Restoration in a postmine environment: Using ecophysiological techniques to improve the establishment of framework Banksia woodland seedlings Stephen M Benigno BSc This thesis is presented for the degree of Doctor of Philosophy School of Plant Biology The University of Western Australia 2012
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Banksia woodland background · Banksia woodland community (Fig 1.1) is the center of distribution to several large genera including Banksia trees (Fig 1.3), Leucopogon shrubs, and
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Restoration in a postmine environment:
Using ecophysiological techniques to improve the
establishment of framework Banksia woodland seedlings
Stephen M Benigno BSc
This thesis is presented for the degree of Doctor of Philosophy
School of Plant Biology
The University of Western Australia
2012
ii
iii
DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK
PREPARED FOR PUBLICATION
This thesis contains work prepared for publication, some of which has been co-authored. The publications arising from this thesis are original work undertaken by the student (Stephen Benigno), with guidance from his three supervisors (Jason Stevens, Kingsley Dixon, and Deanna Rokich) and university employee Greg Cawthray. The bibliographical details of the work and where it appears in the thesis are outlined below.
Chapter 2 - Benigno S, Dixon K, Stevens J, Ecophysiological adaptations of three woody
mediterranean tree seedlings to the postmine stresses of drought and soil compaction
In prep, divided into two manuscripts for publication under the titles:
1) Biphasic drought is key to seeding establishment in sandy mediterranean-type soils
2) Interplay of drought and soil compaction lead to catastrophic decline in
phreatophytic species in mediterranean-type environments
Chapter 3 - Benigno S, Dixon K, Stevens J, Increasing soil water retention with native-
sourced mulch improves seedling establishment in postmine mediterranean sandy
soils (abridged version published in Restoration Ecology under the same title,
October 2012 DOI: 10.1111/j.1526-100X.2012.00926.x)
Chapter 4 - Benigno S, Dixon K, Cawthray G, Stevens J, Soil physical strength rather than
excess ethylene reduces root elongation in mechanically impeded sandy soils
(published in Plant Growth Regulation under the same title, April 2012, DOI:
10.1007/s10725-012-9714-2)
Data collection and analysis, presentation, and writing of this thesis are entirely my own. Drs. Stevens, Dixon, and Rokich, as supervisors of this thesis, oversaw the design and implementation of the experiments, and provided reviews and recommendations for the written manuscripts. Greg Cawthray was not a formal supervisor for this project, but provided enough guidance to warrant inclusion with the work undertaken in Chapter 4.
Table of Contents Statement of Candidate Contribution ................................................................................... iii Table of Contents ................................................................................................................... v Abstract ................................................................................................................................ vii Acknowledgements ............................................................................................................... ix Abbreviations ......................................................................................................................... x
Chapter One: General Introduction
Perth’s Banksia Woodlands: Ecological Background and Need for Restoration .................. 1 Restoration in Postmine Sites ................................................................................................ 5 The Integration of Ecophysiology and Restoration Ecology ................................................. 8 Study Species ......................................................................................................................... 9 Thesis Aims and Outlines .................................................................................................... 10
Chapter Two: Ecophysiological adaptations of three woody mediterranean tree seedlings
to the postmine stresses of drought and soil compaction Introduction .......................................................................................................................... 13 Materials and Methods......................................................................................................... 15
Chapter Four: Soil physical strength rather than excess ethylene reduces root elongation
of Eucalyptus seedlings in mechanically impeded sandy soils Introduction .......................................................................................................................... 81 Materials and Methods......................................................................................................... 83
Chapter Five: General Discussion and Future Research Seedling Establishment in Postmine Conditions ................................................................. 99 Increasing Seedling Survival through Ecophysiological Analysis .................................... 101 Future Research Directions ................................................................................................ 104 Conclusion ......................................................................................................................... 107
treatment), or 4) well-watered with non-compact soil (control). Thirty-two pots, evenly
split between compact and non-compact soil in drought and no drought conditions, did not
contain a seedling to allow for soil impedance measurements. An equal amount of pots
containing compact and non-compact soil were arranged in a completely randomized
design within two separate frames in an unheated glasshouse (Fig 2.2). A single seedling
was transplanted into each pot, and vermiculite was placed over top of the soil surrounding
the seedling to reduce water evaporation from the soil. Seedlings were acclimated in the 1.0
m pots for three months and during this time all pots were watered daily from overhead
sprinklers. Clear plastic sheeting was hung above and along the sides of both frames to
prevent water entry from outside sources. Drought was imposed on half the seedlings by
capping off the overhead sprinklers immediately above one of the frames. This first period
17
of drought lasted for 60 days (first drought phase), after which a re-watering period
occurred for 120 days (recovery phase), and was followed by a second and final 60 days of
drought (second drought phase). The pots in the second frame were watered daily
throughout the entire experiment from overhead sprinklers. At 24 days and 201 days after
seedlings were transferred into the PVC pots, 59 mL of nutrient solution was added to all
seedlings. The nutrient solution was developed specifically to simulate the nutrient poor
soils of southwest Australia, ensuring adequate nutrition was administered in the following
amounts (μM): 400 NO3−, 204 K+, 200 Ca2+, 154 SO4
2−, 54 Mg2+, 40 Fe-EDTA, 4 PO43−,
2.4 BO33−, 0.24 Mn2+, 0.10 Zn2+, 0.030 MoO4
2−, 0.018 Cu2+ (Poot and Lambers 2003).
Measurements
Seedling Survival
Forty-five seedlings from each treatment and species were marked for survival
counts throughout the experiment. Counts were taken five times during the first drought
phase, eight times during the recovery phase, and four times during the second drought
phase. Mortality was defined as a no longer functioning seedling, as derived from
chlorophyll fluorescence values (Fv/Fm) of 0, and if the seedling had completely brown and
brittle stems and leaves.
Physiology and Morphology
Five seedlings from each species and treatment were randomly selected to be
measured non-destructively throughout the entire course of the experiment. Measurements
took place 0, 15, 30, 45, and 60 days after the beginning of the first drought phase and
second drought phase, five days after the start of the recovery phase, and three additional
times throughout the recovery phase. A small plastic tie was secured around a petiole of the
most recently formed, fully expanded and undamaged leaf at the beginning of the
experiment and served as a marker for future gas-exchange and fluorescence
measurements. In the occurrence that one of the original five seedlings died during the
experiment, a random functioning seedling from the same species and treatment was
chosen as a replacement.
18
Fig 2.2 One of two frames containing 1.0 m long PVC pipe pots, each with an individual seedling of B.
attenuata, B. menziesii, or E. todtiana.
A Li-Cor® 6400 gas-exchange analyzer (LI-COR, Inc. Lincoln, NE, USA) assessed
the rates of stomatal conductance (gs), photosynthesis (A), transpiration (E), and
intercellular carbon (Ci). Measurements were taken between 0830 and 1100 h local time,
approximately two hours after the start of the photoperiod. Mid-morning and light-saturated
gas-exchange measurements represent the plant average daily values under drought stress
(Vadell et al. 1995). The Li-Cor was set at a reference level for all measurements taken
throughout the experiment (Flow Rate to the Sample Cell: 300 μmol s-1, Reference Cell
CO2: 400 μmol CO2 mol-1, Artificial Light Source: 6400-02 Red/Blue LED PAR 1,700
μmol m-2s-1). Five measurements on each leaf were performed within ten seconds after the
Li-Cor displayed a steady photosynthetic rate. Carboxylation efficiency (ε) was calculated
as the ratio of photosynthesis to intercellular carbon (A/Ci) (Farquhar and Sharkey 1982),
and has been shown to reflect Rubisco activity within leaves (von Caemmerer 2000).
Using the same recently formed, fully expanded leaf measured by the Li-Cor,
chlorophyll fluorescence was recorded by a PAM Fluorometer (Heinz Walz GmbH,
19
Effeltrich, Germany) (Fig 2.3). Measurements were taken between 1100 and 1400 h local
time. The sample leaf was dark adapted with a lightweight plastic clip for 15 minutes prior
to measurement. The maximum PSII photochemical efficiency (Fv/Fm) was calculated, and
a rapid light curve was performed immediately afterward on the same leaf without
removing the clip. The rapid light curve progressively increased from 0 to 1152 µmol
photons m-2 s-1 over nine light pulses. Maximum electron transport rate (ETR) was
consistently achieved at 822 µmol e- m-2 s-1 for each species, and the ETR and non-
photochemical quenching (NPQ) values were recorded at this PAR intensity.
Fig 2.3 Using a pulse of light to measure the chlorophyll fluorescence of a B. attenuata seedling leaf.
Eight destructive harvests were performed at intervals of 0, 21, 49, 77, 126, 175,
203, and 231 days after the first drought phase was initiated. Five seedlings from each
treatment and species were randomly selected for each harvest. Fluorometry and gas-
exchange measurements were recorded using the same calibrations described above. Xylem
water potential was measured at both predawn (ΨPD, commencing 2 hrs before sunrise) and
midday (ΨMD, 1200-1400 h local time). Water potential was measured from shoots
containing two fully expanded leaves using a Scholander-type pressure chamber (Model
1000 Pressure Chamber Instrument®, PMS Instrument Company Albany, OR, USA). After
removal from the seedling, the shoot was immediately sealed in a zip-lock plastic bag until
the measurement was taken, no longer than 15 minutes after removal (Turner 1988). The
seedling diurnal range of water potential (ΨD) was calculated as ΨD = ΨMD - ΨPD (Whitlow
20
et al. 1992). The regulation of apparent soil-to-leaf hydraulic conductance (KL) was
calculated as KL = -E / (ΨMD - ΨPD) (Aranda et al. 2005). The total area of fresh leaves
along with the total length and average diameter of fresh roots were analyzed on a back-lit
flatbed scanner with a resolution of 0.2 μm using a digital image analyzer (WinRHIZO
Pro®, V. 2007d, Regent Instruments Canada, Inc.). Maximum root depth within the pot was
recorded, and the downward root elongation rate was calculated according to the relative
growth rate equation from McGraw and Garbutt (1990). Specimens were then dried for one
week at 70 C, and the dry weights of leaves and roots recorded. The specific leaf area
(SLA) of seedlings was calculated as the ratio of leaf surface area to leaf dry weight
(Wilson et al. 1999).
Soil properties
On the days of a destructive harvest, soil moisture measurements were recorded in
each pot at depths of 0, 20, 40, 60, 80 and 100 cm, with another moisture reading taken at
the greatest depth of the root in the pot. To maintain soil stratigraphy, tubes were carefully
emptied onto a plastic sheet. An MPM-160-B Moisture Probe Meter® (ICT International
Pty Ltd) was fully inserted into the soil for each measurement. The moisture meter was
previously calibrated to obtain a quadratic polynomial equation (y = -2e-7x2 + 4e-4x; R2 =
0.98) to convert millivolts (x) to gravimetric moisture content (y) (MP406 Moisture Probe
Operation Manual). The gravimetric moisture content was converted to volumetric soil
water content (VSW) using the bulk density of the soil (Black 1965).
Compaction levels in the pots were tested using a soil penetrometer (Rimik CP40II
Cone Penetrometer®, RFM Australia Pty Ltd, QLD, Australia [Cone Diameter 12.83mm;
Area 130 sqmm]) at three times throughout the experiment to a depth of 60 cm (maximum
depth of penetration). The first measurement was taken prior to any water withholding,
while the remaining two measurement rounds were recorded after the first and second
drought phases. Particle density, total porosity and air-filled porosity for each treatment
were calculated for non-compact and compact soils (Table 2.1) (ASTM D854-92 1992).
Statistics
R statistical software (version 2.13.0) was used to perform the statistical tests in this
study. Binomial generalized linear models compared seedling survival between treatments
and species. General linear models compared the physiology and morphology of the
seedlings and the differences in soil moisture and physical properties between treatments,
21
while Tukey’s post hoc test performed pairwise comparisons between treatments when
significant effects were indicated. Data from each phase in the drought treatment (first
drought, recovery, and second drought) was statistically compared against data from the
control treatment measured on the identical dates. ANCOVAs compared differences in the
slopes of the regression lines. SigmaPlot 12 (Version 12.0.0.182, Systat Software, Inc.) was
used to fit regression lines. The homogeneity of the variances was tested by residual plots
and logarithmically or square-root transformed to achieve a normal distribution when
necessary, and all data is presented as untransformed means.
Results
Soil properties
The soil used in this experiment has a particle density of 2.5 g cm-3, and increasing
bulk density reduced the total porosity and air-filled porosity, but increased volumetric soil
water content (Table 2.1). Average volumetric soil water content during the experiment
throughout the entire 100 cm depth of the pot significantly differed between treatments, but
not by species (P < 0.001 treatment, P = 0.965 species). Compared to the control, total
volumetric soil water content significantly increased 16% in the compact treatment and
decreased 52% in the drought treatment (P < 0.001), but did not differ in the drought
compact treatment (P = 0.989) (Table 2.1).
Table 2.1 Characteristics of the soil averaged throughout the entire 100 cm PVC pot for each treatment (n =
106). Compact and drought compact treatments were subjected to a mechanical force to increase bulk
density. Drought and drought compact treatments were subjected to two intervals of water withholding.
Differences in water content are indicated as letters, P < 0.05
Bulk
Density Particle Density
Air-Filled Porosity
Total Porosity
Volumetric Water Content
(g cmˉ³) (g cmˉ³) (%) (%) (cm³ cmˉ³) Control 1.55 2.50 31 40 0.094 a Compacted 1.80 2.50 17 28 0.112 b Drought 1.55 2.50 34 40 0.062 c Drought Compact 1.80 2.50 19 28 0.093 a
Soil water content in the pots containing seedlings was greatest at the depth of 100
cm in every treatment due to the well-drained properties of the soil used in this study (P <
0.001) (Fig 2.4). The drought treatment contained significantly less water than the control
at each 20 cm interval tested throughout the pots (P < 0.001) (Fig 2.4). The drought
22
compact treatment contained less water than the control in the top 20 cm of the soil, but the
amount of soil water gradually increased at deeper intervals within the pots, containing
greater amounts of water than the control at depth of 80 and 100 cm (P < 0.001) (Fig 2.4).
The compact treatment held significantly more water than the control at most depth
intervals throughout the pot (Fig 2.4).
Soil water available to the roots in the drought and drought compact treatments
during the first drought phase was significantly lower than the control (P < 0.001) (Fig 2.5).
During the recovery phase, soil water content in the drought and drought compact
treatments was not significantly different from the control (P ≥ 0.063), but significantly
decreased again during the second drought phase (P < 0.001) (Fig 2.4). The rate and
amount of water lost from the soil in first drought phase was not significantly different to
the second drought phase within the drought treatment (P ≥ 0.209). A 34% decrease in soil
water was recorded 21 days after the beginning of the first drought phase, while a 41%
decrease was recorded 21 days after the beginning of the second drought phase (Fig 2.5). A
47% decrease in soil water content was recorded after 49 days for both the first and second
drought phases (Fig 2.5).
Mechanical impedance of the soil in the drought treatment was not significantly
different from the control at all depths measured (P ≥ 0.733) (Fig 2.6). Soils of the compact
treatment became significantly greater than the control at depths lower than 10 cm (P ≤
0.014), and soils of the drought compact treatment became significantly greater than the
control at depths of 8 cm (P ≤ 0.020) (Fig 2.6). Soils of the compact and drought compact
treatments were unable to record measurements deeper than 22 cm and 20 cm, respectively,
due to high impedance values. Soils in the drought compact treatment had significantly
higher impedance values than the compact treatment at depths of 18 and 20 cm (P ≤ 0.001)
(Fig 2.6).
23
Depth (cm)
0 20 40 60 80 100
Volu
met
ric S
oil W
ater
Con
tent
(cm
3 cm
-3)
0.00
0.05
0.10
0.15
0.20
0.25ControlCompactDroughtDrought Compact
a
b
c c
ab
ca
aab
a ab
cab a
b
c
b
a
b
c
b
Fig 2.4 The average volumetric water content at 20 cm depth intervals within pots. Error bars represent the
standard error of the mean (n = 106). Differences at depth between treatments are indicated as letters, P <
0.05. Soil bulk density in the control and drought treatment = 1.55 g cm-3, compact and drought compact
treatments = 1.80 g cm-3.
Days0 25 50 75 100 125 150 175 200 225
Volu
met
ric
Soil
Wat
er C
onte
nt(c
m3
cm-3
)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Control CompactDroughtDrought Compact
First Drought Recovery Second Drought
*
*
**
*
* **
*
*
*
*
Fig 2.5 The average volumetric soil water content measured to the average depth of roots over 228 days.
Error bars represent standard error of the mean (n = 15), differences from the control are indicated as ‘*’ P <
0.05. The first drought, recovery, and second drought phases are divided by dashed lines. Control and
compact treatments were not subjected to water withholding. Soil bulk density in the control and drought
treatment = 1.55 g cm-3, compact and drought compact treatments = 1.80 g cm-3.
24
MPa0 1 2 3 4 5
Dep
th(c
m)
-60
-50
-40
-30
-20
-10
0 Control Compact Drought Drought Compact
Fig 2.6 The soil penetration resistance within the pots, averaged from three separate intervals during the
experiment. Error bars represent standard error of the mean (n = 9). Soil bulk density in the control and
drought treatment = 1.55 g cm-3, compact and drought compact treatments = 1.80 g cm-3.
Seedling survival
Seedling survival in the drought treatment remained at 100% in all species
throughout the entire first drought phase (Fig 2.7). Seedling deaths in the drought treatment
were first observed during the recovery phase for all species (Fig 2.7). In the drought
treatment, 76% of B. attenuata, 84% B. menziesii and 71% E. todtiana remained alive at
the end of the recovery phase, and mortality was not significantly different from the control
treatment at this point in the experiment (P = 0.366 B. attenuata; 0.579 B. menziesii; 0.276
E. todtiana). Seedling deaths were recorded within 16 days after the commencement of the
second drought phase in all species (Fig 2.7). Within 48 days of the start of the second
drought phase, 100% of seedlings in the drought treatment died (Fig 2.7). No significant
differences in survival were recorded between species throughout the experiment (P ≥
0.860).
Mortality of ≥ 50% was recorded in all species after 49 days in the drought compact
treatment (Fig 2.7). On the final day of the first drought phase (60 days of withholding
water), a survival rate of 16% for B. attenuata, 24% B. menziesii and 20% E. todtiana was
significantly less than the control (P < 0.001) (Fig 2.7). Five days into the recovery phase
25
100% mortality occurred in B. attenuata and E. todtiana with only 7% B. menziesii
surviving, and the remaining B. menziesii died by day 16 of the recovery period (Fig 2.7).
No deaths occurred in the control, while 4% of B. menziesii and 2% of E. todtiana
died in the compact treatment over the course of the experiment, and did not result in any
significant differences (data not shown).
%
0
50
100
Days
0 25 50 75 100 125 150 175 200 225
a) Banksia attenuata
%
0
50
100
a) Banksia menziesii
Days
0 25 50 75 100 125 150 175 200 225
%
0
50
100
a) Eucalyptus todtiana
b) Banksia attenuata
b) Banksia menziesii
b) Eucalyptus todtiana
Soil WaterPhotosynthesisFv/FmSurvival
Fig 2.7 The photosynthetic rate, effective quantum yield of PSII (Fv/Fm), and volumetric soil water content
expressed as a percentage of the control (n = 5), and the survival percentage (n = 45) for B. attenuata, B.
menziesii, and E. todtiana in the a) drought treatment and b) drought compact treatments. The first drought,
recovery, and second drought phases are highlighted by the increases and decreases of the shaded area. Soil
bulk density in the drought treatment = 1.55 g cm-3, drought compact treatment = 1.80 g cm-3.
26
Control
Physiology
Comparative physiology can determine fitness between species (Ackerly 2000;
Austin et al. 2009), and unstressed seedlings of B. attenuata consistently functioned at
higher physiological rates than B. menziesii and E. todtiana, while B. menziesii functioned
at relatively higher rates than E. todtiana (Table 2.2). B. attenuata photosynthetic rate was
43% greater than B. menziesii (P < 0.001) even though the gs of the two Banksias were not
significantly different, possibly as a result of a 47% increase in ε and a 78% increase in
ETR in B. attenuata (P ≤ 0.002) (Table 2.2). The 358% reduction in average gs in E.
todtiana was most likely responsible for the lower rates of A and E compared to the two
Banksias (P < 0.001) (Table 2.2). Throughout the experiment, E. todtiana maintained an
average 53% and 74% greater NPQ than B. attenuata and B. menziesii, respectively (P <
0.001) (Table 2.2). Average Fv/Fm was 0.823 in B. attenuata, 0.809 in B. menziesii, and
0.735 in E. todtiana. The Fv/Fm of the Banksias were not significantly different from each
other (P = 0.974), but were both significantly greater than E. todtiana (P < 0.001).
Seedling ΨPD of unstressed B. menziesii was more negative than B. attenuata by
16% (P = 0.016) and by 25% than E. todtiana (P = 0.001) (Table 2.3). ΨMD in B. attenuata
and B. menziesii was significantly more negative than E. todtiana by an average of 18% (P
≤ 0.006) (Table 2.3). The ΨD of B. attenuata was significantly greater than B. menziesii by
31% and E. todtiana by 38% (P < 0.001) (Table 2.3). No significant differences were
observed in average KL between the two Banksia species (P = 0.351), and the KL of E.
todtiana decreased by 50% and 40% from B. attenuata and B. menziesii, respectively (P =
0.011 B. attenuata; P < 0.001 B. menziesii) (Table 2.3).
Morphology
Unstressed B. menziesii seedlings had 39% and 72% more leaf surface area than
unstressed B. attenuata and E. todtiana, respectively (P ≤ 0.003), while E. todtiana
exhibited a 38% greater SLA than B. attenuata (P = 0.028) (Fig 2.8). The root to shoot ratio
of E. todtiana was 84% and 71% higher than B. attenuata and B. menziesii, respectively (P
< 0.001 (Fig 2.8) and the total root length of both E. todtiana and B. menziesii was 33%
greater than B. attenuata (P ≤ 0.008) (Fig 2.8). The percentage of fine roots
27
Table 2.2 Seedling physiological values ± standard error of the mean throughout 228 days for the control (n = 60), compact (n = 60) and drought (n = 55) treatments.
Drought compact treatment values were averaged over 60 days during the first drought phase (n ≥ 25). Soil bulk density in the control and drought treatment = 1.55 g
cm-3, compact and drought compact treatments = 1.80 g cm-3. Differences from the control treatment are indicated as ‘*’ P < 0.05, ‘**’ P < 0.01, ‘***’ P < 0.001.
Differences between species within the control treatment are indicated by letter P < 0.05.
Fig 2.10 The relationship between A vs E, A vs gs, and gs vs ε in B. attenuata, B. menziesii, and E. todtiana in the drought treatment. The slope, y-intercept,
R2 and P values were derived from regression lines transformed to achieve normality. Data is presented as untransformed
34
35
gs above 0.05 mol H2O m-2 s-1 for the initial 45 days of the first drought phase (Fig
2.11a).The average rate of gs dropped below 0.05 mol H2O m-2 s-1 in all three species when
water was withheld past 45 days in the first drought phase (Fig 2.11b).
Stomatal conductance rate was reduced significantly during the initial 45 days of the
first drought in the two Banksias (P ≥ 0.001) (Fig 2.11a). The decreases in gs in these two
Banksias species corresponded to decreases in E (P < 0.001), and only the rate of A in B.
menziesii was reduced significantly during this time (-31%, P = 0.002) (Fig 2.11a). The gs
of E. todtiana remained constant during the initial 45 days of the first drought phase and
maintained rates of E and A (Fig 2.11a). A 40% increase in ε was observed in E. todtiana
(P = 0.012) and the ETR of all three species increased during the first 45 days of drought
(+29% in B. attenuata P = 0.029; +38% in B. menziesii P = 0.038; +24% in E. todtiana P =
0.003), while no changes in NPQ were recorded (P ≥ 0.421) (Fig 2.11a). After 45 days of
withholding water during the first drought phase the gs of all three species dropped below
the severe drought threshold of 0.05 mol H2O m-2 s-1 (Banksias P < 0.001; E. todtiana P =
0.045) and resulted in the decreases of A (P ≤ 0.033) and E (P ≤ 0.011) (Fig 2.11b).
Significant reductions in ETR, NPQ and ε were recorded after 45 days of the first drought
(Fig 2.11b). Only the ε of B. menziesii was not significantly reduced during this time (P =
0.452) (Fig 2.11b).
Significant changes in hydraulic conductance were observed regardless of drought
severity (gs > or < 0.05 mol H2O m-2 s-1) during the first drought phase in all species (Fig
2.12). The ΨPD decreased in all species (P ≤ 0.001), and the ΨMD of E. todtiana decreased
120% (P < 0.001) while the ΨMD of B. attenuata and B. menziesii was not significantly
affected during the first drought phase (P ≥ 0.058) (Fig 2.12). Significant reductions in KL
were observed in both B. attenuata and B. menziesii (P ≤ 0.023) with no decreases in E.
todtiana recorded (Fig 2.12). The ΨD was unaffected during the first drought phase in all
three species (P ≥ 0.106) (Fig 2.12).
Recovery phase
The physiology of seedlings from the drought treatment were defined to have
recovered when values recorded during the recovery phase were not significantly less than
those from the control treatment (P ≥ 0.05). Recovery was observed in every physiological
trait for B. attenuata, B. menziesii, and E. todtiana, and recovery was found to occur when
values from seedlings in the drought treatment attained values ≥ 70% compared to the
36
control treatment (Table 2.4). Physiological traits from seedlings in the drought treatment
were shown to surpass those of control values during the recovery phase (Table 2.4). The
exact number of days for recovery was dependent on species and specific physiological
trait, and the A, gs, E, ε, ETR, and NPQ of the seedlings in the drought treatment of all three
species had recovered within 17 days after re-watering (Table 2.4). The average KL, ΨPD,
ΨMD, and ΨD of each species attained values significantly similar to the control values
within 17 days after the recovery phase commenced (P ≥ 0.05, data not shown).
Table 2.4 The highest percentage of recovery from seedlings in the drought treatment as compared to the
control treatment, measured during the recovery period. Numbers in parentheses are the maximum days after
re-watering for seedlings in the drought treatment to statistically recovery to control treatment values (n = 5).
Photosynthesis (μmol CO2 m-2 s-1)
Stomatal Conductance (mol H2O m-2 s-1)
Transpiration
(mmol H2O m-2 s-1)
B. attenuata 118% (17) 121% (17) 158% (5) B. menziesii 87% (5) 84% (5) 111% (5) E. todtiana 76% (5) 108% (17) 122% (17)
Carboxylation
Efficiency (mol mol-1)
ETR
(μmol e- m-2 s-1)
NPQ
B. attenuata 134% (17) 126% (5) 164% (5) B. menziesii 83% (5) 357% (17) 177% (5) E. todtiana 78% (5) 175% (5) 74% (5)
37
ControlDrought
A(
mol
CO
2 m
-2 s
-1)
0
2
4
6
8
10
12
14
g s
(mol
H2O
m-2
s-1
)
0.0
0.1
0.2
0.3
0.4
ETR
(µm
ol e
- m-2
s-1
)
0
10
20
30
40
50
B. attenuata
B. menziesii
E. todtiana
NPQ
0
1
2
3
(m
ol m
ol-1
)
0.00
0.01
0.02
0.03
0.04
0.05
B. attenuata
B. menziesii
E. todtiana
*
*
*
*
*
*
*
*
*
* **
***
**
*
* *
*
B. attenuata
B. menziesii
E. todtiana
*
**
**
*
*
**
*
*
*
E(m
mol
H2O
m-2
s-1
)
0
1
2
3
4
5
* **
*
**
*
a)
a)
a)
a)
a)
a)
b)
b)
b)
b)
b)
b)
c)
c)
c)
c)
c)
c)
Fig 2.11 Seedling physiology in the control and drought treatment during a) the initial 45 days of the first
drought phase when droughted seedlings experienced moderate to severe drought stress (gs > 0.05 mol H2O
m-2 s-1) (n = 15), b) the last 15 days of the first drought phase when droughted seedlings experienced very
severe drought stress (gs < 0.05 mol H2O m-2 s-1) (n = 5), and c) the initial 45 days of the second drought
phase when droughted seedlings experienced very severe drought stress (gs < 0.05 mol H2O m-2 s-1) (n = 15).
Error bars represent standard error of the mean, differences from the control are indicated as ‘*’ P < 0.05.
38
Second drought phase
During the second drought phase severe drought occurred much quicker, the
average rate of gs dropped below 0.05 mol H2O m-2 s-1 in all three species within 15 days of
withholding water. None of the three species were recorded with gs values greater than 0.05
mol H2O m-2 s-1 during the second drought phase (Fig 2.11c). Physiological traits that were
unaffected during the initial 45 days of the first drought phase were significantly reduced
during the same duration in the second drought phase (Fig 2.11c). Significant decreases in
both gas-exchange and chlorophyll fluorescence was observed in the two Banksia species
(P ≤ 0.018), and only ETR was unaffected during the second drought phase (Fig 2.11c).
The ETR, NPQ and ε of E. todtiana decreased significantly (P ≤ 0.025) while gs, A, E were
unaffected (Fig 2.11c).
The ΨPD and ΨMD of all species decreased significantly during the second drought
phase, (P ≤ 0.010), and ΨD was unaffected in all three species during this time (P ≥ 0.474)
(Fig 2.12). The KL of both Banksias was reduced significantly during the second drought
phase (P ≤ 0.029), but the KL was not affected significantly in E. todtiana (Fig 2.12).
B. attenuata B. menziesii E. todtiana
D
(MP
a)
0.0
0.2
0.4
0.6
0.8
B. attenuata B. menziesii E. todtiana
M
D(-
MP
a)
0.0
0.5
1.0
1.5
2.0
2.5
*
**
*
P
D(-
MP
a)
0.0
0.5
1.0
1.5
2.0
2.5Control 1st Drought Recovery 2nd Drought
* * **
*
*
KL
(mm
ol H
2O M
Pa-1
m-2
s-1
)
0
2
4
6
8
10
12
14
** *
*
Fig 2.12 ΨPD, ΨMD, ΨD, and KL of B. attenuata, B. menziesii, and E. todtiana seedlings in the control and
drought treatment during the first drought, recovery, and second drought phases. Error bars represent standard
error of the mean (n ≥ 5), differences from the control are indicated as ‘*’ P < 0.05.
39
Morphology
The drought treatment did not cause significant reductions in either leaf surface area
or SLA in any of the three species (P ≥ 0.125) (Fig 2.8). An increase in root to shoot ratio
was observed in B. attenuata (40%) and B. menziesii (36%) (P < 0.001), with no change in
E. todtiana (P = 0.999) (Fig 2.8). Total root length increased 33% in B. attenuata (P =
0.001), with no significant differences in B. menziesii or E. todtiana (P ≥ 0.371). An
increase of 12% was observed in the fine root percentage of both in B. attenuata and B.
menziesii (P ≤ 0.049), and no difference was observed in E. todtiana in the drought
treatment (P = 0.234) (Fig 2.8). Average root diameter was reduced 18% to 0.61 mm in B.
attenuata and 20% to 0.60 mm in B. menziesii (P < 0.001) while the root diameter of E.
todtiana in the drought treatment remained unchanged from the control at an average 0.60
mm (P = 0.084). The roots of all species reached the bottom of the pot (1.0 m) within 132
days after transplanting, and root depth after 104 days was not significantly different from
the control.
Compact treatment
Physiology
Compact soil had no effect on the physiological responses of B. attenuata, B.
menziesii, or E. todtiana, and the average physiological values over the course of the
experiment were not significantly different from the control values (Table 2.2). Seedling
hydraulic conductance (ΨPD, ΨMD, ΨD, and KL) was unaffected in the compact treatment
(Table 2.3).
Morphology
The compact treatment did not significantly affect the aboveground morphology of
any of the three species, with no differences in either leaf surface area or SLA observed (P
≥ 0.540) (Fig 2.8). The root to shoot ratio of seedlings in compact treatment decreased
36%, 33%, and 32% in B. attenuata, B. menziesii, and E. todtiana, respectively (P ≤ 0.003)
(Fig 2.8). The total root length was reduced 29% and 44% in B. attenuata and B. menziesii,
respectively (P ≤ 0.002), but was unaffected in E. todtiana (P = 0.635) (Fig 2.8). Fine root
percentage decreased in B. menziesii by 17% (P < 0.001) and was not significantly different
in B. attenuata or E. todtiana (P ≥ 0.335) (Fig 2.8). The average diameter of roots in the
compact treatment increased 19% to 0.88 mm in B. attenuata, and increased 26% to 0.96
40
mm in B. menziesii (P < 0.001), but the average root diameter of 0.76 mm in E. todtiana
was not significantly different from the control (P = 0.315). Maximum root depth was
reduced in the compact treatment by an average of 71% between all species before the roots
of the control could encounter the bottom of the pot 104 days after transplantation (P <
0.001) (Fig 2.8). After 304 days of growth in the 1.0 m pots, maximum root depth was 34
cm in B. attenuata, 35 cm in B. menziesii, and 42 cm in E. todtiana, with significantly
greater depth recorded in E. todtiana over both Banksias (P ≤ 0.011).
The root elongation rate in all three species decreased over the course of the
experiment in the compact treatment (P < 0.001) (Fig 2.14). From 104 days after
transplantation to 314 days after transplantation, the root elongation rate slowed 50% in B.
attenuata, 44% in B. menziesii, and 52% E. todtiana (Fig 2.13). On day 104, E. todtiana
exhibited a faster downward elongation rate than B. attenuata and B. menziesii (P ≤ 0.005),
and there was no difference between B. attenuata and B. menziesii (P = 0.937) (Fig 2.13).
On day 314, there was no difference in the downward root elongation rate between the
three species in the compact treatment (P ≥ 0.640) (Fig 2.13).
B. attenuata B. menziesii E. todtiana
Dow
nwar
d R
oot E
long
atio
n R
ate
(cm
m-1
day
-1)
0.000
0.002
0.004
0.006
0.008
0.010
0.012
After 104 days growthAfter 314 days growth
a a
b
c cc
Fig 2.13 Root elongation rates of B. attenuata, B. menziesii, and E. todtiana seedlings in the compact
treatment (soil bulk density = 1.80 g cm-3) after 104 days and 314 days after planting in 1.0 meter long pots.
Error bars represent standard error of the mean (n = 10), differences between species and treatment are
indicated as letters, P < 0.05.
41
Drought compact treatment
Physiology
All physiological traits during the first drought phase was significantly lower than
the control in all species (P < 0.001) (Table 2.2). Hydraulic conductance (ΨPD, ΨMD, ΨD,
and KL) of all species was significantly less in seedlings subjected to the drought compact
treatment (P < 0.001) (Table 2.3).
Morphology
The leaf surface area of B. attenuata and B. menziesii decreased 39% and 47%,
respectively (P ≤ 0.007), and did not significantly change in E. todtiana (P = 0.925) (Fig
2.8). The SLA of B. menziesii decreased 28% (P < 0.001), and did not significantly differ in
B. attenuata or E. todtiana (P ≥ 0.427) (Fig 2.8). Root to shoot ratio decreased 22% in B.
attenuata (P = 0.024), 29% in B. menziesii (P = 0.007), and 31% in E. todtiana (P = 0.017)
(Fig 2.8). The percentage of fine roots and the average root diameter did not differ from the
control treatment in any of the species (P ≥ 0.390) (Fig 2.8). Total root length decreased
30% in B. attenuata, and 53% in both B. menziesii and E. todtiana (P ≤ 0.001) (Fig 2.8).
Maximum root depth of seedlings in the drought compact treatment was restricted by an
average of 72% between all species before the roots of the control could encounter the
bottom of the pot 104 days after transplantation (P < 0.001) (Fig 2.8).
Discussion
Seedling survival in response to postmine conditions
The species in this study utilized different water-relation strategies to cope with
drought; both B. attenuata and B. menziesii seedlings are “drought-avoider water-savers”
with isohydric behavior, while E. todtiana seedlings are “drought-tolerant” (anisohydric)
(Levitt 1980; Tardieu and Simonneau 1998). There have been conflicting reports as to
whether isohydry or anisohydry is the more favorable strategy to survive drought, and there
is no justification for the environmental or evolutionary importance of possessing one
strategy over the other (Beis and Patakas 2010; Franks et al. 2007; McDowell et al. 2008).
This is supported by the similarities in survival between species and drought-coping
strategies in the drought or drought compact treatments. Reconstructed postmine soils do
not distinguish between isohydric and anisohydric species, and the heavily compact soil
disrupts natural physiological behavior by restricting maximum depth, placing the seedlings
42
at a greater susceptibility to drought stress when grown in compact soil (Kozlowski 1999;
Unger and Kaspar 1994; Zisa et al. 1980). Therefore these different drought-coping
mechanisms did not convey a survival advantage and allow one species to outperform the
others under postmine conditions.
Seedling response to drought
Evidence for species-specific drought-coping strategies
In this study, small decreases in soil moisture resulted in large decreases in both the
stomatal conductance and transpiration rate of leaves in the two Banksias. Immediate
stomatal closure allowed the seedlings to maintain a stable xylem water potential, and has
been described as the first line of defense in response to drought (Sperry and Pockman
1993; Tyree and Sperry 1989; Yordanov et al. 2000). The Banksias therefore exhibit
isohydric behavior to drought, preventing cavitation-induced seedling mortality (Tardieu
and Simonneau 1998). However this behavior can be detrimental to carbon acquisition over
prolonged droughts, given that stomatal conductance is highly correlated with drying soil
(Aranda et al. 2005; Jarvis and Davies 1998; Medrano et al. 2002b). Drought-induced
stomatal closure places the two Banksias at risk of carbon starvation because CO2
absorption and fixation is reduced throughout the entire drought period (Jarvis and Davies
1998; Medrano et al. 2002b). E. todtiana exhibited a different approach to drought by
employing an anisohydric behavior: operating at lower water potentials while maintaining
stomatal conductance rates at or near control levels for a longer period of time during
drought (Tardieu and Simonneau 1998). While anisohydry allows E. todtiana to withstand
lower water potentials, species are known to manage their xylem water potentials near the
point of complete hydraulic failure (Sperry et al. 2002). This places E. todtiana at a greater
risk of hydraulic failure during prolonged drought, but allows the seedlings to maintain
relatively greater carbon gain over an extended amount of time during drought (McDowell
et al. 2008).
Ecophysiological adaptations to drought
The observed relationship between photosynthesis and transpiration provides
evidence that the three species exhibit a range of WUE values, with B. attenuata achieving
the greatest WUE of the three species during water deficit. Higher WUE in dry
environments may be an adaptive trait for seedling survival, but the ecological advantages
43
could be compromised if these seedlings are grown in environments with seasonal
precipitation, where the competition for water is strong during the dry season (Cohen 1970;
DeLucia and Heckathorn 1989). Sands and Nambiar (1984) found that the productivity of
Pinus radiata seedlings transplanted in deep sands was greatly reduced in the first year of
planting because their shallow roots were unable to out-compete weeds for water. After a
second summer, these seedlings had developed a 2 m taproot that was able to access deeper
soil moisture (Sands and Nambiar 1984). Therefore a high WUE is only beneficial if a
conserved source of soil water is available for absorption later in the season (Cohen 1970),
i.e. groundwater access by a deep taproot. Thus there is a need for Banksia and Eucalyptus
seedlings to develop a deep taproot to extract water at depth, otherwise they would be
forced to compete for water in the same root zone as species that do not conserve water use,
placing them at a disadvantage when dry summer occurs.
While photosynthesis was regulated by stomata during drought, non-stomatal
limitations to photosynthesis were also observed in all species. In B. menziesii and E.
todtiana, proportional decreases in both photosynthesis and carboxylation efficiency were
observed as stomata gradually closed during drought, indicating that stomatal and non-
stomatal limitations are of similar importance to photosynthesis when the seedlings are
under moderate drought stress (Medrano et al. 2002b). Stomatal limitations were more
dominant in moderately drought-stressed B. attenuata seedlings, as smaller decreases in the
stomatal conductance of B. attenuata resulted in greater decreases in photosynthesis. Non-
stomatal limitations do not appear to be a dominant factor for B. attenuata until stomatal
conductance drops below 0.05 mol H2O m-2 s-1. Decreases in non-stomatal limitations to
photosynthesis may be a sign of increased water stress, and may indicate that the entire
photosynthetic processes of B. menziesii and E. todtiana are more readily damaged by
drought than that of B. attenuata, where there is integrated down-regulation of
photosynthesis (Medrano et al. 2002b).
Stomatal control over water loss could have allowed seedlings to maintain leaf area
throughout drought (Metcalfe et al. 1990; Rambal 1993). Changes in belowground biomass
in the Banksias, such as a higher root to shoot ratio, a greater percentage of fine roots, and a
higher total root length are advantageous in maintaining water status during droughts
(Ewers et al. 2000; Hacke et al. 2000; Hund et al. 2009). The root plasticity of the two
Banksias species increases the likelihood of survival during drought by maintaining a stable
water status (Tschaplinski et al. 1994). Root structure was maintained in E. todtiana
44
throughout the drought phases, but the morphology of this species appeared to be more
naturally suited to drought conditions, with relatively low leaf area, and a high root to shoot
ratio, total root length, and percentage of fine roots. All three species have relatively low
SLA that was not affected by drought stress, but differs between species, with B. attenuata
possessing the lowest SLA and E. todtiana the highest (Garnier et al. 2001; Li et al. 2005;
Poorter and Jong 1999). A low specific leaf area suggests a higher degree of sclreophylly
that may be beneficial for seedlings under water deficits by making leaves less prone to
wilt, allowing continuous water flow and photosynthetic rates (Hoffmann et al. 2005;
Marron et al. 2003; Turner 1994).
Both the Banksias and E. todtiana maintained a stable ΨD throughout the first and
second drought phases. This strategy has been classified as “isohydrodynamic”, and was
first described in E. gomphocephala, an anisohydric tree species that also inhabits the
geographic range of the three study species (Franks et al. 2007). To continually extract
water, seedlings must endure daily pressure variation in xylem conductance: from highly
stressed (more negative) during the day to relatively relaxed at night (Halvorson and Patten
1974). The isohydrodymanic behavior of both Banksia species and E. todtiana provides
overnight recovery of the seedling hydraulic conductance when evaporative demand is
lower (Gebrekirstos et al. 2006). When the seedlings cannot re-saturate overnight, ΨPD
values would approach ΨMD values, leading to xylem cavitation and a loss of water
absorption (Franks et al. 2007; Gebrekirstos et al. 2006). A larger and more stable ΨD
throughout dry periods could suggest greater daily recovery from drought stress and a
higher tolerance to long-term drought (Halvorson and Patten 1974; Sucoff 1971).
Seedling response during multiple droughts and re-watering
The physiological recovery during the prolonged re-watering period did not
guarantee seedling survival over multiple droughts. Despite the similarities in the rate of
water depletion between the first and second drought phases, B. attenuata, B. menziesii and
E. todtiana seedlings were more susceptible to a second round of drought. The regulation of
seedling physiology during times of drought was observed in all species, albeit to different
degrees, however physiological damage occurred more quickly and intensely during the
second drought phase. Although a full reversal of physiological damage was witnessed
during the recovery phase after the first drought, it is possible that irreversible damage
45
within the seedlings led to the accelerated mortality rates observed during the second
drought phase than the first.
Despite adjustments in gas-exchange, fluorescence, and hydraulic conductivity over
multiple droughts, the specific drought-coping strategies employed by the species in this
study appear be ill-equipped to deal with multiple rounds of drought. The “drought-avoider
water-saver” and “drought-tolerant” strategies are regarded as unsuitable to withstand
multiple rounds of drought during the investigation of drought-preconditioning techniques
for three species of woody Mediterranean seedlings (Vilagrosa et al. 2003). During a
second round of drought, a “drought-avoider water-saver” species (Juniperus oxycedrus)
and “drought-tolerant” species (Quercus coccifera) did not display benefits to
preconditioning, while a “drought-avoider water-spender” (Pistacia lentiscus) exhibited
increases in aboveground biomass, leaf water content, Fv/Fm, and higher water potentials
(Vilagrosa et al. 2003). Similar beneficial behavior was observed in the “water-spender”
carob (Ceratonia siliqua) (Lo Gullo and Salleo 1988), and may be induced by the greater
sensitivity of “water-spenders” to an initial round of drought (Vilagrosa et al. 2003).
However, this strategy requires an adequate soil water source to compensate for
maintaining a constant leaf water content and may not be suitable for soils with a deep, or
inaccessible water table (Lo Gullo and Salleo 1988). Therefore the “drought-avoider water-
saver” species of B. attenuata and B. menziesii, and the “drought-tolerant” E. todtiana,
most likely can only survive multiple droughts by accessing a reliable groundwater source
after the first summer.
Damage to the photosystem
Given that mortality did not begin to occur until water was restored during the
recovery phase, the seedlings appeared to have crossed a damage threshold during the first
drought phase where their inherent physiological capacity to recover is compromised.
Incomplete recovery after re-watering can be reflective of a damaged photosystem
(Miyashita et al. 2005), as the risk of photodamage increases as the stomata close and
excess light is unable to be utilized by the photosynthetic process, over-energizing and
damaging the photosynthetic apparatus (Kitao et al. 2003). Prior to severe drought (gs >
0.05 mmol m-2 s-1), all three species had higher ETR without adjusting NPQ, suggesting
that the dissipation of excess photochemical energy and maintenance of a higher fraction of
open centers in PSII is achieved not through thermal dissipation but through higher electron
46
flow (Cavender-Bares and Bazzaz 2004). However, extending the drought past 45 days
resulted in a breakdown in the mechanisms used to protect the photosystem as CO2
availability was reduced and seedlings shifted towards photorespiration (Medrano et al.
2002a). Photorespiration is not as efficient as photosynthesis in utilizing electrons and
because of this ETR decreases in response to the abundance of energy (Fig 2.11b) (Stryer
1988). At this point during water deficit, NPQ is expected to protect against photoinhibition
and photodamage by thermally dissipating any additional light energy, as low CO2
availability is known to induce trans-thylakoid ΔpH within chloroplasts, promoting an
increase in NPQ (Cavender-Bares and Bazzaz 2004; Cousins et al. 2002; Muller et al.
2001). Instead of increasing, NPQ was negatively affected by the prolonged drought, and
no relationship was observed to exist between drying soil and NPQ in any of the species.
Even without a reliance on NPQ, PSII efficiency (Fv/Fm) remained high in seedlings
of all species that survived over the first drought and recovery phases, even as
photosynthesis fluctuated in response to available water. PSII can be highly drought
resistant, and complete and permanent photoinhibition is not common even during severe
drought (Epron and Dreyer 1993; Medrano et al. 2002a; Yordanov et al. 2000).
Nevertheless, damage to the photosystem was recorded in specific individuals of all species
during the first drought and recovery phases, as seedlings that were defined as dead
measured an Fv/Fm value of 0. Previous reports have stated that recovery of photosynthesis
in some species is not possible if stomatal conductance reach values lower than the severe
drought threshold (gs < 0.05 mmol m-2 s-1) (Flexas et al. 2006; Quick et al. 1992). This was
not the case within our study species, as the recovery of stomatal-regulated physiology was
witnessed in seedlings even when stomatal closure exceeded severe drought values. It is
more likely that once extreme photodamage occurs in these seedlings, recovery of seedling
physiology after re-watering is not possible.
The severe drought experienced within 15 days during the second drought phase
lead to early decreases in photosynthesis, carboxylation efficiency, ETR, and NPQ, while
Fv/Fm approached 0 in all species. Such an intense and rapid decrease in physiology was
sufficient to kill the remaining seedlings of all species 48 days into the second drought
phase. Given Fv/Fm damage was only recorded late in the second drought phase, complete
photoinhibition in these species is most likely a response to an accumulation of damage to
other physiological traits within the seedlings. Nevertheless, the efficiency of PSII remains
intact until the seedling is on the verge of death and therefore should not be used as an early
47
indicator of the health of these specific seedlings under drought (Adams and Demmig-
Adams 2004; Bukhov and Carpentier 2004; Zivcak et al. 2008).
Damage to the hydraulic system
Within five days immediately following the first drought, quick recovery of
transpiration rates to control levels was observed in the two Banksias, while recovery of
transpiration took place within 17 days in E. todtiana. If seedling hydraulic conductance
did not have time to recover prior to an increase in transpiration, this would indicate that
water transpired by the leaves is being released from storage within cavitated xylem vessels
(Sperry and Pockman 1993). This could cause a positive feedback, further increasing the
loss of hydraulic conductivity, creating embolisms and cavitation with death following
soon after, and could explain the deaths recorded during the recovery phase (Sperry et al.
2002). However due to the destructive nature of water potential measurement on seedlings,
hydraulic properties were only able to be recorded 17 days after re-watering, and thus the
recovery of conductance in relation to leaf transpiration immediately following re-watering
could not be determined.
After the first drought phase, increased rates of gas-exchange during the recovery
phase restored KL in the two Banksia species, most likely due to factors such as the growth
of new roots or embolism recovery aided by predawn xylem relaxation (stable ΨD values)
(Franks et al. 2007; Gebrekirstos et al. 2006). However, a greater loss of hydraulic transport
was present in the two Banksia species during the second drought phase as evidenced by
relatively larger decreases in both midday water potential and soil-to-leaf hydraulic
conductance (KL). Hydraulic constraint has been shown to a signal for triggering stomatal
closure (Tyree and Sperry 1989; Sperry et al. 1998), and can also be species specific
mechanism (Aranda et al. 2005). E. todtiana was able to exhibit greater resistance to a loss
of KL throughout both the first and second drought phases and the recovery phase,
potentially due to the maintenance of relatively low transpiration rates compared to xylem
water potential.
Seedling response to simultaneous drought and increased soil compaction
Seedling root elongation was severely restricted by compact soil in all species, and
maximum root depth is generally considered to be an essential trait in determining water
extractive capabilities from the soil (Araki and Iijima 2005; Passioura 1988). The amount
48
of water available to the seedling roots during the first drought phase in this treatment
dropped below 0.03 cm3 cm-3, a value where water becomes unavailable to plants grown in
soil of the type used in this study (Carbon et al. 1982). Even though available water was
located at lower depths of the pots, the seedlings were unable to access this water because
of severely restricted roots. This lack of accessible water caused a complete failure of
hydraulic conductance in all species in a relatively short amount of time, leaving no
potential for recovery.
The isohydric nature of the two Banksia species can place them at risk of carbon
starvation during prolonged drought, while the anisohydric E. todtiana is more likely to
experience xylem cavitation (McDowell et al. 2008). However, it is possible for isohydric
and anisohydric species to experience both carbon starvation and hydraulic collapse, and
both will only occur when the intensity and length of drought is severe (McDowell et al.
2008). It is possible that a combination of both carbon starvation and xylem cavitation was
the cause of seedling mortality, given the rapid and extreme collapse in all physiological
values occurring during the first drought phase. The conditions of increased soil
compaction and drought, and therefore postmine soils, place B. attenuata, B. menziesii, and
E. todtiana seedlings at risk of both carbon starvation and xylem cavitation, when they are
only physiologically designed to accommodate one factor.
Belowground morphology was severely negatively affected by simultaneous
drought and soil compaction, with lower root to shoot ratio, total root length, and maximum
root depth recorded in all species. The modification of root morphological traits to aid soil
water uptake during drought (increased root to shoot ratio, total root length, and maximum
root depth) were unable to occur due to the extreme soil compaction (Ewers et al. 2000;
Hacke et al. 2000; Hund et al. 2009). Leaf area in the two Banksias was reduced, possibly
as an extreme strategy to reduce transpiration due to such rapid water loss, or from reduce
leaf growth through a lack of water (Metcalfe et al. 1990; Rambal 1993).
Seedling response to increased soil compaction
Despite the extreme restriction of root elongation and other belowground
morphological traits, seedling physiology was not impeded by compact soil, as increased
soil mechanical impedance by itself does not usually damage seedling function (Kozlowski
1972; Kozlowski 1999; Marquez 2010). Water available to the roots was occasionally
greater than that of the control, as water infiltration is not as rapid in soils with high bulk
49
densities (Kramer and Boyer 1995). Given the high physiological and hydraulic
performance of these seedlings in this treatment, seedlings can become established in
heavily compact soils if sufficient water was supplied (Marquez 2010; Zisa et al. 1980).
However, such water availability would need to occur through adulthood in this restoration
setting.
Fig 2.14 The roots of an E. todtiana seedling growing in a) compact soil (bulk density of 1.80 g cm-3) and b)
non-compact soil (bulk density of 1.55 g cm-3) after 132 days of transplanting in 1.0 m long pots. The
seedling grown in non-compact soil extended to the bottom of the pot.
In response to compact soil, the two Banksia species exhibited an average thicker
root diameter, which is a previously document morphological response to increased soil
compaction (Bengough and Mullins 1990; Materechera et al. 1992; Materechera et al.
1991). This response is thought to aid root elongation through mechanically impeded soil: a
root that expands radially can maneuver soil particles away from the root tip and expand
50
axially into the open space (Atwell 1993). Indeed, differences between the ability of species
to penetrate through compact soil have been related to root thickness, but this principle has
not been confirmed (Bengough and MacKenzie 1994; Bengough and Mullins 1990; Clark
et al. 2003; Materechera et al. 1992; Materechera et al. 1991; Misra et al. 1986). If an
increase in root thickness was the main driver or only reason in aiding elongation through
compact soil, the rate of root elongation of the Banksias would be faster than E. todtiana.
Although E. todtiana initially has a greater root elongation rate over the first 104 days after
transplanting the seedlings in the 1.0 m pots, over time the roots of all three species
elongated at similar rates, most likely due to the increase in soil compaction at greater
depths within the pots.
Root elongation is known to become inhibited at soil strengths of about 2 MPa
(Aggarwal et al. 2006; Allmaras et al. 1988), and this strength was encountered at
approximately the 15 cm depth within the pots. Maximum depth of the seedlings 304 days
after transplanting elongated past 15 cm in all species, and although root elongation rate
slowed throughout the experiment, it is apparent that these species can penetrate this soil
type at strengths greater than 2 MPa. The maximum root depth of E. todtiana, while greater
than both Banksias, is relatively shallow considering the unrestricted roots of these
seedlings in a natural setting can reach depths of ~1.5 m in the first year of growth (Rokich
et al. 2001).
Conclusion
In seasonally dry environments, drought-resistant mechanisms are critical during the
seedling establishment stage, and should be considered in restoration programs. Here we
define specific strategies to cope with drought for Banksia and Eucalyptus from the
biodiverse sandplains of southwest Australia. Ecological evidence from adult trees favors
one of the species in the study in field experiments (B. menziesii) (Groom et al. 2000; Muir
1983). Under drought-stressed conditions the seedlings of these species appear to behave
differently, as witnessed in previous studies (Donovan and Ehleringer 1991; Ehleringer and
Sandquist 2006). B. attenuata seedlings appear to be the most adapted to drought, with
higher WUE, an integrated down-regulation of photosynthesis, a low SLA, and adaptive
root architecture. However, these drought-adapted traits did not convey a survival
advantage. The physiological differences between species during drought were most
apparent in gas-exchange and water relation parameters (isohydric Banksia species versus
51
anisohydric E. todtiana); there was relatively little difference between species with regards
to chlorophyll fluorescence, or energy and heat dissipation values. This suggests that
isohydry and anisohydry, while using different physiological mechanisms, are equally
effective at coping with drought (McDowell 2008). The ability to offset photodamage may
be a more important factor in establishing seedlings under low water conditions.
When focusing strictly on seedling survival, it would be disadvantageous to design
restoration practices around one particular species, as found in this study from two
Banksias and one Eucalyptus species. The framework species method (Blakesley et al.
2001) is not applicable for restoring this particular mediterranean-type ecosystem in a
postmine setting, given there was no apparent survival favorite among these three dominant
overstorey species. A biphasic pattern of drought, as well as imposing simultaneous
drought and soil compaction was shown to be equally antagonistic to survival during the
seedling stage of these three phreatophytic evergreen trees, and must be addressed before
restoring the land from a postmine setting to its historical state. To resist and survive
drought, these species need to have unrestricted access to water during the critical seedling
stage within two years. Methods to supply seedlings with water, or more importantly,
alleviate compact soil and aid root development, are imperative to successfully restore
postmine sites in this biodiverse mediterranean-type environment.
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53
C H A P T E R T H R E E
Increasing soil water retention with native-sourced mulch improves
Banksia seedling establishment in postmine mediterranean sandy soils
Introduction
In the biodiversity hotspot of southwest Australia, up to 90% seedling mortality in
postmine restoration occurs during a seasonal drought, with the causes behind this decline
in mediterranean-type ecosystems such as southwest Australia (Schar et al. 2004), restricted
root development exposes seedlings to greater risk of water stress (Thompson et al. 1987),
and could further exacerbate the high mortality rate (Rokich et al. 2001). Soil restoration to
facilitate natural root development is therefore essential for successful restoration of a
postmine environment (Bradshaw 1997).
Sand quarries are often abandoned with minimal restoration effort, especially sites
mined prior to 1980 (Schroeder 1997). In southwest Australia, the sand quarry extraction
process reduces the overall soil profile from over 30 m to less than 5 m above the season
high groundwater level. Only the top 10 cm of the entire profile is retained for restoration
purposes as this topsoil contains many of the propagules (2621 seeds m-2 (Rokich et al
2000)), supporting organisms (fungal symbionts) and nutritional benefits required for
successful plant establishment. Removal of the natural soil profile has important
ramifications for plant growth in restoration and reconstruction of soil profiles to mimic
natural soil function may be important in optimizing seedling establishment and survival in
restoration.
Reconstructed soils can be manipulated to improve seedling establishment
(Bradshaw 1997), and soils can be mechanically ripped prior to seedling introduction to
reduce soil compaction and enhance root elongation (Ashby 1997). However, compaction
values in postmine sandy soils of the Bassendean dunes in southwest Australia are known
to revert to pre-ripped, root-inhibiting values (Rokich 1999); an uncommon occurrence in
soils that are predominately sand (Harper & Gilkes 1994). Unlike natural hardsetting, the
54
increased soil strength resulting from this “cryptic compaction” is not markedly reduced
when soils are re-wetted (Harper & Gilkes 1994; Rokich 1999), and techniques to alleviate
this phenomenon have not yet been trialed.
The application of soil amendments can be used to restore postmine soil by
overcoming loss of soil structure, restoring hydrological balance and mineral nutritional
capacity. However the effect varies with amendment type and interaction with the soil
environment, dependent on the biotic or abiotic characteristics of the degraded soil (Wong
2003). Broadly speaking, amendments are classified into organic or inorganic amendments.
Organic amendments, such as mulch or manure can resist compaction forces by stabilizing
soil aggregates or diluting the profile with a material of lower bulk density, as well as
increasing soil water retention (Barzegar et al. 2002). Inorganic amendments have been
more specifically designed to meet plant nutritional (eg. fertilizers), or
physical/hydrological (eg. porosity and drainage) requirements (Babalola & Lal 1977).
Here we examine the impacts of soil amendments and their underlying effects on seedling
morphology, physiology, and survival to understand and improve native seedling
establishment past the critical establishment stage in postmine sandy soils. Native mulch
and gravel are utilized as amendments to alleviate seedling response to the seasonal drought
of a mediterranean-type ecosystem and the “cryptic compaction” phenomenon. The
amendments are hypothesized to: 1) reduce soil strength and increase root elongation rate,
2) improve soil water retention and lessen the effects of drought on seedling function, and
3) replace soil nutrients lost during the mining process. Improvements in soil and seedling
health from the amendments will be reflected in greater seedling establishment over two
years, increasing the success of restoring postmine sandy soils in a mediterranean-type
environment.
Materials and Methods
Study Site
The experiment was conducted over two years (May 2009 – April 2011) within an
operational sand quarry 30 km northeast of Perth, Western Australia (Figs 3.1), where
mining operations removed 20 - 30 m of soil from the profile to 3 m above the water
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Fig 3.1 Satellite image from 2009 showing an overhead view of the sand quarry where the experiment was conducted. Differently-aged restoration areas, starting from
1999 onwards, are labeled by color. The experimental sites used in this study, both restoration and natural, are depicted on the image.
Fig 3.2 During mining operations within the study site. The scale and extent of soil removal (20-30 meters)
can be seen as mining vehicles extract the siliceous sands.
Fig 3.3 a) Pre- and b) postmine landscape of the Banksia woodlands within the Swan Coastal Plain.
56
57
table prior to the start of the experiment (Figs 3.2 & 3.3). The site is located within the
Bassendean dunes, characterized by low-nutrient, leached acidic podzols with high acidity
and low water holding capacities (Bolland 1999; Dodd & Heddle 1989; McArthur 1991).
Over 80% of the average annual rainfall of 800 mm is recorded between May and October,
and temperatures fluctuate between an average 35°C (February) and 5°C (July) (Bureau of
Meteorology 2012; Dodd & Heddle 1989) (Fig 3.4). Prior to mining, the native flora
consists of a dense shrubby heathland with a diverse mid-storey of woody shrubs and
herbaceous groundcover scattered with relatively few species of trees (Fig 3.3) (Dodd et al.
1984).
Winter Spring Summer Autumn Winter Spring Summer Autumn
Rai
nfal
l(m
m)
0
20
40
60
80
100
120
140
160 Rainfall April 2009 - April 2011Average Yearly Rainfall
Fig 3.4 Average rainfall at Pearce RAAF, 10 km north/northeast of the study site, indicating lower rainfall
during the two years of the experiment. Source: Bureau of Meteorology 2012.
Experimental Design
Three replicate sites (23.5 m x 9.5 m) were located within the area of the quarry
undergoing restoration. These ‘restoration sites’ each contained a control, organic
amendment, and inorganic amendment treatment plot, 6.5 m x 9.5 m, separated by a 2 m
buffer (Fig 3.5b). Three 6.5 m x 9.5 m replicate ‘natural sites’ were located in undisturbed
remnant Banksia woodland, separated at least 50 m by native woodland and cleared of
above-ground vegetation (Fig 3.5c). The organic amendment consisted of native brush
cleared from the quarry prior to soil extraction and crushed to create a mulch (≤ 5 cm size).
58
‘Blue metal’, a crushed basalt gravel of 10 mm size, was used as the inorganic amendment.
Blue metal is commonly used as a drainage medium consisting of magnesium and calcium
silicates with small quantities of potassium, phosphorus, and trace elements (Coventry et al.
2001). A front-end loader deposited 5 m3 of the organic or inorganic amendment over the
corresponding plot (Fig 3.5a). A 4-pronged traxcavator plowed the amendments to a depth
of 0.5 m to achieve a final concentration of 12% amendment to soil volume. This plowing
process was repeated in the control plot to maintain disturbances between treatments at the
restoration sites. Topsoil was then applied using leading practice: 10 cm of fresh topsoil
stripped from an adjacent intact Banksia woodland system after tree removal and was
immediately spread over each restoration site and ripped to a depth of 0.5 m using a
bulldozer operated tyne (Rokich 1999). The topsoil is characterized chemically as being
relatively high in nutrients compared to deeper horizons, although still considered nutrient
impoverished (extractable P < 2 mg/kg and K 15 mg/kg) and physically, as a loamy sand
(94% coarse sand, 1% clay) (McArthur 1991).
Fig 3.5 a) Spreading the organic and inorganic amendment in the restoration sites. b) Labeled restoration sites
prior to mixing the amendments to a depth of 50 cm. c) A natural site cleared of aboveground vegetation.
59
Study Species and Sowing
Banksia attenuata R.Br and B. menziesii R. Br are dominant, phreatophytic, and
evergreen trees of the region, routinely utilized as framework species in local restoration
programs (Dodd et al. 1984). Local provenance seeds were hand-sown 2 cm under the soil
surface in early May 2009. Seeds were sown randomly 0.25m apart, along 14 rows within
each plot. The rows were separated alternatively by 0.25 m and 0.5 m to allow access to
seedlings with minimal disturbance, and a 1-m buffer perimeter surrounded the rows on all
sides of each plot to assure seedlings grew within amended soil. A continuous 2 m × 6.5 m
rectangular area within each plot remained devoid of seeds to allow for soil-specific
measurements without disturbance to the seedlings.
Measurements
Soil Moisture
Soil moisture was recorded at four locations within each plot every two months
using a moisture probe meter (MPM-160-B, ICT International Pty Ltd). At each of the four
locations, four measurements were taken at depths of 0, 25, and 50 cm. A coarse white sand
obtained from the quarry was used to calibrate the moisture probe. The quadratic
polynomial equation (y = -2e-7x2 + 4e-4x; R2 = 0.98) was determined from the calibration
curve and converted the millivolts (x) to gravimetric soil moisture content (y)
(Anonymous).
Soil moisture was matched with rainfall data acquired from the nearest Bureau of
Meteorology site (Pearce RAAF), 10 kilometers north of the study site (Fig 3.4). Rainfall at
this site is characterized by a typical mediterranean-type climate, with 86% of rainfall
occurring during the winter/spring months of May to October (Dodd & Heddle 1989).
Soil Chemical and Physical Properties
The “cryptic compaction” was measured by recording soil impedance every two
months with a Rimik CP20II Cone Penetrometer (RFM Australia Pty Ltd, QLD, Australia,
Cone Diameter 12.83 mm; Area 130 sqmm). A minimum of ten readings were taken in
each plot at random locations, with measurements every 2 cm to a depth of 50 cm. Four
0.25 kg soil samples at depths of 0, 25, and 50 cm were collected at the end of the
experiment and analyzed by CSBP Ltd to test for pH and electrical conductivity (EC) in a