Grazing and light modify Silene latifolia responses to nutrients and future climate

with impacts on plant performance [47–49]. To cope with drought induced stress, plants may profit from traits that minimize water loss such as smaller SLA [7,50–52]. Some studies have examined the interactive effects of climate change and nutrient enrichment in grassland sys- tems [53–55], but only a few have investigated how climate change and nutrient enrichment interact with herbivory [56]. We used transplants of a widely distributed and well-studied European grassland species, Silene latifolia [57,58; hereafter Silene], to study plant responses to interactions among climate, nutrients, grazing and light. We chose Silene as our representative of typical forbs in temperate grasslands because, besides being common and widely distributed, it is palatable to herbivores [59,60], important for pollinators [61], and has also been shown to respond to environmental changes [62] and substantially contributes to the local recruitment from seed in our study sys- tem (Jessen & Eskelinen, personal observation). We therefore expect this species to be highly responsive to resource manipulations and herbivory, and changes in its performance should reflect to community dominance relationships. We grew Silene individuals under full-factorial combination of nitrogen, phosphorus and potassium (NPK) fertilization, light addition and herbivore (sheep) exclusion, and replicated these treatments under ambient and future cli- matic conditions. We directly manipulated light in the field, using modern LED lamps that

mimic natural sun light. Our future climate of seasonally altered temperature and precipitation mimicked projected climate for Central Europe [63]. We measured two common traits (SLA and foliar C:N) linked to resource-use strategies [64–66], competitive ability [19], and palat- ability to herbivores [46,67]. As a measure of performance, we collected data on plant above- ground biomass and plant height. We asked the following questions: 1) Are the effects of nutrient and light additions, future climate, or their interactions on Silene performance greater in the absence of grazers? 2) Does increased nutrient and light availability, future climate, or their interactions affect Silene foliar traits? We made the following predictions: 1a) Nutrient and light additions will increase Silene height and biomass more in the absence of grazers, because competition for these resources is greater under dense vegetation (i.e., without graz- ing). 1b) This pattern will further depend on future climate which can either amplify or reduce competition for nutrients and light, depending on whether it increases or decreases the bio- mass of surrounding vegetation. 2a) Light addition will dampen the effect of nutrient addition on Silene SLA and foliar C:N since fertilization-induced light limitation is relaxed. 2b) Future climate will interact with fertilization and light addition on Silene SLA and foliar C:N, because it can either amplify or reduce competition for light, depending on whether it increases or decreases the biomass of surrounding vegetation. Material and methods Study site

Our experiment is embedded in the Global Change Experimental Facility (GCEF) at Bad Lauchsta¨dt Research Station (51˚22060 N, 11˚50060 E), Germany. In this area the long-term mean annual precipitation is 489 mm and the mean annual temperature is 8.9˚C [63]. The soils are fertile Haplic Chernozems [63,68]. The GCEF combines research on different land- use types with ambient and future climate regimes in a split-plot design [63]. For this study we used ten extensively grazed pastures within the GCEF which represent species-rich grassland vegetation of dryer regions of Central Germany [63]. These pastures are grazed two to three times a year by a herd of about 20 German black-headed sheep, depending on the amount of vegetation biomass [63]. In 2018, 2019 and 2020 grazing was limited to two grazing events per year because of extreme drought conditions [69,70]. Each pasture has a size of 16 x 24 m and is grazed by the sheep with high intensity for 24 hours [63]. This short term, high intensity PLOS ONE Transplant performance and trait responses to abiotic and biotic changes PLOS ONE | https://doi.org/10.1371/journal.pone.0276789 November 8, 2022 3 / 16