HARVEST METHOD:
The methods for setting up each point frame plot are described below in the section 'POINT FRAME PIN-DROP METHODS'. The methods here describe how each shoot was harvested from the plots which were used for the point frame method.

Six shoots were harvested from each of 19 1m x 1m point frame plots which were dominated either by Salix pulchra or Betula nana tall, shrub species. The "shoots" described here are branch clippings between eight to ten inches long. Whenever possible they were selected for appearing relatively healthy, intact leaves, and each shoot was taken from a different plant. As these shoots were used for shoot-level and leaf-level measurements, shoots with bi-furcated stems -- or two stems from the same branch/height were cut -- one for leaf-level and the second for shoot-level analyses.

Before being cut, we measured the distance from the point frame to the highest tip on the shoot as well as the distance from the point frame to the shoot five inches from the tip. This way, we could approximate the angle of the shoot relative to the ground. Using the same criteria as for the pin-drop measurements, we measured the distance from the top of the shoot to the soil. In addition, the row number and pin-hole number nearest to the shoot's location with respect to the point frame was also recorded .

We then measured the leaf area index (LAI) of the shoot by holding an LAI-2000 (Li-Cor Inc., Lincoln, Nebraska, USA) in the exact location where the shoot had been and taking the average of three readings (one above, three below). For these measurements we used the one-quarter cut out and took care to always hold the instrument level and with the technician's body casting a uniform shadow over the instrument's eye, with the technician standing between the instrument and the sun. On occasion when it was raining during the shoot harvest, the area near where the shoot had been was marked with flagging tape, and the the LAI measurement was taken at a later date, using the height from frame/distance from ground meausrements as well as the row number and pin hole measurements as a guide.

Once cut, the shoot was placed immediately into water and transported to the lab. Once in the lab, the end of each shoot was clipped under water to ensure that there were no air bubbles in the stem that would inhibit the flow of water. Shoots were then allowed to sit at ambient room conditions (~20-25 degrees Celsius) until the the photosynthetic rates could be measured.

AREA CALCULATIONS:
Area calculations were made using the open source software Image J, available at http://rsbweb.nih.gov/ij/download.html. The procedures for each kind of area calculation are outlined here:

SHOOT SILHOUETTE AREA: Digital image capture of intact stem and leaves
1. Arrange the shoot on a white background (e.g. blank paper or foam board) as it sat in the LiCor conifer chamber. Add a note of sample number and date.
2. Place a ruler that you can later use as a reference. Ensure ruler/stem do not overlap.
3. Use a tripod to hold the camera in a steady position and as close as possible to the stem, keep this distance constant throughout the samples if possible to avoid the need to re-calibrate Image J.
4. Take a digital image of the shoot in very bright light conditions, using diffuser or reflector panels as needed to minimize shadows. [Shadows can be mistakenly analyzed in the software as plant tissue if not careful.]
5. Download the images in the computer, save them as TIFF files.
 
SHOOT TISSUE AREA: Image capture of shoot tissues (leaf, petiole, stipule, organ, green/brown stem) using a scanner
 1. Pluck the sample with forceps, arrange leaves, petioles, stipules, green and brown stems and inflorescences separately, grouped by type onto the white background of a plastic board. Place a ruler that you can later use as a reference. Add a note of sample number and date.
2. Press flat with the clear plastic cover. Ensure ruler/plant parts are not overlapping.
3. Place plastic side down on scanner (ensure no parts have overlapped when inverting plastic board).
4. Scan the images to the computer, save them as TIFF files.
 
BOTH SILHOUETTE AND TISSUE AREA ANALYSES:
In the Image J software program:  
1. Open leaf image via Select File → Open→ your file name
2. Convert scanned colour image of leaf to greyscale:
3. Image→Type→8 bit
4. Set measurement scale: Draw a line over a 50 mm section of the ruler then Analyze →Set Scale
 5. In Set Scale window enter 50 into the 'Known Distance' box and change the 'Unit of Measurement' box to mm , check 'Global' (the Global option will apply the same scale to all the images you open after this, if you took the photos from different heights you might need to repeat this procedure every time)
6. Draw a new line and confirm that the measurement scale is correct.
7. Threshold the leaf image: Image→ adjust→ threshold
8. Play with sliders to include all of leaf in red and click ‘Apply’
 9. Calculate area of entire silhouette area and area of each tissue type:
Enclose the region of the shoot or tissues that you wish to calculate the area for with the rectangular selection tool, then Analyze → Analyze Particles
 10. Select the options of display masks and display results.
Area of measured pixels is displayed. Check that only your leaf is included in the outline (not the ruler or extraneous tissue types).
The data of the leaf area is displayed on a new window ‘summary’, this data can be copied into Excel or the data can be saved directly as an Excel file. Keep summary window open between picture analyses to maintain a record of measured areas.
 11. Save new black and white image as well as its original colour photo with an appropriate file name.

DRYING AND WEIGHING:
Once digital images of each shoot and tissue had been made, each plant tissue was separated, counted, and placed in a labeled coin envelope. These samples were then dried for a minimum of three days at 60 degrees Celsius prior to being weighed on a four-point balance with glass enclosure.

Plant tissues were weighed in aggregate, thus the average mass included in these data are the total tissue mass divided by the count of that tissue (e.g. number of leaves, petioles, etc). In the same manner, plant tissure area was calculated in aggregate, thus the average area per leaf/petiole/stipule is based on the total area of that tissue divided by the count of that tissue.

At the start of this project, we did not take care to distinguish leaves, petioles, or stipules. While not thought to make a significant difference in the area or mass calculations for most samples, we did not want to include the petioles in the CHN analyses. Thus, for those samples that had removeable petioles (all B. nana, most S. pulchra, but no Vaccinium spp. or L. palustre samples), the petioles were clipped after drying and the leaves re-weighed. In some cases the mass of the leaves with petioles removed was greater than the original mass; in these cases, the original mass was preserved. [The original mass of all samples weighed is listed in the ORIGINAL LEAF MASS W/ PETIOLE data-column.]

NOTE: The area used for correcting the LiCor A/Ci and light curve data included leaves, petioles, and stipules.

CHN ANALYSIS:
Grinding: All leaf samples were dried in an oven at 60°C before grinding. Samples were small enough to grind the entire sample without subsampling. Leaves were ground using the Retsch MM 200 for 3 minutes or until a talcum powder consistency was achieved.

Weighing: After grinding, samples were stored in glass scintillation vials and dried again at 60°C for at least 36 hours. Once samples were removed, vials were tightly re-capped. When not in use, vials were stored in dessicators. 3.5-4.5 mg of each sample was weighed into a 10x12 mm tin capsule. A standard calibration curve was created using increasing amounts of aspartic acid (from about 0.2 mg to 5.0 mg). A chemical standard, acetanilide, and an organic sample, apple leaf, were run after the standard curve. Every ten samples, an aspartic acid check standard and a duplicate of an already-packed sample were run.

CN analysis: CN analysis was run between 10/4/2012 and 11/15/2012 by Rachel Rubin using the ThermoScientific 2000 at the Ecosystems Center, MBL, Woods Hole, MA. Duplicate sample values were averaged (mass, %N and %C) before inclusion into final results. The CHN data is available in the file "2012_GS_ITEX_PF_CHN_Data"

POINT FRAME PIN-DROP METHODS:
We preferentially selected tall shrub canopies dominated either by Betula nana or Salix pulchra, that is canopies that were greater than 75 cm height. Care was taken to select fairly uniform canopies, that is avoiding the edge of a shrub stand or areas where the canopy had a large gaps, suggesting the area may have been disturbed.

We used point frames constructed from a 1.1 m x 1.1 m aluminum square with holes in each corner to accomodate steel rod posts used as the legs of the point frame. In this way, the frame could rest upon the four leg posts that had been hammered into the ground and remain adjustable in each corner. The frame had a level on each side, and great care was taken to ensure that the frame was (a) unable to be pushed deeper into the ground and, (b) level on all four sides prior to taking measurements. These factors were important to the measurement to have accurate data regarding the distance from the frame and the overall height of each point sampled in the canopy.

The aluminum frame had numbered, regularly spaced holes on two opposite sides in order to accomodate a metal bar that could be placed across the frame and locked into place. [These holes on the frame are the row numbers.] The bar that was placed across the frame similarly had numbered, evenly spaced holes in order to accomodate a pin--a long (100-200cm) metal rod with a diameter of ~3.175 mm. [The holes on this bar are the pin hole numbers.] Measurements were only ever taken from odd row numbers, and alternated even/odd pin hole numbers with each row; in this way, for every plot 25 evenly spaced locations were sampled covering an area of one square meter.

The length of the pin was marked every half-centimeter so that the distance could be read easily. Measurements were made by lowering the pin through a pin hole and, once encountering a leaf or stem, recording the following: row#, pin hole#, hit#, and the species hit. If the object hit was not a leaf, the plant tissue was noted; the diameter of each stem hit was estimated in millimeters, and the length of every graminoid blade hit was recorded from the point at which it was hit to the tip. As the primary species of interest for this project were for a select number of species (B. nana, S. pulchra, S. glauca, S. reticulata, V. uliginosum, V. vitis, L. palustre), species that were not the target of interest were classified as functional groups--e.g. graminoid spp., forb, moss.

The last pin-hit recorded for each pin hole was always at the "soil" which was considered to be the transition between the green and brown plant material, often in a mossy layer.

ITEX Manual, updated 2011

http://www.blackwellpublishing.com/plantsci/pcecalculation/
Sharkey, T.D., Bernacchi, C. J., Farquar, G.D. and Singaas, E.L., 2007. Fitting photosynthetic carbon dioxide response curves for C3 leaves. Plant, Cell and Environment, 30: 1035–1040. doi: 10.1111/j.1365-3040.2007.01710.x

ITEX Manual, updated 2011