Electronic Supplementary Material
Competitive outcomes between marsh halophytes shift across stress
gradients: Greenhouse and field experiments
Hem Nalini Morzaria-Luna‡*. Department of Botany. University of Wisconsin-Madison.
Madison, WI, USA.
Joy B. Zedler. Department of Botany and Arboretum. University of Wisconsin-Madison.
Madison, WI, USA.
‡ Current
address: Marine Resources Assessment Group Americas Inc., 2725 Montlake
Blvd., E. Seattle, WA 98112. HemNalini.MorzariaLuna@noaa.gov. Ph: (206)861-7605.
Fax: (206)860-3394
*Corresponding author
Text S1. Environmental conditions in experimental units.
We measured water salinity in each unit, and soil surface interstitial salinity, soil
redox potential (Eh), and pH in each pot throughout the experiments to assess whether
our experimental treatments created the intended stress gradient. Soil interstitial salinity
was measured on the top 5 cm of soil as previously described. Five Eh measurements
per pot were made at 5 cm depth following the wet and dry hydroperiods using platinum
electrodes and a calomel reference electrode. Eh was calculated by adding the potential
of the calomel electrode against a standard hydrogen electrode (244 mV) to each
millivolt reading (Faulkner et al. 1989) and was not adjusted for pH. Concurrent with soil
Eh, we measured pH by mixing ~10 g of soil with 10 ml of DI water and using a Barnant
20 meter (Barnant Co., Barrington, IL. USA. Model 559-3800). At the time of harvest,
we collected soil samples to measure soil interstitial salinity in the bottom 5 cm of each
pot (as for surface salinity) and organic content of a pooled sample from the bottom and
top of each pot (as loss on ignition, (Ball 1964). We dried soil samples at 60°C, ground
them in a mortar and sieved with a 2-mm mesh, then burned 5 g in a furnace at 375°C
for 16 h and re-weighed.
To evaluate whether environmental conditions (i.e. Eh, soil surface salinity, water
salinity, and pH) drove species’ responses, we used generalized linear models to
perform regression analyses of available variables (means per pot) vs. total biomass.
The Akaike information criterion was used on the linear models to select the variables
that best explained the structure of the data (Venables and Ripley 2002).
a. Additive experiment (4 month)
We measured water and soil surface salinity monthly on October 2, November
10, December 12, 2002 and January 12, 2003. Eh and pH were measured at the end of
a dry and wet hydroperiod on October 31 and November 10. The soil surface in low and
medium water levels was hypersaline (95.5±1.4 g l-1 and 69.7±3.9 g l-1) and higher than
in high water (35.9±2.8 g l-1; Table S1; F =211.6, P <0.001). Salinity in the bottom of
pots, measured at the end of the experiment, was on average 38.6±1.4 g l -1 lower than
surface salinity. Over the course of the experiment surface salinity decreased ≤ 80%
from initial levels (F =88.2, P <0.001). Water salinity was negatively related to soil
surface salinity (F =4.0, P <0.001). Eh was oxidizing (>400 mV) following both
hydroperiods, weakly reducing in high water levels (382.7±7.6 mV) and oxidizing in
medium and low water (437.1±7.3 and 471.2±6.9 mV; Table S2). Soil pH increased with
water level (low 7.1±0.1 to high 7.5±0.5; Table S2; F =15.7, P =0.0004). Soil organic
content was negatively related to water levels (low 31.8±0.9 to high 26.3±0.5; Table S3;
F =8.9, P =0.001). Total biomass per pot was positively related to water salinity, which
increased as soil surface salinity decreased (F =19.4, P =0.0004). This pattern differed
from the replacement experiment, where total biomass was positively related to soil
surface salinity.
a. Additive experiment (1 year)
We measured water and soil surface salinity on October 2, November 10,
December 12, 2002, January 12, 2003, March 14, June 6, and August 31, 2003. Eh, and
pH were measured at the end of dry and wet hydroperiods on October 31 and
November 10, 2002 and August 24 and September 10, 2003. Soil surface salinity was
negatively related to water level (F =96.7, P <0.001; Table S1). The soil surface was
hypersaline in low and medium water levels (87.4±2.8 and 72.7±3.8 g l-1), and salinity
decreased in high water (32.0±3.0 g l-1). Salinity in the bottom 5 cm of pots measured at
the end of the experiment was 42.7±8.2 g l-1 lower than surface salinity (F =5.5, P
=0.04). Soil surface and water salinity decreased 25-75% over the course of the
experiment (soil F =12.3, P <0.001; water F =186.2, P <0.001). Water salinity showed a
positive relationship with water level (F =17.1, P =0.003). Eh was weakly reducing
following the wet hydroperiod (398.3±5.9 mV; F =40.2, P <0.001) and in high water
(378.2±7.9 mV; F =30.4, P <0.001; Table S2); in other treatments Eh was oxidizing
(>400 mV). pH increased with water level (low 6.9±0.1 to high 7.2±0.04; F =7.01, P
=0.03; Table S2). Soil organic matter was on average 22.5±0.6% and did not differ
among water levels (Table S3). Total biomass per pot was negatively related to water
salinity, which increased as soil surface salinity decreased (F =7.3, P =0.03).
b. Field additive experiment (4-month)
We assessed soil surface salinity and organic matter infrequently. Marsh-plain
soils are often too dry to allow direct tests of interstitial water salinity, so we collected
soils cores (5-cm depth) on December 12 and May 19, dried them at 60°C, and then
added DI water to make a uniform saturated soil paste (Richards 1954). We measured
soil salinity as in the greenhouse. Soil-paste salinities can be lower than interstitial soil
salinities (PERL 2001). We determined organic content as loss on ignition of soil
samples collected December 12, 2002. We saw no difference in surface soil salinity
between low and high elevations in our field experiment (Table S1). However, salinity
was higher in May (77.4±5 g l-1) than in December (62.5±3.2 g l-1; F =6.5, P =0.02). Soil
organic matter was higher at low elevation (30.5±2.5%) than high elevation (18.4±1.7%;
Table S3).
Text S2. Detailed results from experimental treatments.
Greenhouse experiments
We collected fruits produced by Triglochin throughout each experiment and
recorded the number of spikes and fruits per spike (each fruit has ~6-8 seeds) (Table
S4). After harvesting, we milled shoots and roots separately to pass a 40 mesh (420-µm
openings). In each experiment, we analyzed a sub-set of tissue samples corresponding
to extremes in treatment combinations for total N and organic content. Total N was
determined by Kjeldahl digestion at the UW Soil and Plant Analysis Lab. Organic
content in plant tissue was determined as loss on ignition by burning one gram of tissue
at 500°C for 4 h and re-weighing to determine organic content. Percent organic content
was used to correct above- and belowground biomass to ash-free weight.
Field experiments
Plant material collected at the end of the experiment was washed, dried, and
milled to determine tissue total N and organic matter content as described above.
Data analysis
For each experiment, we assessed treatment effects by comparing root:shoot
ratio and tissue total N. In the greenhouse, we also examined seed production, soil
organic matter, soil salinity, Eh, and pH. The factors used in the model were N supply,
water level (or planting locations in the field experiment), and planting (monoculture and
mix). We assessed normality of the residuals using a Q-Q plot and homogeneity of
variance with Levene’s test. When necessary, we transformed count data using x’ =√x +
⅜ and log-transformed shoot:root ratios to stabilize the variance. We used linear mixed
effects models with interactions in the greenhouse experiments to test the effects of
treatment factors on root:shoot ratio, and tissue N content. The field experiment was
analyzed using a two-way ANOVA with interactions; block was specified as a factor.
ANOVA posthoc comparisons were analyzed using Tukey’s Honest Significant
Difference (HSD) test on a model with no random effects. Fruit counts were analyzed
using a Poisson generalized linear model (GLM). All data were analyzed using R
version 2.0.1 (R Development Core Team) and the packages MASS (Venables and
Ripley 2002) and nlme (Pinheiro et al. 2004).
Means±1 standard error are reported in the text. Hereafter, species grown in
monotype are referred to as Salicorniamono and Triglochinmono; those grown together are
abbreviated Salicorniamix and Triglochinmix.
a. Additive experiment (4-month)
Triglochin monoculture produced more fruits than plants grown in mix
(563.7±129.1 vs 98.2±38.3 fruits pot-1; Table S5). There was a significant effect of N
supply and water levels on Triglochin seed production (Table S5). In general, Triglochin
seed set was greater in fertilized than unfertilized pots and greater with high than low
water (P <0.001).
For both species, the ratio of below- to aboveground biomass was highest when
no N was added (Table S6; F =7.9, P <0.001). The root:shoot ratio of Triglochin
(monoculture 0.5 and mix 0.6) was higher than the ratio of Salicornia (0.1 monoculture
and mix; F =126.9, P <0.001). There was no effect of water level treatments on
root:shoot ratio.
Our analysis of tissue N content in a subset of samples (Fig. S2; both N-supply
treatments in low and high water levels) showed the same pattern for shoot and root
samples. When plants were grown in mix, N content was higher in fertilized pots relative
to unfertilized; there was no difference between fertilized and unfertilized pots when
species were grown in monotype (F =48.21, P <0.001). Nitrogen in tissue of plants
grown in mix increased 20-35% with N addition. Overall, the ranking in N concentrations
between planting treatments was Triglochinmono, 17.9±1.3>Triglochinmix,
16.9±1.1>Salicorniamono, 12.8±0.5=Salicorniamix, 12.3±0.7 mg g-1. The differences
between planting treatments were significant only for fertilized plants (F =48.2, P
<0.001). Only Triglochin responded to water level, its N content was greatest in high
water for both planting treatments (F =3.7, P =0.02). We found that Salicornia had
greater shoot than root N content in both planting treatments. While Triglochinmono root
N content was higher than shoot content, there was no difference for Triglochinmix (F
=32.3, P <0.001).
b. Additive experiment (1 year)
Triglochin produced on average 287.6±67.6 fruits pot-1 in the monoculture and
355±49.3 fruits pot-1 in the mix; and fruit production was a function of water level, N and
planting treatments (Table S5; P <0.001). The root:shoot ratio of Triglochin in both
planting treatments (monoculture 1.7 and mix 1.8) was higher than for Salicornia (0.4
monoculture and mix; F =88.8, P <0.001). There was no effect of water level on
root:shoot ratio of either species (Table S6).
Shoot N content was higher for Triglochin than Salicornia in both planting
treatments (Triglochinmono 1.0±0.1 and Triglochinmix 0.9±0.1 >Salicorniamono 0.7±0.03
and Salicorniamix 0.6±0.1; Fig. S3; F =22.8, P <0.001); there was a significant interaction
with water level (F =3.16, P <0.02). In general, N content was higher in shoot tissue
from plants grown in high water levels relative to medium and low (F =42.12, P <0.001);
we observed the same pattern in root tissue (F =3.1, P <0.001). For both species, root
N content of monocultures was higher than for mix (Salicorniamono 1.3±0.1 and
Triglochinmono 1.0±0.04 >Salicorniamix 0.8±0.1 and Triglochinmix 0.8±0.1; F =23.5, P
<0.001). Salicornia root N was higher than shoot N content in both planting treatments;
there were no differences between shoot and root N for Triglochin (F =24.0, P <0.001).
c. Field experiment (1 year)
Root:shoot biomass ratios for Triglochinmono (2.1) and Triglochinmix (2.9) were
higher than for Salicorniamono (0.6) and Salicorniamix (0.5; F =54.3, P <0.001) and higher
than in greenhouse experiments. Note that few Triglochin shoots remained at the time
of sampling. Overall, root:shoot ratios were higher without N added than with N (Table
S6; F =14.0, P <0.001). The root:shoot ratio was also greater at high elevation than low
elevation (Table S6; F =14.0, P <0.001).
Shoot N content was consistently higher in plots with N added than without (Fig.
S4; F =61.7, P <0.001). Triglochin shoot N content was higher than for Salicornia
(Triglochinmono 3.4±0.02 and Triglochinmix 3.0±0.2 >Salicorniamono 1.3±0.1 and
Salicorniamix 1.3±0.1; F =23.5, P <0.001). Salicorniamono root N also increased with N
addition, and there was no significant effect of N supply on Salicorniamix or either
Triglochin planting treatment (F =23.6, P <0.001). Overall, Triglochin root N
concentration was higher than that of Salicornia (Triglochinmono 1.3±0.1 and Triglochinmix
1.1±0.1 >Salicorniamono 0.8±0.1 and Salicorniamix 0.7±0.1; F =23.5, P <0.001); there was
a significant interaction between N-supply and planting treatment (F =23.6, P <0.001).
There was no effect of elevation on either shoot or root N content. For both species,
average N content was higher in shoots than roots (F =277.2, P <0.001).
References
Ball DF (1964) Loss-on-ignition as an estimate of organic matter and organic carbon in
non-calcareous soils. Journal of Soil Science 15: 84–92.
Faulkner SP, Patrick WHJ, Gambrell RP (1989) Field techniques for measuring wetland
soil parameters. Soil Science Society of America Journal 53: 883–890.
PERL (2001) 2001 Annual report on ecosystem monitoring. Tijuana River Estuary
National Estuarine Research Reserve. Pacific Estuarine Research Laboratory
Pinheiro J, Bates D, DebRoy S, Sarkar D (2004) nlme: Linear and nonlinear mixed
effects models. R package version 3.1-52.
Richards LA (1954) Diagnosis and improvement of saline and alkali soils. Agriculture
Handbook No. 60, United States Department of Agriculture
Venables WN, Ripley BD (2002) Modern applied statistics with S. Springer, New York
Table S1. Soil salinity (mean±SE g l-1) measured in the top (surface) and bottom 5 cm
of each pot between water level treatments and planting locations. Surface salinity was
measured throughout the experiment and bottom salinity at the end of the experiments;
n =sampling dates. Salinity in the greenhouse experiments is interstitial salinity and field
salinity is soil paste. (***) indicates significant variables (P <0.0001).
Water level
n
High
4-month greenhouse experiment
Bottom
1 15.4 ± 1.1
Surface
4 35.9 ± 2.8
1-year greenhouse experiment
Bottom
1 21.3 ± 3.0
Surface
8 32.0 ± 3.0
1-year field experiment
Locations
Low
Surface
2 64.4 ± 3.9
Medium
Low
20.7 ± 2.2 19.8 ± 2.5
69.7 ± 4.0 95.5 ± 1.4
5.9 ± 0.5 9.4 ± 1.2
72.7 ± 3.8 87.4 ± 2.8
High
75.6 ± 4.8
Table S2. Soil redox potential (mV) and pH from the two greenhouse additive experiments. Data are means±SE between
water level, planting treatments and hydroperiods (see Fig. S1). Measured following wet and dry hydroperiods; n
=sampling dates.
Experiment
Factor
n
Planting
Monocultures
Salicornia
Triglochin
Mix
Water level
Low
Medium
High
Tide
Spring
Neap
4-month
Redox (mV)
pH
2
1-year
Redox (mV)
4
432.6 ± 7.9
426.1 ± 7.3
432.3 ± 8
7.3 ± 0.1
7.3 ± 0.1
7.3 ± 0.1
410.0 ± 8.3
43.1 ± 8.1
433.8 ± 8.4
7.2 ± 0.1
7.1 ± 0.1
7.0 ± 0.1
382.7 ± 7.6
437.1 ± 7.3
471.2 ± 7.0
7.6 ± 0.1
7.1 ± 0.1
7.1 ± 0.1
378.2 ± 7.9
444.7 ± 8.1
455.0 ± 7.6
7.2 ± 0.04
7.2 ± 0.1
6.9 ± 0.1
410.6 ± 6.1
450.1 ± 6.4
7.47 ± 0.1
7.12 ± 0.1
453.7 ± 7.1
398.3 ± 5.9
7.0 ± 0.1
7.2 ± 0.04
pH
Table S3. Soil organic matter (mean±SE %) from the greenhouse additive experiments between water level, N-supply,
and planting treatments. Organic content obtained by loss on ignition for samples collected at the end of the experiment.
N was added monthly in the 4-month experiment and once (15 g N m-2) at the start of the 1-year experiment.
Experiment
4-month
N addition (g m-2)
15
0
Water level
Salicornia monoculture
Low
26.8 ± 2.7 30.2 ± 0.2
Medium
29.7 ± 1.1 24.3 ± 0.6
High
35.0 ± 2.21 30.9 ± 1.4
Triglochin monoculture
Low
24.3 ± 1.6 30.1 ± 1.17
Medium
25.3 ± 4.3 29.2 ± 1.7
High
31.1 ± 3.2 28.8 ± 2.4
Mix
Low
25.4 ± 0.4 25.6 ± 1.0
Medium
23.8 ± 2.6 23.9 ± 1.8
High
24.3 ± 2.9 24.8 ± 3.4
1-year
20.9 ± 1.4
21.6 ± 1.7
24.5 ± 2.2
21.4 ± 1.1
22.4 ± 1.0
26.8 ± 1.9
21.7 ± 2.1
19.6 ± 1.0
24.1 ± 2.8
Table S4. Organic tissue content of plants grown in the greenhouse and field additive
experiments. Table indicates percent organic matter (mean±SE) determined by loss on
ignition. There were significant (P <0.05) differences between planting treatments in
every experiment except the 1-year greenhouse experiment.
Monoculture
Mix
Species Salicornia Triglochin Salicornia Triglochin
Experiment
4-month
greenhouse
1-year greenhouse
1-year field
70.2 ± 2.1
74.5 ± 1.4
67.8 ± 2.5
75.9 ± 2.4
73.8 ± 1.2
62.9 ± 1.9
82.2 ± 1.6
74.9 ± 1.3
79.6 ± 3.2
73.6 ± 2.6
80.5 ± 3.4
74.8 ± 1.6
Table S5. Triglochin fruit production (means±SE fruits pot-1, n =3) in the greenhouse additive experiments. Results
shown for plants grown in monotype and mix, between water level by N-supply treatments. N was added monthly in the 4month additive experiment, and once at the start of the 1-year experiment. Significant factors (P <0.05) are indicated
following the name of the experiment.
Planting treatment
Monoculture
-2
N supply (g m )
0
15
4-month experiment
[Water level, N supply, and planting]
Low water level
61 ± 34.1
869.7 ± 31.1
Medium
70 ± 13.4
893 ± 113.6
High
95.3 ± 28.1
1393.3 ± 177.9
1-year experiment
[Water level and planting]
Low water level
262.3 ± 72.1
Medium
280.3 ± 204.6
High
320.3 ± 83.5
Mix
0
15
20.7 ± 12.1
0
36.7 ± 20.5
178 ± 97.9
59.3 ± 52.5
294.4 ± 170.9
453 ± 20.5
276 ± 95.6
336 ± 107.3
Table S6. Root:shoot ratios (mean±SE, n =3) from the greenhouse experiments. Data
for plants grown in monoculture and mix, between water level and N-supply treatments.
N was added monthly in the 4-month additive experiment and once (15 g N m -2) at the
start of the 1-year additive experiment. There were significant (P <0.05) differences
between N-supply treatments in the 4-month experiments and among planting
treatments in all experiments.
Water level
4-month additive experiment
Salicornia
Low
Medium
High
Triglochin
Low
Medium
High
Salicornia
Low
Medium
High
Triglochin
Low
Medium
High
N addition (g m-2)
Monocultures
15
0
15
Mix
0
0.1 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
0.3 ± 0.01
0.2 ± 0.01
0.2 ± 0.02
0.1 ± 0.04
0.1 ± 0.03
0.1 ± 0.01
0.3 ± 0.1
0.2 ± 0.03
0.3 ± 0.1
0.2 ± 0.02
0.2 ± 0.01
0.2 ± 0.02
0.8 ± 0.01
0.7 ± 0.04
0.6 ± 0.03
0.4 ± 0.1
0.5 ± 0.1
0.4 ± 0.04
0.6 ± 0.02
0.7 ± 0.1
0.6 ± 0.02
0.2 ± 0.01
0.3 ± 0.03
0.3 ± 0.02
0.4 ± 0.1
0.3 ± 0.03
0.3 ± 0.01
0.6 ± 0.02
0.7 ± 0.1
0.6 ± 0.02
0.6 ± 0.02
0.7 ± 0.1
0.6 ± 0.02
Figure S1. Hydrograph depicting the water level treatments = high (–), medium (–), and
low (▬), in the 4-month and 1-yr greenhouse experiments. In each treatment there were
two hydroperiods; each regime was repeated for two weeks.
Figure S2. Tissue N content from the greenhouse additive experiment (4-month). Data
are means for Salicornia virginica (Sv) and Triglochin concinna (Tc) in monotype (mono)
and in mix pots (mix); error bars are SE. Data for water level treatments by N supply, 15
g m-2 and no addition (0). Biomass is dry weight per plot.
Figure S3. Tissue N content from the greenhouse additive experiment (1-year). Means
for Salicornia virginica (Sv) and Triglochin concinna (Tc) in monotype (mono) and in mix
pots (mix); error bars are SE. Data for water level treatments, low (L), medium (M) and
high (H). N was added once as 15 g m-2 at the start of the experiment. Biomass is dry
weight per plot.
Figure S4. Tissue N content in the field additive experiment (4-month). Data are means
for Salicornia virginica (Sv) and Triglochin concinna (Tc) in monotype (mono) and in mix
plots (mix); error bars are SE. Data for locations of different elevation on the marsh
plain: low and high (0.6±0.01 and 0.8±0.02 m NGVD) by N-supply treatments, 15 g m-2
and no addition (0). Biomass is dry weight per plot.