Trinity College Botanic Garden long-term monitoring program

When End of the growing season (July, not later than mid-August)

Leaf selection

1.1.1 Select 3 leaves per tree

1.1.2 Per each leaf

• Tag leaf with a ribbon a code that will work as leaf ID (e.g. the first leaf measured on Arbutus unedo will be AU01)
• Make note of leaf orientation (the N/S/E/W direction the leaf is facing)
• Make note of GPS position of the tree (if new tree from the ones already used)

Porometry

1.2.1 1.2.1 Take 1 porometry reading x each leaf x each tree

• Once per day between 08:30 h and 14:00h
• Over 3-5 consecutive days. Repeat approximately at the same time every day (to account the natural day-to-day variability in gs for each species under ambient conditions) following same order of trees and leaves measured
  Note

  Equipment-related:Calibrate every day or if temp changes of ca 15 degreesMeasure at the prevailing microclimate condition of that dayMeasure in automatic mode - measures continuously and derive best estimate for leafLeaves-related:Measure at the interveinal areolae at mid-lamina of the abaxial surface. In the case of compound leaves, measure the terminal leaflet; for larger leaves clamp the sensor as far onto the leaf as possible, taking care to avoid damage to the leaf marginMeasure leaves at the the same position in the crown (e.g. base level, arm reach), to normalise for WUE and gs changes with tree heightMeasure always at the same position (e.g. right blade of leaf that face me)During the measuremets:Minimise interfering with the leaves (look away or wear mask to avoid changing CO2 conditions locally, move the leaf as little as possibleName the reading with leaf ID

1.2.2 1.2.2 At each tree measure and take note also of

• Temp (°C), RH% at each tree with thermo-hygrometer
• Solar radiation at each leaf with ceptometer's small sensor
• Any other condition met while taking the measurements (e.g. sudden wind, moving the leaves too much)

Download results

• Convert in .csv format
• Clean data (variable names lowercase, no spaces, keep only relevant info, add meatadata taken in the field i.e. temp, rh, notes). Final dataset should look like " Expected results" below

Download metadata

• Take weather data from the garden MET station, readings from the 30 days before the monitoring (monitoring days included)
• Clean data. Final dataset should look like " Expected result" below

When During porometry, after taking stomatal conductance readings. It may not be possible to take LAI for each tree, depending on their local conditions (e.g. for trees that are shaded by other trees)

LAI readings

TBD

When Immediately after the last day of porometryCollection needs to be after a period of 5 to 10 days of no rain and low winds, to avoid particulates to being washed away

Leaf selection

Same 3 leaves x tree used for porometry

Leaf collection and dehydration

2.2.1 Pick leaves and tag them with the same code used previously

2.2.2 Press in plant press for approx. 2 weeks, changing newspapers regularly or Oven dry or Oven dry at 50-60 °C for 2 days

2.2.3 Sort dried leaves in separate envelopes reporting leaf ID

Elemental analysis in the lab

TBD

Water Use Efficiency

TBD

When Collection must be immediately after the last day of porometry, or soon after. It has to be after a period of 5 to 10 days of no rain and low winds, to avoid particulates to be washed away

Leaf selection

• 3 leaves x tree , choosing leaves close to the ones used for porometry/ elemental analysis (same branch, similar dimension and orientation if possible)

Leaf collection and dehydration

3.2.1 Pick leaves and tag them with the same code used previously + progressive numbering (i.e. sample ID that will be used in Scanning Electron Microscopy (SEM) later)

3.2.2 Wrap leaves in lint-free tissue to avoid contamination from paper fibers or other sources while pressing

3.2.3 Press in plant press for approx. 2 weeks, changing newspapers regularly or leave the press in oven at 50-60 °C for 2 days. Leaves need to be pressed so that can be imaged correctly under the SEM later

3.2.4 Sort dried leaves in separate envelopes reporting leaf ID and sample ID, keeping them wrapped in lint free tissue

Samples preparation in the Centre for Microscopy and Analysis lab

3.3.1 Cut 1 sample of 0.5 x 0.5 cm ca from each leaf with single edged blade

3.3.2 Prepare SEM stubs with conductive tabs

3.3.3 Using tweezers, mount 1 sample x stub and press firmly with lint-free tissue

3.3.4 Paint the edges of the sample with carbon paint (pure graphene) to prevent curling while in the SEM

3.3.5 Mark the bottom of the stub with the sample ID

3.3.6 Leave samples mounted on the stubs to dry out for 1-2 days

3.3.7 Carbon coat samples at 20 nm thickness

Analyses using TESCAN MIRA3 TIGER SEM

3.4.1 Set microscope settings

3.4.2 Map samples

For each sample, pick 3 sub-sample areas at random at low magnification and increase resolution at 800 X

For each sub-sample

• Take micrographs using Secondary Electron light (SE) (optional)
• Map samples with Energy Dispersive X-Ray Spectroscopy (EDS) using Back Scattered Electron light. Map will be performed using the integrated AZtec software
• Label sub-samples as A, B, C (so e.g. sample 1, will have 1A, 1B, 1C)

3.4.3 Save project in double copy

Analyses using offline computer

3.5.1 Download saved project and open with AZtec

3.5.2 Set feature analysis settings

For each sub-sample

3.5.2 Select PM on the sample surface by tresholding image to select features by their appearance (PM will be distinguishable from the background, can be selected highlighting lighter part of the spectrum for heavy metals, darker for carbon-based particulates)

3.5.3 Adjust filter settings to better select PM (e.g. use erosion filer if feature is bigger than actual PM when observed in the background). If filter settings still highlight features not corresponding to any actual PM, manually delete the features that do not match

3.5.4 Extract data on highlighted features, showing morphological and chemical information as in "Expected result" below

3.5.5 Copy-paste data on spreadsheet

Download results

• Convert in .csv format
• Clean data. See "Expected results" below

When Collection must happen immediately after the last day of porometry, or soon after. It must be in dry weather to avoid molds in specimens

Selection of the specimen

4.1.1 Select and pick 1 specimen x tree for the actual herbarium sheet + 2/3 extra leaves (or leaflets) to preserve for any future analysis

4.1.2 Label each specimen annotating species name

4.1.3 Separately, make note of

• Species name
• Specimen ID , as "TCBG sample-number" e.g. "TBCG1". Each specimen ID must be unique, so latest specimens have to continue the numbering of the herbarium sheets made in previous years
• Date
• Collectors
• Coordinates (can be taken from previous specimens)
• Location in the garden (e.g. walled garden, west arboretum etc. can be taken from previous specimens)
• Notes , including branch height when on the tree, ecological notes

4.1.3 Place specimen in plastic bag for transport (ideally one bag per specimen)

Preparation of specimens

4.2.1 Dehydrate plants in plant press within 12 hours from collection. Store the plant press in a dry place for approx. 2 weeks, changing newspapers regularly

4.2.2 Once ready, drop the press with all the specimen in the Herbarium freezer inside the Botany Department for at least 72 hours, to kill any residual mold that could contaminate the specimen that are already in the Herbarium.

Making of herbarium labels

Print labels with information noted in the field

Note

Tip Copy information in a spreadsheet as it follows: And use the herblabel R package (using "KUN") to print labels instead of creating them one by one. Final result should look like

Making of herbarium sheets

4.3.1 Mount specimens on herbarium sheets, making sure to

• Stitching them instead of using glue. Use glue only if necessary (e.g. specimen is too heavy, there are too many leaves) so that each specimen can be removed and used for future analyses
• Storing the extra leaves in a separate paper bag attached to the sheet
• Printing and attaching herbarium labels reporting the information noted on the field

4.3.2 Store herbarium sheet in dedicated box in Trinity Herbarium

Citation

TBD