Plant functional traits of the 100 most abundant species in the Pasoh forest reserve 50-ha forest dynamics plot, Malaysia

We determined crown traits, leaf traits, and wood density for tree species from the 50-ha Forest Dynamics Plot in the Pasoh Forest Reserve (2.92 °N, 102.31 °E). We targeted the six largest individuals by DBH of the 100 most abundant species in the 2005 census of the 50-ha plot. We obtained crown and leaf trait data for 436 of the targeted trees and all 100 targeted species. Crown traits included height and mean crown diameter. Leaf traits included number of leaflets for compound leaves, leaf area, leaf fresh mass, leaf dry mass, leaf dry matter content, leaf dry mass per area, leaf lamina thickness, leaf dry tissue density, and leaf nitrogen and phosphorus concentrations. We also determined wood density for 73 of the most abundant species in the 50-ha plot that previously lacked wood density data. 

Methods
We determined crown traits, leaf traits, and wood density in 2008 for tree species from the 50-ha Forest Dynamics Plot in the Pasoh Forest Reserve. The Pasoh Forest Reserve is administered by the Forestry Research Institute Malaysia (FRIM) and is located near the village of Sempang Pertang in Negeri Sembilan state in Peninsular Malaysia (2.92 °N, 102.31 °E). Elevation ranges from 70 to 90 m above sea level across the 50-ha plot. Temperatures average 26.3 °C, annual rainfall averages 1,896 mm, and minimum mean monthly rainfall is 96 mm in January. The reserve supports species-rich, dipterocarp forest, and 862 tree species have been recorded in the 50-ha plot (https://forestgeo.si.edu/sites/asia/pasoh).

The 50-ha plot was first censused between 1986 and 1988 and was recensused in 1990/91 and approximately every fifth year thereafter. At each census every free-standing tree is permanently tagged, measured for diameter at breast height (DBH), identified to species, and mapped to the nearest 0.5 m following protocols described by Condit (1998). Manokaran et al. (1992) describe the first census in detail.

Leaf and crown traits

We targeted the six largest individuals by DBH of the 100 most abundant species in the 2005 census of the 50-ha plot. Unfortunately, 70 of these large trees were duplicated in the 2005 census data and our field data forms. So, we only targeted 530 individuals. We obtained leaf and crown trait data for 436 trees because 92 trees had died and two trees had broken off above breast height.

Crown traits included height and mean crown diameter (both in meters). We measured height with a telescoping pole for trees less than 15.2 m tall and with a laser range finder and clinometer for taller trees. We calculated the heights of taller trees as the sum of the height of the observer’s eye and the product of the distance and the sine of the angle to the uppermost leaves. We measured crown diameters as the mean distance between the center and the edge of the crown in the four cardinal directions. Note, the center of the crown was projected onto the ground and was often displaced from the tree’s trunk. Heights and crown diameters approach species-level maximum values because (1) we targeted the six largest individuals of the 100 most abundant species in the 50-ha plot and (2) there were 894 individuals of the 100th most abundant species in the 2005 census.

We collected leaves from the uppermost foliage of each tree, using a pruning pole to approximately 10 m and a shotgun for taller trees. The shotgun often shattered petioles so all measurements are for leaf lamina. We sealed freshly collected leaves in ZipLock bags; placed them on ice; transported them back to the laboratory; measured field fresh mass and leaf area within 24 hours (usually within six hours), and then dried the leaves to constant mass at 60 °C before measuring dry mass. We measured two leaves from each tree. The shotgun often failed to bring down entire compound leaves so we counted the number of leaflets and recorded the area of one leaflet for each compound leaf.

Leaf traits included number of leaflets for compound leaves, leaf area (cm2) as lamina area for entire simple leaves and as lamina area of one leaflet for compound leaves, leaf fresh mass (g), leaf dry mass (g), leaf dry matter content (LDMC; proportion), leaf dry mass per area (LMA; g m-2), leaf lamina thickness (microns), and leaf dry tissue density (g cm-3). For compound leaves, leaf area can be estimated as the product of leaflet number and leaflet area. LDMC equals leaf dry mass divided by leaf fresh mass. We measured leaf lamina thickness with a micrometer (Mitutoyo Corporation, Japan). We calculated leaf tissue density as leaf dry mass divided by the product of leaf area and leaf thickness.

Leaf traits vary with light levels. We therefore recorded exposure to sunlight on a widely used five-point scale developed by Dawkins and Field (1978). Exposure equals 5 for trees whose crowns emerge above the canopy. Exposure equals 4 for trees whose crowns form part of the continuous canopy. Exposure equals 3 for subcanopy trees with more than 10% of their crown area exposed to direct sunlight when the sun is directly overhead. Exposure equals 2 for subcanopy trees that only receive direct sunlight from canopy gaps displaced to one side and whose foliage is often oriented towards this gap. Finally, exposure equals 1 for subcanopy trees that only receive direct sunlight as short sun flecks. Leaves collected from trees with exposure scores of 3, 4, or 5 were in direct sunlight. Leaves collected from trees with exposure scores of 1 or 2 received very little direct sunlight.

We determined mean species-level leaf tissue nitrogen (%) and leaf tissue phosphorus concentrations (%) for sun leaves exposed to direct sunlight and shaded leaves with minimal exposure to direct sunlight. Sun leaves had exposure values of 3, 4, or 5 (see previous paragraph). Shade leaves had exposure values of 1 or 2 (see previous paragraph). To determine mean values for each species and exposure category, we pooled equal masses of oven-dried leaf tissue from the appropriate trees. FRIM laboratory protocols for nitrogen and phosphorus determinations can be found at the end of this document. Unfortunately, the original laboratory report includes just one significant digit for many phosphorus concentrations.

Wood density

We targeted the 100 most abundant species in the 2005 census of the 50-ha plot that lacked wood density values but collected wood samples outside the 50-ha plot. We systematically searched forest near the Pasoh Field Station with FRIM botanists Ranger Sha’ri and especially Mr. Abu to locate target species. For shrubs and treelets that rarely reach 10 cm DBH, we collected one 5-cm segment of an approximately 10-mm diameter branch. For larger species, we collected one core from individuals larger than 10-cm DBH with a 5-mm diameter increment borer. We sealed freshly collected samples in ZipLock bags; placed them on ice; transported them back to the laboratory; measured fresh volume by water displacement within 24 hours (usually within six hours), and then dried the samples to constant mass at 60 °C before measuring dry mass. We broke cores into pieces less than 5 cm long, calculated the specific gravity of each piece as oven-dried mass divided by fresh volume, and estimated wood density as an area-weighted average, where area refers to the annulus represented by each piece, assuming a circular trunk (Wright et al. 2010).

References

Condit, R. 1998. Tropical forest census plots. Springer-Verlag and R. G. Landes Company, Berlin.

Dawkins, H. C., and D. R. B. Field. 1978. A Long-Term Surveillance System for British Woodland Vegetation. Department of Forestry, Oxford University, Oxford, Great Britain.

Manokaran, N., J. V. La Frankie, K. M. Kochummen, E. S. Quah, J. E. Klahn, P. S. Ashton, and S. P. Hubbell. 1992. Stand table and distribution of species in the 50-ha research plot at Pasoh Forest Reserve. Pages 1-454. FRIM Research Data, Forestry Research Institute Malaysia, Kepong, Malaysia (https://forestgeo.si.edu/sites/default/files/pasoh.pdf).

Wright, S. J., K. Kitajima, N. J. B. Kraft, P. B. Reich, I. J. Wright, D. E. Bunker, R. Condit, J. W. Dalling, S. J. Davies, S. Diaz, B. M. J. Engelbrecht, K. E. Harms, S. P. Hubbell, C. O. Marks, M. C. Ruiz-Jaen, C. M. Salvador, and A. E. Zanne. 2010. Functional traits and the growth-mortality trade-off in tropical trees. Ecology 91:3664-3674.

Laboratory protocol for nitrogen determination

TEST METHOD MKT 14 : Determination of Total Nitrogen by Distillation Method

14.1        Method Adopted

Kjedahl digestion method and determination of total nitrogen by distillation process.

14.2        Background

A classical Kjedahl method is applied during digestion in which concentrated sulphuric acid is used to convert nitrogen into ammonium form. In order to raise the temperature and at the same time promote the oxidation of organic matter, sodium sulphate is added together with catalyst (selenium). Total nitrogen is then determined by distillation method using HCl solution for titration.

14.3       Apparatus

·        Analytical Balance

·        Whatman Filter paper no. 2 or equivalent

·        Glass funnel

·        Conical Flask 100 ml

·        Specimen vial 100ml

·        Pipette 10 ml

·        Distillation unit Buchii K-314

·        Titrator  Jencons Digitrate or Burette

·        Block digestor

14.4        Reagent

14.4.1    Reagent for Digestion

·        Concentrated  Sulphuric Acid  98% . Analytical grade

·        Catalyst – A well ground mixture of sodium sulphate and selenium (100:1)

14.4.2    Reagent for Distillation

·        Sodium hydroxide 30% : Dissolve 300 g of  NaOH in 200 ml distilled water and make up to 1 litre in volumetric flask.

·        Boric acid 3%. Dissolve 30 g of boric acid in distilled water and make up to 1 litre in volumetric flask.

·        Indicator.  Dissolve 0.10 g of methyl red and 0.05 g of methylene blue in 100 ml ethyl alcohol.

14.4.3      Reagent for Titration

·        0.01 N HCl. Pipette 0.83 ml of concentrated HCl in 400 ml distilled water and make up to 1L in volumetric flask.

14.5    Procedure

14.5.1    Digestion

1.      Weigh  0.50 g  of oven dried soil into a digestion  tube. For foliar sample, 0.05 of oven dried sample is used.

2.      Add in 1 g of catalyst.

3.      Add 2.5 ml conc. Sulphuric acid. Place the tubes on vortex mixer and mix thoroughly.

4.      Digest for 2 hours or more at 350 C until  yellow clear solution  is obtained.

5.      Allow the digest to cool in a fume cupboard for a few minutes.

6.      Add 10-15 ml distilled water before the tubes getting cold to prevent solidification.

7.      Remove the tubes and add in distilled water. Put the tubes on a vortex mixer and mix to dissolve all particles.  Repeat the process and diluted to 100 ml. Leave overnight to enable the undigested particles to settle.

8.      Filter the solution into a specimen vial.

14.5.2    Distillation

1.         Warm up the distillation unit for 30 minutes. Switch on the tap water connected to cooling coil.

2.         Pour  NaOH 30% solution into the labeled NaOH container.

3.         Pipette a 10 ml of sample solution into distillation tube. Insert the tube into the distillation unit.

4.         Press NaoH button 4 times to add estimated 12 ml  NaOH inside the digestion tube

5.         Add 10 ml of  3% of boric acid into conical flask follow by  2-3 drops of indicator. Place the conical flask beside the digestion tube to collect the distillate.

6.         Distill the sample for approximately 4 minutes.  This is done by turning the steam tap to on for approximatele 4 minutes.

7.         Then turn the steam tap to off .

8.         Proceed to titration.

14.5.3    Titration

1.                  Titrate the solution in conical flask with 0.01 N HCl. The color should turn pink for end point. Result of  nitrogen is calculated based on formula given.

14.6    Calculation

% N  = [  (Amount of HCL in ml  - Blank ) x 0.00014 x 100 x 10 ] ÷ weight of sample

1 ml of 0.01 N HCL = 0.00014 g nitrogen

100 = percentage

10 = volume of sample solution used in distillation process

14.7    Reference

1.      MS 678 : 1980 . Methods for Soil Chemical Analysis. Part  II :      Determination of soil Nitrogen     A:       Distillation Method

 Laboratory protocol for phosphorus determination 

TEST METHOD MKT 19: Determination of  Phosporous in Plant by Yellow Vanadate Method

19.1        Method Adopted

Plant samples are digested by microwave technique and phosphrous is determined by yellow method on UV-Visible Spctrophotometer

19.2        Background

The plant material is digested by acid oxidation under closed pressurized vessel in microwave oven. Digestion in sealed Teflon vessel under high pressure efficiently oxidized organic matter of the sample by a digestion reagent directly hated with microwave energy. Combination of microwave heating and acid digestion under pressure in Teflon vessel provides fast heating, reducing digestion time and allowing contamination control.

Phosphorus in sample digest is then determined colorimetrically using the formation of the yellow vanado-molybdate complex by UV-Visisble Spectrophotometer at 420 nm wavelength.

19.3       Apparatus

·        Oven

·        Analytical Balance

·        Whatman Filter paper no. 42 or equivalent

·        Glass funnel

·        Volumetric flask 50 ml or 100ml

·        Conical Flask 50 ml

·        Plastic bottle

·        Teflon vessel

·        Microwave oven

19.4        Reagent

19.4.1      Reagent for Digestion

·        Concentrated HNO3  65%

·        Hydrogen Peroxide 30 %

19.4.2      Reagent for Colorimetric Determination

·        Reagent 1

1.5 % Ammonium Vanadate in 1.5 N Nitric acid : Add 95.5 ml of concentrated nitric acid to about 800 ml of distilled water. Cool the solution and add 0.5 g of ammonium vanadate. Shake well to dissolved and then makeup to 1 Litre with distilled water.

·        Reagent 2

1.5 % Ammonium Molydate  : Dissolve 30 g of ammonium molybdate in distilled water and make up to 2 litre. Filter the solution.

19.4.3      Reagent for Standard Phosporous

·        Standard Stock Solution 1000 ppm

Dry potassium dihydrogen phosphate at 105 C for 2 hours. Weigh accurately 0.43948 g into 600 ml beaker. Dissolve with approximately 300 ml distilled water. Transfer into 1 Litre volumetric flask and makeup to the mark.

·        Standard Stock Solution 100 ppm

Pipette 25 ml of  1000 ppm into 250 ml volumetric flask. Make up to the mark with distilled water.

·        Working Standard

Pipette 0, 5, 10, 20 and 30 ml of 100 ppm standard stock solution into five 100 ml volumetric flasks.. Dilute each solution with distilled water to about 50 ml. Add 3 ml conc. Nitric acid and 2 ml hydrogen peroxide. Makeup to the mark. These solutions each contain 0, 5, 10, 20 and 30 ppm P respectively.

19.5          Procedure

19.5.1    Digestion

9.      Oven dry plant sample at 105 C for 2 hours.

10.   Allow to cool in a dessicator.

11.   Weigh accurately 0.3000 + 0.05g into Teflon vessel.

12.   Introduce the vessel into safety shield.

13.   Add in 3 ml conc. Nitric acid and 2 ml hydrogen peroxide. Swirl the vessel to homogenize the sample with acid.

14.   Close the vessel and introduce into rotor segment, then tighten using torque wrench.

15.   Insert the rotor segment into the microwave oven and connect the temperature sensor.

16.   Run the microwave programme :

Step 1: Ramp from 30 C to 120 C (5 mins.)

Step 2: Hold at 120 C for 5 mins.

Step 3: Ramp from 120 C to 180 c for 10 mins.

Step 4: Hold at 180 C for 10 mins.

17.   Ventillation time is set for 20 mins. to allow the sample to cool down.

18.   The solution is transferred into 50 ml or 100 ml volumetric flask. Distilled water is used to rinse the vessel

and makeup to final volume.

11.  The solution is filtered through filter paper before analysed P determination             

19.4.2      Phosphorus Determination by Yellow Vanadate Method

1.      Pipette 5 ml of working stand solution into 50 ml conical flask.

2.      Add in 15 ml of Reagent 1 followed by 10 ml of Reagent 2.

3.      Swirl the flask to mix the solution. Leave for 30 minutes for color formation.

4.      The same procedure is followed for sampl solution.

5.      Measure the color intensity of the standard solution on UV-Visible Spectropotometer at 420 nm wavelength. This to obtain calibration curve. Then analye the sample solution.

The procedure is very hazardous. Always use safety glasses and chemical and heat-resistant gloves.

19.5      Calculation

%   =  (Conc. – Blank) x DF ÷ sample weight  x final volume

where ,     Conc.    = reading from ICP;           Blank        = blank reading       ; DF      = dilution factor (if any)

19.6       Reference

R. Thomas White, JR & Gareth E. Douthit. Use of microwave oven and nitric acid-hydrogen peroxide digestion to prepare botanical materials for elemental analysis by inductive couple plasma emission spectroscopy. J. Assoc. Off. Anal. Chem. Vol. 68, No. 4. pp 766-769 (1985).