Experimental study

2.1. Selection of sites
The same conditions as for the comparative part, however, only one site is needed for the experiment. If you can do more that is of course welcome! During the experiment, the sites should be managed as it is common in the area, i.e. if the grasslands are mown once or twice a year, then the experimental site should also be mown. In this case make sure to plan your measurements to take place at peak plant biomass.

2.2. Setting up the experiment
The experiment will be a randomized block design with three blocks, 8 treatments, and three replicates per treatment (N = 24 total experimental plots, Fig. 2). Each experimental plot will be 5 x 5 m in size, separated from the other plots by a 1m walkway.  Each 25m2 plot will be subdivided into four 2.5 x 2.5 m subplots (A, B, C, D), with one dedicated to the core sampling, one to additional site-specific studies and two for future network-level research (e.g. exclusion of oomycetes, warming treatment,…). The subplots should be randomly assigned to the different uses. The subplot dedicated to the core sampling will further be divided into four 1m x 1m small plots, with the one located closest to the centre designated for the assessment of species composition (cover). The other three small plots will be designated for destructive sampling such as the assessment of herbivore and pathogen damage, and biomass harvest (see Fig. 4).
To quantify the impact of different consumer groups, they will be excluded (reduced) using biocides. Treatments will involve the removal of consumer groups alone, i.e. insects, mollusc, and fungi, in all possible two-way combinations, all consumer groups together and a control, giving a total of 8 treatments (Fig. 4).


Fig. 4: A) In 8 large plots (5 x 5m) eight consumer exclusion treatments will be established. Insects (I), molluscs (M), and fungi (F) will be excluded using biocides, as well as all two-way combinations, all consumer groups together or no consumers.  B) These 8 treatment combinations will be replicated in three blocks. C) One experimental plot will be subdivided into four 2.5 x 2.5 m subplots, and one dedicated to the long-term core sampling, one for site-specific projects, and two for future net-work studies. The core sampling subplot will be further divided into four 1 x 1m small plots, with the one located closest to the centre designated for the assessment of species composition (cover). The other three will be designated for the assessment of herbivore and pathogen damage (plant images) and biomass harvest (orange rectangles) and will rotate every year.

 2.3. Treatment applications
To control insect herbivores, we will use Lambda-Cyhalotrhin (e.g. Karate Zeon, Syngenta), which is a broad spectrum, non-systemic insecticide frequently used in herbivore exclusion studies and with few non-target effects. It will be sprayed four times during the growing season. To control foliar fungi a combination of azoxystrobin and difenoconazole (azoxystrobin inhibits fungal mitochondrial respiration, Difenoconazole interrupts the synthesis of ergosterol, a fungal cell membrane component, e.g. a mix of Score Profi and Ortiva, Syngenta), will be spayed four times during the growing season. To control molluscs molluscicide pellets based on ferric phosphate (e.g. Limax Ferro, Syngenta) will be applied four times during the growing season. It might be that in some countries some biocides are not approved. If this is the case, please contact us and we will discuss alternative products that can be used. Biocides may not wipe out infestation, but they do significantly reduce enemy attack on plants and are so far the only experimental approach to assess the importance of invertebrate herbivores and pathogens in natural plant communities.

2.4. Measurements per site – Baseline data (see comparative part)
To characterize the different sites around the globe, several measurements of soil conditions, and invertebrate communities and plant traits will be taken. This allows us to link consumer impact to several drivers (latitude, altitude, soil fertility, above- and belowground consumer and predator abundance and characteristics, diversity and biomass), and to shed light at the context dependency of biotic interactions. The baseline measures are the same as those described in the “comparative part”. Randomly select ten of the 24 plots in which you will assess the invertebrates, herbivore and pathogen damage, plant traits and the soil characteristics (in one of the three small plots dedicated to the destructive sampling). You should record the plant species composition and harvest the biomass in all of the 24 plots, though, as baseline plot measures. Take these measures before the application of the treatments. This means that data from the experimental field sites can be used in several analyses immediately. 

In addition to the measurements described above, plant traits of all species at a site should be measured. This is important to test whether the response of plants to enemy exclusions follow patterns predicted by defense-deployment strategies (e.g. growth defense-tradeoff). For each plant species, five individuals per site should be randomly sampled, and their height, SLA and LDMC assessed. As you will already have measured traits on most of the species from a site within the baseline measurements, you only need to measure plant traits of the species that you haven’t measured yet. These measurements can also be done at a later stage but should be completed after the fourth year.

2.5. Annual measurements per plot
Plant species composition
Before the start of the experiment, and once annually, estimate the percent plant cover per plant species in the subplot dedicated to the core sampling, in the small plot located closest to the centre (see Fig. 4, cover). Cover measures follow the same protocol as for the baseline data. In systems in which species composition shifts strongly within the year or which have a two-times mowing regime, we recommend that species composition is assessed twice, once in spring/early summer and once in late summer. This allows us to account for differences in phenology and to capture the maximum cover of each species.

Herbivore damage and fungal infection
Measure the herbivore damage and fungal infection per plot in one of the three small plots dedicated for destructive sampling, using the same method as described for the comparative part. You also need to record the cover of the plant species in the small plot in which you measure herbivore damage and fungal infection. This is important to estimate community weighted mean herbivory and pathogen infection levels. This means that cover measures will be done on two subplots per plot. Each year, the measurements should be done in a different small plot (but never use the small plot designated to the core plant cover estimation, see Fig. 3).

Above- and belowground biomass
To quantify consumer impact on productivity (top-down control), clip the aboveground plant material to 5 cm above ground level, in two 10cm x 50 cm strips of each core sampling subplot, in one of the three small plots (but not the one you will use for the damage assessment). Each year, the biomass harvest should be done in a different small plot (see Fig. 3).
Collect the total aboveground biomass, dry and weigh it. Sampling should be done at peak biomass production (the timing of peak biomass will vary between sites and will be defined by local researchers for their system). If your site has a two-times mowing regime, biomass should be collected twice per year to better estimate site productivity (before the cuts). Root biomass will be measured as standing root biomass in year 3 of the experiment: take a soil core of 5cm diameter, 30 cm deep, and sort to separate roots.
Send the dry biomass samples to the project coordinators. We will then measure several leaf characteristics (leaf N and P, fibre content etc.).

We will soon provide more detailed instructions and explanations of all the tasks!

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