95 = 22.1–28.6) for this covariate did not change the outcome (results not dominated by Calluna vulgaris (8.7%, CI95 = 6.6–10.8) shown). We thus only kept a linear effect of the day of and Rhododendron ferrugineum (6.5%, CI95 = 5.0–8.0). sampling in our models. Non-leguminous forbs (NLF; 15.6%, CI95 = 13.1–18.1), Because the response variables and most of our pre- leguminous forbs (LF; 9.1%, CI95 = 7.3–11.0) and legu- dictors presented a trend over time (see Results, Fig. 1, minous woody (LW; 8.3%, CI95 = 6.4–10.2) comprised Fig. S2 and Table S1, Supporting Information, for an the rest of the diet, mainly represented by the LW Cytisus overview of temporal variation in the tested parame- spp. (8.2%, CI95 = 6.3–10.0) and the LF Trifolium spp. ters), we were confronted with an inflated probability of (5.2%, CI95 = 5.0–6.5). detecting spurious correlations and confounding effects (Graham 2003). Therefore, we also detrended these pre- dictors (GDD, abundance of chamois, sampling date, age Relationship between diet quality and diet and fADF) by regressing them on year and calculating the composition residuals (see Grosbois et al. 2008 for a detailed expla- The PLS multivariate regression was built using 2 la- nation). Again, we also standardized these variables and tent vectors that explained 72.9% of the variance in diet used beta/linear regressions to assess the consistency of composition and 45.6% of the variance in diet quality. the previous model averaging using the detrended vari- The examination of the 2-dimensional regression coeffi- ables in place of the raw covariates and by including the cients showed that fecal nitrogen was positively related linear effect of year in all models to account for time to forbs consumption (LF: 0.241, NLF: 0.281) and nega- trends in the variables. The results can be found in Sup- tively related to graminoids (−0.141) and woody plants porting Information S1 and Table S2. (LW: −0.119; NLW: −0.151). At the same time, fecal All statistical analyses were performed using R soft- lignin was positively related to woody plant consump- ware version 3.6.3 (R Core Team 2020) and the accepted tion (LW: 0.190; NLW: 0.283) and negatively related to significance level was fixed at P < 0.05. The R-packages graminoids (−0.453). “mgcv” (version 1.8-23; Wood 2006) and “plsdepot” (ver- sion 0.1.17; Sanchez 2012) were used to create the vari- able FNc and the PLS multivariate regression between Effects of environmental and biological diet composition and quality, respectively. The R-package parameters on diet composition and quality “MuMIn” (version 1.43.6; Barton 2018) was used for model averaging and the package “car” (version 3.0-0; Model-averaging provided strong support for an ef- Fox & Weisberg 2011) was used to calculate the VIF fect of sampling day and abundance on all components © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, 83 Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. J. Espunyes et al. Table 1 Averaged estimates of the standardized environmental and biological parameters on the diet composition and quality of Pyrenean chamois Parameter Response variable Relative importance Averaged estimate SE z-value P-value GDD fN 1 0.139 0.039 3.52 <0.001 Woody plants 0.30 −0.006 0.096 0.06 0.94 Graminoids 0.47 −0.013 0.105 0.13 0.89 Forbs 0.35 0.011 0.091 0.125 0.90 Abundance fN 1 −0.145 0.038 3.76 <0.001 Woody plants 1 0.418 0.093 4.49 <0.001 Graminoids 1 −0.270 0.097 2.77 0.005 Forbs 1 −0.265 0.091 2.90 0.003 DoY fN 1 0.366 0.033 10.89 <0.001 Woody plants 1 −0.319 0.091 3.49 <0.001 Graminoids 1 −0.247 0.085 2.88 <0.001 Forbs 1 0.60 0.07 7.56 <0.001 Age fN 0.85 0.064 0.029 2.19 0.027 Woody plants 0.44 0.101 0.077 1.31 0.19 Graminoids 0.82 −0.173 0.074 2.33 0.019 Forbs 0.28 0.045 0.068 0.66 0.51 Sex fN 0.30 0.062 0.066 0.94 0.35 Woody plants 0.23 0.056 0.173 0.32 0.75 Graminoids 0.23 0.035 0.173 0.203 0.83 Forbs 0.23 0.001 0.142 0.01 0.99 fADF fN 1 −0.194 0.029 6.63 <0.001 Abundance × GDD fN 0.35 −0.040 0.042 0.95 0.34 Woody plants 0.09 −0.097 0.117 0.83 0.40 Graminoids 0.23 0.161 0.112 1.43 0.15 Forbs 0.12 −0.104 0.114 0.91 0.36 † Relative importance represents the sum of the AICc weights across all the models where the predictor occurs. GDD, the accumulated growing degree during April; DoY, the day of sampling; fADF, the fecal acid-detergent fiber content of the diet composition and quality of Pyrenean chamois models, none of the VIF computed were >2, providing (Table 1). Similarly, the importance of GDD in explain- no evidence for collinearities among predictors (Table S4, ing variation of fN over time was highly supported, Supporting Information). whereas this covariate had no effect on diet composi- Model-averaged parameter estimates indicated that the tion. Among individual characteristics, only age had an consumptions of woody plants and graminoids were neg- effect on graminoid consumption and diet quality. Mod- atively related to sampling day. On the contrary, the con- els including other factors (sex and interaction between sumption of forbs and diet quality was positively related abundance and GDD) had much less support and high to this parameter. uncertainties around their model-averaged parameters es- Both GDD (slope ± SE = 2.15 ± 0.93, R2 = timates. For each response variable, we considered the 0.19, P = 0.030) and abundance of chamois (slope ± model including significant model-averaged parameter SE = −47.19 ± 3.92, R2 = 0.87, P < 0.001) showed estimates as the best model to compute the variance in- trends over time, characterized by earlier onsets of spring flation factors. For every variables included in the best and decreasing estimates of population size since 1992 84 © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. Spring advance and herbivore diet Figure 2 Effect of the abundance of Pyrenean chamois on their diet composition (adjusted for the day of sampling in the case of woody plants and forbs, and adjusted for the day of sampling and the age in the case of graminoids). Lines show the beta regressions, grey points represent raw data and black points represent values averaged for a given abundance estimate. (Fig. 1). These shifts contributed to changes in diet the timing of spring vegetation growth and the seasonal composition and quality (with similar effect size of both availability in high-quality plants (Post & Stenseth 1999; factors for models on diet quality; Table 1). Decreasing Zweifel-Schielly et al. 2012; Espunyes et al. 2019a; abundance of Pyrenean chamois was associated with a Duparc et al. 2020; see also Fig. S1, Supporting Infor- decreasing consumption of woody plants (−56% between mation). This is particularly expected in medium-sized extreme abundance values; Fig. 2) and a concomitant in- ungulates like chamois in which species’ energy require- crease in diet quality (Fig. 3). By contrast, the consump- ments and digestive abilities make them more selective tion of graminoids and forbs increased with decreasing for high-quality food (Clauss et al. 2003). In our study, abundance of chamois (+87% and +143% between ex- we observed that Pyrenean chamois increased their treme abundance values, respectively). Increasing values consumption of forbs throughout spring but decreased of GDD in April, and the related advance of spring on- the consumption of graminoids and woody plants. This sets, also resulted in a marked increase in fecal nitrogen feeding process is directly related to the pattern of dietary (+22% for the most extreme values; Fig. 3) without any quality as indexed by fecal nitrogen. FN is considered effect on diet composition (Table 1). Finally, old animals in many studies as a reliable indicator of phenology and of both sexes had a diet composed of less graminoid than quality of plants eaten by grazing and mixed-feeding young animals, and of higher quality (Fig. 3). herbivores (Festa-Bianchet 1988; Hodgman et al. 1996). Accordingly, our results show an increase of fecal nitrogen content along the season related to an increasing DISCUSSION consumption of forbs and a decreasing consumption of Our long-term study shows that the ongoing advance- graminoids and woody plants. In highly seasonal envi- ment of spring onset (Fig. 1a) is leading to an earlier ronments, growing plants present seasonal changes in access to highly nutritious early-growing plant stages, nitrogen concentrations. Deciduous plants allocate sub- increasing diet quality in Pyrenean chamois. However, stantial quantities of nitrogen into new leaves during early these earlier spring onsets had no effect on the diet growing stages (i.e. spring; van Soest 1994). However, composition, this latter being rather related to population evergreen plants allocate it gradually throughout the densities. Thus, while consuming the same proportions growing season and in lower quantities, making decid- of plant groups, chamois obtained a more nutritious diet uous plants more appetizing (Jaeger et al. 1999; Körner during earlier springs. 2011). In fact, in our study, the consumption of forbs Herbivore species living in highly seasonal environ- throughout spring varies much more than the other plant ments are expected to make foraging choices that track groups, supporting the importance of its phenology in © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, 85 Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. J. Espunyes et al. Figure 3 Variation in fecal nitrogen (fN) of chamois according to (a) growing degree day (GDD, adjusted for Age, fADF, abundance and day of sampling), (b) abundance of chamois (adjusted for Age, fADF, GDD and day of sampling), (c) age (adjusted for fADF, GDD, day of sampling and abundance), and (d) day of sampling (adjusted for age, fADF, GDD and abundance). Lines and surrounding colored areas show the linear regression and their 95% confidence intervals. Grey points represent raw individual values while black points represent yearly (a, b) or age-specific (c) means. its consumption. Similar findings have been reported populations and species (Herfindal et al. 2006; Ozgul in an Alpine population of chamois where animals sys- et al. 2010; Garel et al. 2011; Tveraa et al. 2013). tematically selected a low proportion of the available However, this is not always the case as Pettorelli et al. biomass composed of high-quality plants at a specific (2007) determined that warmer springs also tended to be phenological stage (flowering) throughout the growing associated with fast plant growth and shortened periods season (Duparc et al. 2020). of high quality forage, that in turn negatively affected We detected an earlier onset of the growing-season juveniles growth in 2 alpine ungulate species. Similar over the last decades (Fig. 1a) in line with several studies counterproductive effects may occur in the growth and that provide evidences about climate warming and ad- survival of juveniles if the species is unable to adjust its vances in the phenological traits of organisms (Menzel parturition time to the earlier springs, causing a mismatch & Fabian 1999; Parmesan 2006; Moyes et al. 2011). By between parturition and the optimal foraging period (Post allowing individuals more time to grow and build up & Forchhammer 2008). Plard et al. (2014) detected that reserves, this phenomenon could lead to an enhancement this mismatch led to decreasing dynamics in a roe deer of the performance of herbivores, as reported in other population. Kourkgy et al. (2016) already questioned 86 © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. Spring advance and herbivore diet the ability of Pyrenean chamois to adjust their gestation 2014)—making our results conservative (see Supporting length or conception dates to the earlier onsets of spring. Information S2). Their results showed that females had a much lower Older animals had a higher diet quality than younger adaptive potential to the spring conditions than to au- ones and consumed lower proportions of graminoids. tumn conditions. However, capital breeders, as Pyrenean These results could provide additional support to the the- chamois, should meet the high cost of early lactation ory of an increased experience in plant selection for herbi- mainly through maternal capital, potentially reducing the vores during their life (Froy et al. 2018; Malagnino et al. adverse effects of this mismatch (Kerby & Post 2013; 2021) but could also be explained by a dominance hier- Richard et al. 2017). Furthermore, the impact of climate archy (Fattorini et al. 2018b). However, contrary to the warming during the seasons generally considered as most predictions, sex had no effect on the diet quality and important (spring and winter in alpine/artic ecosystems) composition, despite a sexual difference in the spatial could also be counterbalanced by other consequences behavior and habitat selection of this species (Loison in other seasons traditionally considered as less impor- et al. 1999; Dalmau et al. 2013). Sexual differences in tant (e.g. autumn: Loe et al. 2020). In that sense, more diet quality and composition has rarely been studied in research on the demographic impacts of phenological any chamois, but Pérez-Barberia et al. (1997) observed mismatch and other consequences of climate warming a similar diet composition in both sexes, at least for over seasons in alpine species is needed to determine the Cantabrian chamois (Rupicapra p. parva) in spring. Be- consequences of climate change over the long term. ing highly selective for high-quality palatable plants (Cor- Our findings also show that increasing population latti et al. 2013; Redjadj et al. 2014; Duparc et al. 2020) density negatively affects individual diet quality. As chamois species may be able to feed selectively on high- animal density increases, it is expected that intraspecific quality forage and compensate for sex-specific behavioral competition for resources also increases, affecting the differences. access to the most nutritious plants at the individual We recommend the use of fecal nitrogen to monitor level (Kie et al. 1980; van Beest et al. 2014). It is thus long-term trends in density-dependent and density- fundamental to consider this parameter when evaluating independent environmental conditions through changes the mechanisms and the consequences of global warming in the nutritional state of a given population (Blanchard on the life-history traits of herbivores. Additionally, et al. 2003; Gálvez-Cerón et al. 2013). Furthermore, the abundance of chamois in the area was negatively because we did not detect a sex effect and age has less related with the consumption of forbs and graminoids, effect (2.3 times lower) as compared to densities, the while positively related to the consumption of woody collection of fresh unidentified droppings during seasonal plants. In fact, the consumption of woody plants was field surveys coupled with NIRS analysis could be an mainly driven by the abundance of chamois and the effect inexpensive option to monitor the density-dependent was 2 and 4 times stronger than for the consumption processes in a population as a complement to other indi- of graminoids and forbs, respectively. Lignin is not cators of ecological changes (Morellet et al. 2007; Santos only indigestible, but also interferes with digestion by et al. 2014). This study also highlights the fundamental reducing the rumen turnover rate and the intake capacity need of long-term monitoring programs to understand (Van Soest 1994). Thus, the consumption of graminoids, the ecological processes affecting herbivore performance while negatively related with nitrogen intake, can be in the context of global changes (Festa-Bianchet et al. favorable due to their highly negative relationship with 2017). fecal lignin content. Therefore, intraspecific competition In the current context of climate crisis (Steffen et al. forces chamois to have a lower digestible and protein-rich 2018), understanding the mechanistic effects of these diet and, conversely, the important negative trend over variations on the dietary selection of individuals is an time in chamois abundance has led individuals of this essential step to understand the consequences of cli- population to feed much less on woody plant and more on mate change on the life-history traits of wild populations graminoids and forbs. Note that this pattern could have (Sinclair & Parkes 2008). Projection studies on species been also generated by temporal changes in landscape distribution, food webs and trophic asynchrony are in characteristics (e.g. open-landscape restoration; Cazau crucial need of data on the ecological mechanisms of et al. 2011). However, this is not the case here and, environmental change and associated dietary selection instead, the proportion of grasslands over woody habitats (Thuiller et al. 2017; Veldhuis et al. 2019). By includ- has rather decreased by around 12% during the study ing data on resource use and dietary quality, we believe period —a common process in alpine areas (Venn et al. that such studies may create more realistic and robust © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, 87 Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. J. Espunyes et al. predictions on the impact of the climatic crisis on the life- Theoretic Approach. Springer, New York. https://doi. history traits of wild herbivores. org/10.1007/b97636. Carrascal LM, Galván I, Gordo O (2009). Partial least ACKNOWLEDGMENTS squares regression as an alternative to current regres- sion methods used in ecology. Oikos 118, 681–90. The authors are thankful to all the field assistants and Cazau M, Garel M, Maillard D (2011). Responses of volunteers for their amazing work in trapping Pyrenean heather moorland and Mediterranean mouflon forag- chamois and collecting feces during such a long period. ing to prescribed-burning and cutting. The Journal of They are grateful to Météo France for providing the Wildlife Management 75, 967–72. climate data. This study was funded by the research Charmantier A, Gienapp P (2014). Climate change and partnership program “Approche Intégrée de la Démogra- timing of avian breeding and migration: Evolutionary phie des Populations d’Isard” (N°2014/08/6171) between versus plastic changes. Evolutionary Applications 7, the Office Français de la Biodiversité (formerly Office 15–28. National de la Chasse et de la Faune Sauvage) and the Servei d’Ecopatologia de la Fauna Salvatge (SEFaS). Clauss M, Lechner-Doll M, Streich WJ (2003). Ruminant Johan Espunyes acknowledges a pre-doctoral grant from diversification as an adaptation to the physicomechan- the Government of Andorra (ATC015 AND 2015/2016, ical characteristics of forage. A reevaluation of an old 2016/2017, and 2017/2018). Emmanuel Serrano was debate and a new hypothesis. Oikos 102, 253–62. funded by the Spanish Ministerio de Economía y Com- Cleland EE, Chuine I, Menzel A, Mooney HA, Schwartz petitividad (Mineco) through a Ramon y Cajal agreement MD (2007). Shifting plant phenology in response to (RYC-2016-21120). global change. Trends in Ecology and Evolution 22, 357–65. Clutton-Brock TH, Albon SD, Guinness FE (1989). Fit- REFERENCES ness costs of gestation and lactation in wild mammals. Barton K (2018). MuMIn: Multi-Model Inference. R Nature 337, 260–2. package version 1.42.1. https://CRAN.R-project.org/ Corlatti L, Bassano B, Valencak TG, Lovari S (2013). 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Generalized Additive Models : An In- parameters on diet composition and quality troduction with R. Chapman & Hall/CRC, London. S2 Land-cover evolution Cite this article as: Espunyes J, Serrano E, Chaves S et al. (2022). Positive effect of spring advance on the diet quality of an alpine herbivore. Integrative Zoology 17, 78–92. 92 © 2021 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.