Trait measurements
For leaf trait measurements (listed in Appendix Table A2), 1-3 leaves
each were collected from selected seedlings and immediately stored in a
ziplock bag between moist tissue paper to make sure the leaves were
fully saturated with water (Cornelissen et al. 2003). Five
seedlings from the control group were selected for each species for
trait measurements.
To measure SLA (cm2 g-1) and LDMC
(µm mg-1) (Cornelissen et al. 2003), the fresh
leaf area was measured using the CI-202 leaf scanner (CID bio-science).
After measuring fresh weight, leaves were placed in paper bags and
air-dried for a week, following which they were dried at 60°C for 3 days
to get dry leaf mass. SLA was calculated as the ratio of leaf area to
its dry biomass. LDMC was calculated as the ratio of dry leaf biomass
and fresh leaf biomass.
To measure stomatal traits, the leaf surface was wiped thoroughly to get
rid of dust and excess moisture. Nail polish imprints were prepared and
scanned under 40X magnification in a light microscope. For stomatal
density (SD, number per cm2), four fields of vision
were imaged from each imprint. The number of stomatal apertures were
counted and the image area was measured using the rectangle tool in
ZEISS ZEN2 software (v1.0, RRID: SCR_013672). We chose three of the
clearest stomates from each of the images and estimated stomatal size
(SS, µm2) by drawing an ellipse around the stomata and
stomatal length (SL, µm) using ZEISS ZEN2 (v1.0, RRID: SCR_013672). We
then calculated stomatal area fraction (SAF) for cm2of leaf by taking a product of SD and SS (SD * SS *
10-8). Model results for SAF are similar to SD
(Appendix Figure A3) and therefore we decided to use SD for further
analysis and interpretation.
To measure vein density (VD), leaves were immersed in petri dishes with
cleaning bleach and monitored daily. Once the leaves lost all the
chlorophyll, they were washed with distilled water and stained with
methylene blue diluted 1:100 with distilled water. The samples were put
on a clean slide and imaged under 20X magnification. Xylem diameter (XD,
µm) was measured using an inch-long section of the stem that was cut off
and sectioned immediately into thin slices with a stainless-steel blade.
Five to ten of the thinnest sections were observed under the 40x light
microscope and the diameter of 8-10 of the largest xylem vessels were
measured using the line and circle tools of the software. All
microscopic measurements were performed using a Primostar, Carl Zeiss
microscope and images were taken using the camera accessory Axiocam-105
mounted on the microscope. All images were analyzed using the Zen (3.0
Blue edition) software.
To measure root traits, harvested roots were stored in a freezer until
scanning. The root samples were taken out just before the scan and
cleaned thoroughly to remove any traces of soil and dirt. The roots were
scanned using an Epson Regent LA2400 scanner. The scans were analyzed
using WinRHIZO (Regent Instruments Inc., Canada) for total root length
(RL, mm.), diameter and root surface area (RA, mm2).
Specific root length (SRL, cm. g.-1) was calculated as
the ratio of the total root length and the root’s dry biomass. Root mass
fraction (RMF, g g−1) was calculated as the fraction
of root dry mass and total plant dry biomass. Seasonality index (SI) of
each species was derived from a previous analysis that estimated
species’ affinity to seasonal drought conditions based on their
distribution across the Western Ghats (Krishnadas et al. 2021).
Positive value of seasonality index indicates that the species prefers
less seasonal conditions, i.e., wet associated.