Protocol for Specific Leaf Area (SLA) and Leaf Dry Matter Content (LDMC)

Measure Specific Leaf Area (SLA) and Leaf Dry Matter Content (LDMC) on the FIVE individual plants from up to FIVE species selected for herbivory and pathogen percent damage measurements and plant trait measurements (see main protocols for selecting species and individuals).
Transport the individuals in cool, damp conditions, for example, using wet paper towel in a cooler back to the lab. If your individual plants are severely damaged, take extra plant material to ensure that the leaf trait measurements are on undamaged leaves.
Rehydrate the fresh leaves overnight after bringing them back from the field. To rehydrate the leaves, place the stems of the individual plants into water upon arrival and store the samples in a cool, dark place overnight. If storing longer than 24hr before taking measurements – store in a refrigerator at 5°.
Select FIVE undamaged leaves from each individual. If all five leaves together do not fit into an A4 size for scanning, select less than five. Record how many leaves are selected in the datasheet (between 1-5).
Wipe off any water from the cuticle of the selected leaves and weigh the leaves to obtain fresh leaf weight (units are g).
Scan the five fresh leaves together on a flatbed scanner (see Fig. 1 for examples). Ensure that all leaves sit flat on the scanner with no overlap and the background behind the leaves is clean and a uniform shade (preferably white). Scan the leaf with the petiole included. If your scale is not set for the scanned image, ensure you scan a ruler in order to set the scale for the leaf area when you are doing the calculations. Calculate the leaf surface area using an image program such as ImageJ (see Appendix 1 for protocols for using ImageJ to calculate leaf surface area below). Record the total leaf area of the sum of all the leaves scanned in the datasheet (units are mm²).

**Fig. 1:** *an example of a Canon flatbed scanner (size A4) and an example of an Epson flatbed scanner (size A4)*

Once the fresh leaves have been scanned and weighed, put them in labelled paper envelopes altogether (i.e. place all the leaves that were scanned on the same image together into one envelope). Place the envelopes in a drying oven at 70°C for at least 72hr for the leaves to dry out completely. Once they have dried out, remove the leaves from the oven and let them cool in a desiccator or container with silica gel. This will ensure that the hot leaves will not absorb any moisture as they cool. Once the leaves have completely cooled weigh the dried leaves in their respective groups (i.e. weigh the leaves together that were scanned on the same image together). Record the sum total weigh of all the dried leaves in the datasheet (units are mg).
Once both the leaf surface area and the leaf dry weight are recorded in the datasheet, the formula (Eqn. 1) will calculate specific leaf area (SLA) automatically in the spreadsheet.

**Eqn. 1:** *Calculation for specific leaf area (note the units when you are taking measurements)*

Leaf dry matter content will also be calculated automatically in the spreadsheet as in Equation 2 (Eqn. 2) from the fresh weight (mg) and the dried weight of the leaves (mg).