DOI: 10.5061/dryad.47d7wm3nf
title: Vertebrate grazing can mitigate impacts of nutrient addition on plant diversity and insect abundance in a semi-natural grassland

description: Human-induced nutrient eutrophication is a major threat to grassland
 biodiversity, because it promotes the dominance of fast-growing plants.
 Negative impacts of fertilization on plant biodiversity may be offset by
 grazing by large vertebrate herbivores. However, whether grazers also
 mitigate impacts of nutrient addition on insects is less well understood.
 We use a field experiment to test how plant communities and abundances of
 pollinators and grasshoppers respond to nutrient addition and grazing by
 different assemblages of large herbivores, i.e., access by all herbivores
 (including cattle and horses), access by wild herbivores only (wild boar
 and deer), no access by large herbivores. Plant biomass increased, plant
 diversity decreased and community composition shifted towards lower forb
 cover in response to fertilization, but only in the absence of all
 herbivores. Flower visitation by Hymenoptera (bees and wasps), i.e., the
 most abundant pollinator group, was reduced by nutrient addition only in
 the absence of all herbivores and was positively related to flowering
 plant richness. In contrast, flower visitation by Diptera (e.g.,
 hoverflies) was enhanced by fertilization, but not affected by grazing.
 Orthoptera (grasshopper) abundance was reduced by grazing and enhanced by
 nutrient addition, with positive impacts of fertilization tending to be
 stronger in plots with only wild or no herbivores. The abundance of
 grasshoppers was positively related to grass biomass. We conclude that
 vertebrate herbivores can offset impacts of fertilization on both plant
 and insect communities, making grazing by large mammals an essential tool
 to protect insects, particularly pollinators. Most responses to nutrient
 addition were only apparent in plots without any large herbivores,
 suggesting that wild herbivores alone could already mitigate nutrient
 impacts. We also show that insects with contrasting feeding guilds may be
 favoured by fertilized, ungrazed conditions. Therefore, creating a mosaic
 of patches grazed at different intensities will enhance overall insect
 biodiversity.
description: <strong>Study system</strong>: We set up a field
 experiment with nutrient addition and grazing treatments in a semi-natural
 grassland in the Veluwe area in the Netherlands (52.03 °N, 5.80 °E) (e.g.,
 Kardol et al. 2006, Hannula et al. 2017, Veen et al. 2018). The field site
 is located on a loamy, sandy mineral soil (Kardol et al. 2006). The mean
 annual temperature was around 10.7°C and the mean annual precipitation was
 approximately 840 mm (Veen et al., 2018; Royal Netherlands Meteorological
 Institute (KNMI)). The field has been used for agriculture and was
 abandoned in 1982. Since then, it has been managed by Natuurmonumenten as
 nature reserve and has developed into a grassland with a
 Plantagini-Festucion plant community (Kardol et al. 2006). The grassland
 is visited by naturally occurring herbivores, including roe deer
 (<em>Capreolus capreolus</em>), mainly a browser, and red deer
 (<em>C. elaphus</em>), a mixed feeder, and wild boar
 (<em>S. scrofa</em>), a facultative herbivore. European hares
 and rabbits occur in the area, but at very low densities. In addition,
 domesticated semi-wild herbivores, i.e., Sayaguesa cattle (<em>Bos
 taurus</em>) and wild horses (<em>Equus ferus</em>),
 both grazers, have been introduced as proxies for their extinct ancestor’s
 aurochs and tarpan and roam year-round freely in the area. In the seven
 years prior to our study, the densities of cattle and horses were both
 fluctuating around 2.5 animals (~1875 kg and 1050 kg, respectively) per
 100 hectares; the densities of red deer and wild boar varied between 5-7.5
 animals (i.e., ~110-170 kg) and 2.5-11.5 animals (i.e., ~190-860 kg) per
 100 hectares, respectively. In general, densities of large grazers in
 Europe are relatively low as a result of management, including hunting;
 however in rewilding areas, such as our study site, densities of
 herbivores are generally higher, better resembling historical densities
 (Fløjgaard et al. 2022). <strong>Experimental
 design</strong>: In the fall of 2017, we established a controlled
 nutrient addition × grazing experiment, which is part of the Nutrient
 Network (NutNet) (Borer et al. 2014a). In our experiment, we established
 five independent, randomized blocks. Each block consisted of six plots of
 5 × 5 m, which were grouped in three pairs (Fig. S1). One pair of plots
 was fenced with 2 m high fences to exclude all large, vertebrate
 herbivores (referred to as “exclosure”); one pair of plots was fenced with
 barbed wires allowing access to wild boar and deer while excluding
 domesticated herbivores (referred to as “wild herbivores”); and one pair
 of plots was left unfenced to allow grazing by all herbivores (referred to
 as “all herbivores”). Within each pair, one plot has been fertilized with
 slow-release grains containing nitrogen (N; Cote N (2M) 43% urea,),
 phosphorous (P; Triplesuperphosphate, 45%
 P<sub>2</sub>O<sub>5</sub>) and potassium (K,
 Potassiumsulfate, K<sub>2</sub>SO<sub>4</sub>)
 each year starting in 2018 at the start of the growing season (end of
 March) according to Borer et al. (2014a). For N, P, and K we apply 10 g of
 each nutrient per m<sup>-2</sup> yr<sup>-1</sup>.
 In 2018, at the start of the experiment, all fertilized plots also
 received a mix of micronutrients, equalling 100 g
 m<sup>-2</sup> and including Fe (15%), S (14%), Mg (1.5%), Mn
 (2.5%), Cu (1%), Zn (1%), B (0.2%) and Mo (0.05%) (Borer et al. 2014a).
 All fertilizers were ordered at Telermaat, Boskoop, the
 Netherlands. <strong>Data
 collection</strong>:<strong> </strong>We measured
 aboveground plant biomass and plant community composition at the peak of
 the growing season in July 2017 (starting conditions before establishing
 exclosures) and after three years of fertilization and herbivore exclusion
 in July 2020. Within each plot, we installed a strip of 20 cm × 1 m and
 clipped all aboveground plant biomass. Samples were sorted to separate
 plant litter, monocots, forbs (including legumes), and mosses. All biomass
 was dried until constant weight at 60°C for at least 48hrs and weighed. We
 recorded plant community composition by visually estimating the percentage
 cover of each plant species using the decimal Londo scale (Londo 1976) in
 a 1 × 1 m permanent subplot within each plot. We used cover data to
 calculate plant species diversity as the Shannon index, and we used the
 biomass data to calculate the percentage of forb biomass relative to the
 total biomass of forbs and grasses. We measured flower
 abundance by counting floral units in five 50 × 50 cm subplots per 5 × 5 m
 plot, for each flowering species, except wind-pollinated plants, such as
 grasses. Floral units are defined as approximately 1
 cm<sup>2</sup> with at least one open flower (Carvalheiro et
 al. 2013). When the flowers are larger than 1 cm<sup>2</sup>,
 the number of floral units corresponds to the flower size in
 cm<sup>2</sup>, excluding petals. At the peak of the growing
 season, we also measured the amount of light reaching the soil surface by
 measuring light availability above and below the canopy on a sunny day
 between 11 am and 2 pm using a PAR meter (Li-COR). In each plot, we also
 recorded the area of bare soil by visually estimating the percentage cover
 by bare soil during the plant community assessments. We
 quantified the abundance of different groups of insects in 2020, focussing
 on pollinators and herbivorous insects (specifically grasshoppers). For
 pollinators, we carried out flower visitation surveys between 11:30 hrs
 and 16:00 hrs on days with sunny to partly cloudy conditions, temperatures
 between 18 and 30°C, and a maximum wind speed of 5 Bft. Each 5 × 5 m plot
 was observed for 20 minutes by walking very slowly around the plots to
 minimise disturbance. A flower visitation event was defined as a moment
 when a pollinator made contact with the pollen or nectar source of the
 flower. For each visitation event, the plant species and the pollinator
 species were identified, either directly in the field or via photographs
 taken during the visitation event. A photo library was made for all
 observed pollinator species at the site, and identification was checked
 with taxonomic experts. Observed pollinators belonged to the following
 groups: Hymenoptera (bees and wasps), Diptera (hoverflies), and
 Lepidoptera (butterflies and moths). If an individual pollinator visited
 multiple plant species, only the visitation of the first plant species was
 registered. This method aligns with an unpublished Nutrient Network
 protocol where insects were caught upon visitation for identification in
 the laboratory. We used the pollinator observations to calculate Shannon
 diversity. Grasshoppers (Orthoptera) were sampled on 12
 August 2020 by sweep netting. Samples were collected between 10:00 hrs and
 12:30 hrs. The temperature was circa 30°C and the windspeed was 1 Bft. In
 each plot, 50 sweeps were made with the net in 5 transects that were
 equally spaced and covering the whole 5 x 5 m plot. The transects were
 sampled from the outside of the plot towards the inside to prevent
 grasshoppers from escaping from the plot by the disturbance of the sweep
 netting. The sampling of one plot took approximately one minute. Samples
 were transferred to a plastic bag, which was placed in a -20°C freezer
 within 3 hours after sampling. For each sample, the number of adult
 individuals was counted and identified to species or genus
 level.
description: # Vertebrate grazing can mitigate impacts of nutrient addition on plant
 diversity and insect abundance in a semi-natural grassland
 [https://doi.org/10.5061/dryad.47d7wm3nf](https://doi.org/10.5061/dryad.47d7wm3nf) ## Description of the data and file structure This dataset contains one file with five worksheets. | **Worksheet: Plot and plant variables** |   |   |   | | | :--------------------------------------------------------------------------------------------- | :------------------------------------------------------------------------------------------------- | :---------------------------------------------- | :---------------------------------------------------------------------------------------------------------------------------- | ---------------------------------------------- | | **column name** | **explanation** | **levels** | **explanation levels** | **unit** | | year | year of collection | | | | | plotID\_NutNet | unique plot identification (same as the plot identification used in NutNet for the site veluwe.nl) | | | | | block | randomized block | 1 | | | | | | 2 | | | | | | 3 | | | | | | 4 | | | | | | 5 | | | | nitrogen | nitrogen fertilization treatment | 0 | no nitrogen added | | | | | 1 | nitrogen added every year in spring (rates and type according to NutNet protocols: [www.nutnet.org](http://www.nutnet.org)) | | | phosphorus | phosphorus fertilization treatment | 0 | no phosphorus added | | | | | 1 | phosphorus added every year in spring (rates and type according to NutNet protocols: [www.nutnet.org](http://www.nutnet.org)) | | | potassium | potassium fertilization treatment | 0 | no potassium added | | | | | 1 | potassium added every year in spring (rates and type according to NutNet protocols: [www.nutnet.org](http://www.nutnet.org)) | | | fence | fencing treatment | 0 | no fence = access by all herbivores | | | | | 1 | wire = access by wild herbivores (boar, deer), but not domesticated herbivores (cattle, horses) | | | | | 2 | fence = no access to herbivores &gt; 1 kg | | | nutrient | summary of nutrient treatment | control | no nutrients added | | | | | NPK | NPK added | | | exclosure | summary of exclosure treatment | | no fence = access by all herbivores | | | | | | wire treatment = access by wild herbivores (boar, deer), but not domesticated herbivores (cattle, horses) | | | | | | fence = no access to herbivores &gt; 1 kg | | | total\_cover | total cover vegetation and litter | | | % of plot | | baresoil\_cover | cover of bare soil | | | % of plot | | litter\_cover | cover of litter | | | % of plot | | woody\_cover | cover by woody species | | | % of plot | | moss\_cover | cover by moss | | | % of plot | | cowdung\_cover | cover by cow dung | | | % of plot | | horsedung\_cover | cover by horse dung | | | % of plot | | anthill\_cover | cover by ant hill | | | % of plot | | wildboarrooting\_cover | cover disturbance wild boar rooting | | | % of plot | | rodentrunaways\_cover | covert by rodent run ways | | | % of plot | | rabbitdroppings\_cover | cover by rabbit droppings | | | % of plot | | unknowndisturbance | cover by unknown disturbances | | | % of plot | | par\_ambient | light intensity above the vegetation (ambient level) | PAR | | | | par\_ground | light intensity at the ground level (below the vegetation) | PAR | | | | litter\_biomass | litter biomass | | | g m-2 | | moss\_biomass | moss biomass | | | g m-2 | | graminoids\_biomass | graminoid biomass | | | g m-2 | | forbs\_biomass | forb biomass | | | g m-2 | | legumes\_biomass | legume biomass | | | g m-2 | | woody\_biomass | woody biomass | | | g m-2 | | | | | | | | **Worksheet: vegetation composition 2017** |   |   |   | | | PlotID\_NutNet | unique plot identification (same as the plot identification used in NutNet for the site veluwe.nl) | | | | | Year | year of collection | | | | | Exclosure | summary of exclosure treatment | (explanation see tab: plot and plant variables) | | | | Nutrient | summary of nutrient treatment | (explanation see tab: plot and plant variables) | | | | Other columns represent plant species recorded (column names = latin name  species) | | | | % cover of plot | | | | | | | | **Worksheet: vegetation composition 2020** |   |   |   | | | PlotID\_NutNet | unique plot identification (same as the plot identification used in NutNet for the site veluwe.nl) | | | | | Year | year of collection | | | | | Exclosure | summary of exclosure treatment | (explanation see tab: plot and plant variables) | | | | Nutrient | summary of nutrient treatment | (explanation see tab: plot and plant variables) | | | | Other columns represent plant species recorded (column names = latin name  species) | | | | % cover of plot | | | | | | | | **Worksheet: pollinators** |   |   |   |   | | PlotID\_NutNet | unique plot identification (same as the plot identification used in NutNet for the site veluwe.nl) | | | | | Year | year of collection | | | | | Exclosure | summary of exclosure treatment | (explanation see tab: plot and plant variables) | | | | Nutrient | summary of nutrient treatment | (explanation see tab: plot and plant variables) | | | | Other columns represent pollinator species recorded (column names = latin name  species) | | | | number per plot per 20 minutes | | | | | | | | **Worksheet: grasshoppers** |   |   |   |   | | PlotID\_NutNet | unique plot identification (same as the plot identification used in NutNet for the site veluwe.nl) | | | | | Year | year of collection | | | | | Exclosure | summary of exclosure treatment | (explanation see tab: plot and plant variables) | | | | Nutrient | summary of nutrient treatment | (explanation see tab: plot and plant variables) | | | | grasshopper\_count | number of grasshoppers | | | number per plot per 50 sweeps (with sweep net) | | grasshopper\_biomass | grasshopper biomass | | | gram per plot per 50 sweeps (with sweep net) | | | | | | | | **Worksheet**: **Flowering plant species** |   |   |   | | | PlotID\_NutNet | unique plot identification (same as the plot identification used in NutNet for the site veluwe.nl) | | | | | Year | year of collection | | | | | Exclosure | summary of exclosure treatment | (explanation see tab: plot and plant variables) | | | | Nutrient | summary of nutrient treatment | (explanation see tab: plot and plant variables) | | | | Other columns represent counts of flowering plant species (column names = latin name  species) | | | | number per plot |