TITLE: Trinity College Botanic Garden long-term monitoring program 
AUTHORS: Midori Yajima, Michelle Murray, Jennifer McElwain, Stephen Waldren

[DESCRIPTION]
Botanic gardens hold large, documented, and accessible collections of living plants. These represent unique subsets of taxa from different biogeographical regions growing under common environmental conditions, connecting people to global plant research and conservation efforts while offering a place beneficial for human health and wellbeing. Despite Botanic Gardens being an ideal setting for climate change research, their potential for comparative, long-term studies and outreach in the field is still underutilised. As part of its ten year strategy, Trinity College Botanic Garden (TCBG) aims to tap this potential and establish a programme for long-term (>30 years) monitoring of key physiological performances in its living woody plant collection. The programme will also assess particulate pollution (PM10 and PM2.5) interception by the same trees, pairing climate change and urban green research. Importantly, the project will include the design of a transferable protocol, produce vouchered herbarium specimens as a future historical archive and as a pedagogical tool, and support the garden outreach strategy, so as to nurture its link with both Trinity College Dublin and local communities, ensuring the garden’s legacy into the future.

[GUIDELINES]
These are the guidelines for the monitoring currently in place in TCBG. The trees that are  being monitored to date are listed here. The code used for data manipulation and herbarium label making in previous rounds of monitoring can be found on GitHub (link to come).

The stomatal conductance (gs), leaf area index (LAI), elemental analysis/ water use efficiency (WUE), particulate matter analysis, herbarium specimens making have to be performed at the end of the growing season, which in Ireland corresponds from July, to not later than mid-August.

The data management plan can be found at DMPonline (ID 103451)
Data generated in previous years can be found on Dryad (link to come)

[STEPS]
SECTION: Elemental analysis/ Water Use Efficiency
3. When
Immediately after the last day of porometry

Collection needs to be after a period of 5 to 10 days of no rain and low winds, to avoid particulates to being washed away
SECTION: Stomatal conductance
1. When
End of the growing season (July, not later than mid-August)
SECTION: Stomatal conductance
1.1. 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)

SECTION: Stomatal conductance
1.2. Porometry

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 
 

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)
SECTION: Stomatal conductance
1.3. 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


 


1.4. 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


 


SECTION: Elemental analysis/ Water Use Efficiency
3.1. Leaf selection

Same 3 leaves x tree used for porometry 
SECTION: Elemental analysis/ Water Use Efficiency
3.2. 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 at 50-60 °C for 2 days
2.2.3     Sort dried leaves in separate envelopes reporting leaf ID
3.3. Elemental analysis in the lab

TBD
SECTION: Particulate Matter analysis
4. 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
SECTION: Particulate Matter analysis
4.1. 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)

 

SECTION: Particulate Matter analysis
4.2. 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
SECTION: Particulate Matter analysis
4.3. 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
SECTION: Particulate Matter analysis
4.4. 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 
SECTION: Particulate Matter analysis
4.5. 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


SECTION: Particulate Matter analysis
4.6. Download results

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

 

SECTION: Herbarium sheets
5. 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
SECTION: Herbarium sheets
5.1. 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)
SECTION: Herbarium sheets
5.2. 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. 

SECTION: Herbarium sheets
5.4. 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

 

3.4. Water Use Efficiency

TBD

SECTION: Leaf Area Index
2. 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)
SECTION: Leaf Area Index
2.1. LAI readings

TBD
SECTION: Herbarium sheets
5.3. Making of herbarium labels

Print labels with information noted in the field

 

SECTION: Upload data on Dryad
6. TBD