Sowing method
Seeds of Banksia attenuata R.Br. and B. sessilis (Knight)
A. R. Mast & K. R. Thiele (purchased from Nindethana Seed Company, King
River, Western Australia), were sown on filter paper. One seedling was
transferred into each experimental pot on 22nd May,
2016. According to the supplier, the seeds of B. sessilis were
collected from a coastal population, growing over limestone near Jurien
Bay, Western Australia (30°18 S, 115°3’ E). The
provenance of the B.
attenuata seed was unknown.
Experimental design
A pot experiment was carried out in a glasshouse at the University of
Western Australia, Perth, Australia (31°59ʹ S, 115°53ʹ E) using a
randomised complete block design. Glasshouse temperature fluctuated
between 13 and 33°C over a whole year, and transmission of radiation
into the glasshouse was 60% of natural light. The experiment was
designed to explore why B. sessilis is able to grow across a
wider range of P-impoverished soil types and maintain a greater relative
growth rate (RGR) than B. attenuata by comparing the use and
allocation of P among foliar P fractions in the two species. Three soil
treatments were imposed, based on washed river sand: sand only, sand +
laterite (SLAT), and sand + limestone (SLIM). The substrate total P
availability was sand > SLAT > SLIM (Fig. S1,
Shi et al. , 2020). The pots (100 mm inner diameter x 400 mm tall
PVC cylinder) were lined with plastic bags. For each soil treatment, 3.0
kg substrate was added to the pots. For the SLAT and SLIM treatment, a
100 mm layer of laterite or limestone gravel, respectively, was added 50
mm below the soil surface, and other layers were filled with sand. There
were ten replicates for each species in each treatment. Field capacity
of soils in each treatment was calculated as [(wet mass – dry mass) /
dry mass] × 100%. The pots were watered to a constant weight of 80%
of field capacity three times a week. A 20 ml aliquot of basal liquid
nutrient solution lacking P and containing (per kilogram of soil): 217.5
mg KNO3; 74 mg CaCl2; 140 mg
K2SO4; 80 mg
MgSO4.7H2O; 28.9 mg
MnSO4.H2O; 10 mg
ZnSO4.7H2O; 5 mg
CuSO4.5H2O; 0.7 mg
H3BO3; 0.5 mg
CoSO4.7H2O; 0.4 mg
Na2MoO4.2H2O; 20 mg
FeNaEDTA, was applied to each pot once every second week.
Photosynthesis
measurement
Prior to the final harvest, net
photosynthetic rate (Pn) of attached leaves was measured
between 10:00 and 11:00 on March 7th, 9th 2017 using a red/blue LED
light source (LI-6400, LI-COR Inc., Lincoln, NE, USA). The plants were
watered on the day before the photosynthesis measurement. One mature
leaf of each plant was measured under a photosynthetic photon flux
density of 1500 μmol m-2 s-1 and a
CO2 concentration of 400 μmol mol-1.
The leaves used for photosynthesis measurement were sampled, and the
projected leaf area measured at 200 dpi (Epson 1680, Long Beach, CA,
USA) and calculated (ImageJ 1.4, NIH, Bethesda, MD, USA). Leaves were
then dried at 70 °C for 72 h to measure dry mass (DM).
Harvest
After 50 weeks of growing in pots, a total of 20 fully-expanded leaves
with no visible damage or discolouration were harvested from each plant.
The leaves were immediately scanned at 200 dpi to calculate leaf area
(LA1), submerged in liquid nitrogen and stored at -80°C.
Frozen leaves were freeze dried for seven days (VirTis Benchtop “K”,
New York, USA) and dry mass (DM) was determined (DM1).
The remaining leaves on each plant were harvested and scanned at 200 dpi
to calculate the remaining leaf area (LA2). Total LA =
LA1 + LA2. The remaining leaves, stem
and roots were separated and dried at 70°C for 72 h. The DM was
determined for the remaining leaves (DM2) and for stems
plus roots (DM3).
Total leaf DM =
DM1 + DM2. Total plant dry mass
M2 = DM1 + DM2 +
DM3. Leaf mass per area (LMA) was calculated as total
leaf DM / total LA. Seed weight (W 1) was measured
using four lots of 10 (B. attenuata ) or 30 (B. sessilis )
seeds that were dried (70˚C, 48hr) and weighed before calculating the
average seed weight. Relative growth rate (RGR) was calculated as
(lnM 2-lnW 1) /
(T 2-T 1), whereT 1 and T 2 were the dates
of sowing and harvesting, respectively, expressed in
weeks.
Leaf nutrient analyses
Freeze-dried leaves were ground to a fine powder (Geno/Grinder 2010,
Spex SamplePrep, Metuchen, New Jersey, USA).
A
50 mg sample was used to determine inorganic P (Pi) described by Yanet al. (2019).
The P allocated to nucleic acids, lipids, small metabolites (Pi + other
metabolites) and a residual fraction was determined in a 50 mg portion
of powdered leaves using the
differential solubility method described by Hidaka & Kitayama (2013),
as modified in Yan et al.(2019). Metabolite P is defined here as small metabolite P – Pi.
Phosphorus concentrations in extracts and residues from the above
procedures were measured as in
described by Matusiewicz & Golik (2004) using a molybdenum blue method
(Ames, 1966). Total leaf P is the sum of Pi, nucleic acids, lipids,
small metabolites and residual fraction. Total leaf P was confirmed by
acid digestion of ground leaf material, followed by Pi assay. Total
foliar N concentration was determined by combustion of approx. 30 mg of
dried leaf sample (Vario Macro Combustion Analyser, Elementar
Analysensysteme GmbH, Langenselbold, Germany).
Leaf
area-based P concentration was calculated as Total leaf P mass
concentration×LMA; PPUE was calculated as the ratio of photosynthesis
rate to area-based P concentration; Leaf area-based N concentration was
calculated as Leaf N mass concentration×LMA, and photosynthetic
nitrogen-use efficiency (PNUE) was calculated as the ratio of
photosynthesis rates to area-based N concentration.
Statistics
The differences in means between B. attenuata and B.
sessilis on the same substrate were analysed by t test, while
the differences in means within each species across substrate types were
analysed by one-way ANOVA with 95% confidence intervals. The
relationships of foliar P fractions to total foliar P concentration,
leaf mass per area, relative growth rate (RGR) to nucleic acid
phosphorus (P), foliar nitrogen (N) and foliar N to nucleic acid P were
determined by linear regression analysis, the correlation coefficients
were analysed by Student’s T-test. All statistical analyses were
performed using the SPSS Statistics 19.0 (SPSS Inc., Chicago, US), and
graphed with OriginPro 9.5 (OriginLab Corporation, Northampton, MA,
USA).