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

beginning of the growing season (February–April) the lamps were installed about 10 cm above the smallest plants and uplifted as vegetation grew. We set the lamps to switch on two hours after sunrise and switch off two hours before sunset, and they automatically switched off when the temperature exceeded 28˚C to prevent overheating. In 2020, we switched on the lamps March 16. The light addition treatment added light throughout the active growing season and lamps were only removed during the grazing to prevent the sheep from damaging the lamps. Lamps in the fenced plots were switched off during the grazing. At the end of each growing season (October-November), the lamps were removed to prevent frost damage. Using LED lamps that have a wavelength spectrum corresponding to natural sunlight we mimicked increased sunlight availability in a vegetation gap in a natural grassland, and were therefore able to test the role of light limitation. The sheep grazing on the pastures where our experiment is located are owned by a private shepherd and do graze the pastures freely. We are not taking any kind of measurements from the sheep nor do we or our experimental set up manipulate the sheep. Since no animal testing is involved in our experiment there was no need for approval by Institutional Animal Care and Use Committee (IACUC) or other relevant ethics board. Planting of Silene In August 2019 we added seeds of Silene (local seeds from Saale-Saaten Stolle, Germany) in cultivation trays in a glasshouse. When the plants had grown for four months they were trans-

located into a cold house to allow them to adapt to outside conditions. In November 2019, when the plants had grown for five months, we planted one similar-sized individual into each experimental subplot. As Silene is a dioicous species and its genders have been found to differ in size [74], we allowed the transplants to grow relatively large before planting (i.e., they already had adult-sized large leaves and a flowering stalk). We then selected individuals that had similar-sized leaves and were equal in height, and randomly allotted these to the treat- ments. We are therefore convinced that gender differences should not affect our results and, if something, they could only weaken detecting significant responses to the treatments. In lighted subplots we made sure the transplant was planted directly under the LED lamp. After trans- planting we watered the plants. By planting five-month-old relatively large individuals we also reduced the risk of vole/mice herbivory as smaller/younger plants are often eaten by voles and mice [75,76]. During winter, we further protected the transplants with round cylinders of green mesh (Netzhu¨lle Bestklima, best4forst, Germany) with a height of 18 cm, a diameter of 12 cm and a mesh size of 3 × 3 mm. The mesh was attached to the ground to prevent voles/ mice going in below the mesh. The protective mesh was removed in April 2020 when the growing season and plant growth started. Performance and trait sampling To determine growth of Silene transplants, we measured vegetative height (cm) from the

ground to the highest point of each plant. Height was measured after the plants have been grazed, but had the chance to regrow and compensate since harvesting took place two months after the last grazing event. Measuring height of grazed and ungrazed individuals two months after the last grazing allowed us to specifically test for the net effect of grazing, i.e., how much transplants compensated and were able to recover from grazing, under the combination of light addition, nutrient enrichment and climate change. We chose to measure trait variation in SLA and foliar C:N ratio because these have been shown to be associated with slow/fast resource uptake, responsiveness to light availability and attractiveness to herbivores [28,46,64,67]. For data on SLA (mm-2 mg dry mass) we collected a maximum of three healthy 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 5 / 16