4. Measurements of functional traits
In order to analyze variation of functional traits along micro-environmental gradients, I collected some epiphyte individuals for measurements. There were some criteria when collecting these individuals. First, only mature individuals were collected. Whether an individual was mature or not was judged based on morphology and appearance. For fern species, the existence of reproductive structures (e.g., sori) could be used to judge. The leaves of these individuals should be healthy, not wilted or rolled, and without any spot,
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wound, gall or other signs indicating disease or herbivory. All of the individuals used to measure functional traits were collected from those 24 sampled trees. However, due to the limitation of tree-climbing method, small twigs located away from tree trunks were difficult to access. Therefore, only those individuals growing on trunks or vertical main branches were collected. During collecting, dominant species within each vertical zones were collected if its individuals met the criteria of collection mentioned above. To prevent bias in analyses, number of collected individuals of each species was roughly proportional to their relative abundance in each vertical zone. In a way similar to plot surveys, the positional variables of all collected individuals were recorded for further analyses. These variables included height above ground, inclination angle and aspect of the growing surface and diameter of the substrate.
One mature and healthy leaf was chosen from each collected individual to undergo the measuring procedures below:
(1) Measurement of leaf thickness (LT)
Leaves collected from the field may dehydrate to some degree. Hence, I put each leave in a hermetic plastic bag, sprayed some water inside and made the bag close tightly.
Then the plastic bag was placed in cool and shaded place for at least 12 hours to make the leaf rehydrate. After rehydration, the leaf thickness of each leaf was measured using a
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digital thickness gauge (precision = 0.001 mm) (Digital Micrometers Ltd, Sheffield, UK).
Each leaf was measured four times at different positions, typically at the top left, top right, bottom left and bottom right part separately. Measuring thickness at midrib, primary veins or leaf border was avoided. The thickness values of four positions were averaged to represent the thickness of the whole leaf.
(2) Measurement of chlorophyll content
A SPAD-502 chlorophyll meter (Konica Minolta, Osaka, Japan) was used to measure leaf chlorophyll content. This is a non-destructive method, and the procedure is easier and quicker than leaf extraction approach. Each leaf was measured six times at different positions, typically three times at the left part and other three times at the right part. It was ensured that the sensor of the meter was fully covered by leaf lamina when measuring.
Measuring at midrib, veins, spots or sori was avoided. The number read from SPAD-502 chlorophyll meter is called SPAD value. However, SPAD value is a relative index without unit and not easy to interpret from ecological or physiological perspectives. Hence, SPAD values were converted to chlorophyll content per unit area and chlorophyll content per unit mass based on the formula presented by Coste et al. (2010).
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(3) Scanning the leaf and calculating leaf area
After measuring thickness and SPAD value, the leaf was scanned using a scanner (Epson America, Long Beach, CA, USA). Before scanning, the petiole was cut off. The leaf was expanded and flattened when scanning. A length scale and a note that indicated the ID and species of the leaf was scanned along with the leaf. For those species with small simple leaves, two to three leaves were scanned simultaneously. On the other hand, each leaf was divided into several parts and those parts were scanned separately for those species with large leaves or compound leaves (Figure 14). In this way, overlap or extension outside the range of scanner were prevented. The axes and leaflets (parts with green expanded lamina) of compound leaves were divided and then scanned separately.
For those compound leaves difficult to separate all axes and leaflets (e.g., tripinnate leaves), at least the main axis (rachis) and secondary axes were separate from other parts.
The images produced by scanner were then analyzed with Adobe Photoshop CS6 software (Adobe, San Jose, CA, USA). The conversion ratio was first set based on the length scale. After that, the leaf was selected using selection tool, and the area was calculated by image-analyzing tool. For compound leaves, only the leaflets were selected to calculate leaf area. Area of axes were not viewed as part of leaf area in this study.
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Figure 14. Leaf-scanning procedures
A length scale and a note indicating ID and species were scanned along with the leaf. Two to three small simple leaves were scanned simultaneously (left), while compound leaves had to be divided into several parts (right). For a compound leave, at least the main axis and secondary axes were separate from other parts, and these axes were excluded when calculating leaf area.
(4) Measurement of fresh mass
Fresh mass of the leaf was measured using an electronic balance (precision = 0.0001 g) (Ohaus, Parsippany, NJ, USA). It was ensured that the leaf underwent rehydration procedure mentioned above before measuring. For those large leaves or compound leaves which were divided into several parts when scanning, different parts were weighted separately and their fresh mass were summed up to compute total leaf fresh mass. To be
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consistent with leaf area, only the leaflets were used to compute total fresh mass for compound leaves.
(5) Measurement of dry mass
After measuring fresh mass, the leaf was wrapped with a paper envelope and put into oven, then was toasted at 70°C for 72 hours (Pérez-Harguindeguy et al., 2016), and the dry mass was measured afterwards. Once taken out from the oven, the dried leaf would quickly absorb water from the air, so it was put into a hermetic plastic bag as soon as possible before weighting. The leaf was put into a paper envelope again and kept in safe place afterwards so that it could be examined if necessary.
After undergoing all the procedures above, the leaf thickness, SPAD value, leaf area, fresh mass and dry mass were determined for each leaf. These values were further used to calculate other important functional traits. The calculation formulas of different traits are listed below. Six functional traits were used in data analyses (listed in Table 1).
Specific leaf area (SLA) = Leaf area / Leaf dry mass
Leaf dry matter content (LDMC) = Leaf dry mass / Leaf fresh mass
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Leaf water content per unit area (LWC_area)
= (Leaf fresh mass – Leaf dry mass) / Leaf area Chlorophyll content per unit area (Chl_area)
= (117.1 × SPAD value) / (148.84 – SPAD value) (Coste et al., 2010)
Chlorophyll content per unit mass (Chl_mass)
= Chl_area × SLA
Table 1. Six functional traits used in data analyses
Functional traits Abbreviation Unit
Leaf thickness LT mm
Specific leaf area SLA cm2/g
Leaf dry matter content LDMC g/g
Leaf water content (per unit area) LWC_area g/m2 Chlorophyll content (per unit area) Chl_area μg/cm2 Chlorophyll content (per unit mass) Chl_mass mg/g
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