5.2.2 Seed viability
Viability testing followed procedures outlined by the Association of Official Seed
Analysts (1990), except that the number of seeds per replicate run was increased from
25 to 100 to improve statistical rigour. A small pilot study was carried out before the
main trials to determine statistical power and the number of replicates needed to
detect significant responses to the environmental variables (Zar 1999). These trials
indicated that four replicates, each using 100 seeds, yielded a power of >0.99.
104
Seeds were surface sterilized by placing them in small sealed muslin bags and
plunging the bags in 10 % W/V sodium hypochlorite solutions for 20 seconds and
then rinsing them three times in distilled water. For each viability-trial replicate, 100
seeds were evenly spaced in a grid pattern on a disc of Whatmans #3 filter paper
(Whatman laboratory Division, Maidstone, Kent, England) in a 9-cm diameter petri
dish. Each paper disc was wetted with 8 mL of distilled water and the dish sealed
with laboratory film to reduce moisture loss. A total of four replicates (i.e., 400
seeds) was used for each viability test, which were undertaken in growth cabinets with
daytime temperatures of 20
o
C and night temperatures of 10
o
C. A 12:12 hour
light:dark cycle was used. Light was provided by a bank of fluorescent tubes
designed for hydroponic use, that emitted a PAR of 40
μ
mol m
-2
s
-1
measured
at the
level of the seeds. All replicates were shuffled daily within the cabinet to randomise
placement effects. Germination was measured after 7, 14 and 21 days. The trial was
terminated at 21 days because no additional germination was recorded after Day 14.
Germination was judged by the emergence of the base of the hypocotyl from the testa.
The weight of individual seeds was determined by counting the number of seeds in a 1
mg subsample (Association of Official Seed Analysts 1990). The average number of
seeds was calculated from the mean of the aggregated totals of five subsamples.
5.2.3 Interactive effects of salinity, light and temperature on germination
Seed was sorted to ensure potential “germinability” by removing any obviously
unfilled seeds (i.e., those not containing embryos). Unfilled seeds were identified and
discarded on the basis of their noticeably paler colour and transparency when viewed
105
with back-lighting under a dissecting microscope. As with the seed viability trials,
seed was surface sterilized with 10 % sodium hypochlorite solution for 20 seconds,
then rinsed three times in distilled water.
The set of environmental conditions used for laboratory trials was chosen to mimic
the range of conditions occurring in the field. For example, water-column salinity at
Dowd Morass varies from near fresh (< 0.2 g L
-1
) to over half seawater (19 g L
-1
), so
the suite of laboratory salinities not only covered this range but extended to a full
seawater treatment. Long-term climate records show the mean daily air temperature
at the nearby Sale East military base to range from a daily minimum of 3
o
C in July to
a daily maximum of 25
o
C in January and February (Bureau of Meteorology 1988).
The following conditions were used to assess prime effects and interactions among
the three environmental variables of salinity, light and temperature: i) salinity (0, 1, 2,
4, 8, 16 or 32 g L
-1
, made up with a commercially available sea-salt mixture (Red Sea
Heidelberg Aquarium Supplies, Melbourne, Victoria); ii) darkness (constant darkness
versus a 12:12 hr light:dark cycle at a PAR of 40
μ
mol m
-2
s
-1
); and iii) temperature
(constant 10
o
C, 20
o
C or 30
o
C).
Four replicate petri dishes were used per light/temperature/salinity treatment, with 100
seeds per petri dish. As in the seed viability trials, seeds were arranged in a grid
pattern on a Whatmans #3 filter paper disc and covered with 8 mL of water at the
appropriate salinity for each trial. In total, 168 petri dishes were used and 16,800
seeds tested.
106
5.2.4 Effects of preliminary exposure to salt on germination
Approximately 16 mg of sterilised seed, representing about 100 potentially
germinable seeds, were placed in 50 mL containers filled with various concentrations
(0, 1, 2, 4, 8 or 16 g L
-1
) of reconstituted sea water as described above and soaked in
the dark at 20
o
C for a range of time periods (1, 2, 4, 8 or 16 days). At the end of each
period of preliminary saline exposure, seed in each container were washed three times
with distilled water and sown with distilled water as per the viability tests described
earlier. Germination was recorded on the day seeds were transferred to distilled water
and for each subsequent day until no further germination occurred.
5.2.5 Effects of seed burial and substrate type on germination
Approximately 16 mg of seed were sown in 15-cm diameter pots filled with milled
peat moss (Nature Land Brand, TAS Seaweed Pty. Ltd, Devonport, Tasmania). Pots
were filled with substrate to within 1 cm of the top of the pot and gently tapped down
to even the surface. All pots were thoroughly wetted and then stood in 6 cm of
distilled water in trays and placed in a lightly shaded glasshouse at the Trust for
Nature (Cottlesbridge, Victoria). Seed was scattered evenly over the surface and then
either left uncovered or covered with 1, 3 or 6 mm of just-damp peat moss passed
through a 1-mm mesh-size sieve. All pots were lightly misted with a spray bottle and
plastic sheeting placed over the top of the pots to minimise evaporation. Pots were
checked daily for germination for 21 days, germination being determined by the
emergence of cotyledons. Six replicate pots for each burial treatment were used.
107
To check for the effect of different substrate type on germination, additional
pots were established using three different substrates: i) clay (taken from a freshwater
swamp containing M. ericifolia at Cades Road, Whittlesea, Victoria); ii) washed river
sand; and iii) peat moss. The protocol followed that given above, except that only
surface-sown seed was used.
5.2.6 Statistical analysis
Data were analysed with Analysis of Variance (ANOVA) with the SPSS (version 12;
http://www.spss.com/, verified September 2006) and Systat (version 5;
http://www.systat.com/, verified September 2006) computer packages. Percentage
data were arc-sine transformed before analysis. One-way and three-way fully
orthogonal ANOVA designs were used for analysis. Since all factors considered as
“fixed”, treatment effects were calculated with reference to the MS residual (error)
term (Zar 1999). Post-hoc tests used Bonferonni-corrected probability values.
108
5.3 Results
5.3.1 Viability
The seed viability of M. ericifolia was 6 %. Each milligram of seed contained about
560 seeds, indicating that the average seed weight was 0.0018 mg.
5.3.2 Interactive effects of salinity, light and temperature on germination
Individually, light, salinity and temperature all exerted highly significant ( P < 0.001)
effects on germination (Table 5.1). Since all interaction factors were highly significant
( P<0.001), it is impossible to generalise about individual main effects without
reference to the qualifying effects of other main effects. Nevertheless, some trends
can be detected. For example, at 30
o
C there was a consistent and rapid decrease in
percentage germination with increasing salinity (Figure 5.1). No germination was
observed at a salinity of 16 g L
-1
, and only about 5 % of seeds germinated at 8 g L
-1
at
this temperature. In comparison, nearly 50 % germination was observed for seeds at
30
o
C in fresh water. Seeds at 30
o
C had consistently higher germination at all
salinities in constant darkness than they did in a 12:12 hour light:dark cycle.
At 20
o
C, seeds showed maximum germination success (40-50 %, depending on light
conditions) at a salinity of 1 g L
-1
and germination fell at a roughly constant rate with
increasing salinities. Although about 10 % still germinated at a salt concentration of
16 g L
-1
, no seeds germinated at a salinity of 32 g L
-1
. Unlike the case at the highest
109
incubation temperature, seeds at 20
o
C showed statistically significantly higher
percentage germination in the light:dark cycle than in complete darkness.
The percentage of seeds germinating at 10
o
C was about one half of that at 20
o
C at a
given salinity. The maximum germination at 10
o
C (about 20 %) was observed with
seed kept in the light:dark treatment over the three lowest salinities (i.e., 0 to 2 g L
-1
).
Germination success fell regularly, but slowly, at higher salinities and, as before, no
germination was observed at a salinity of 32 g L
-1
. Nevertheless, about 5 % of seeds
still germinated at 16 g L
-1
at 10
o
C, a result in strong contrast to that found for seeds at
30
o
C. There was a very strong effect of light:dark on seeds incubated at 10
o
C, with
seeds in the dark treatment showing about one-half the germination success of those
kept in alternating light:dark cycles.
110
Table 5.1 Results of three-way ANOVA of primary and interactive effects of
salinity, light and temperature on the germination of M. ericifolia seeds.
Source
SS
df
MS
F-ratio P
Main effects
Temperature
0.424
2
0.212
658.658 < 0.001
Salinity 2.450
5
0.490
1521.19
<
0.001
Light regime
0.025
1
0.025
76.477 < 0.001
Interaction terms
Temp*salinity
0.491
10
0.049
152.520 < 0.001
Temp*light
0.049
2
0.024
75.543 < 0.001
Salinity*light
0.021
5
0.004
12.886 < 0.001
Temperature*salinity
*light
0.082
10
0.008
25.317 < 0.001
Residual
(error) 0.035
108
0.00032
111
Figure 5.1 Effects of temperature, salinity and light regime on the germination of M.
ericifolia seeds. Means are shown (n = 4): standard errors are smaller than the
symbols used.
112
5.3.3 Effects of preliminary exposure to salt on germination
Figure 5.2 shows the effects of preliminary exposure to salt for periods of up to 16
days, followed by exposure to freshwater conditions, on germination success across
six initial salinities, starting with freshwater conditions (Figure 5.2 a) and finishing
with soaking in a saline solution of 16 g L
-1
(Figure 5.2 f). Four main results are
evident from this experiment.
First, seeds could germinate in highly saline solutions, a result confirming the
observations in the previous experiment where seed was exposed to the saline solution
from the beginning of the experiment and not subsequently exposed to freshwater
conditions (Figure 5.1). In all treatments, seeds started to germinate even when they
were soaking in the saline treatment solution, and germination continued after they
were washed, transferred to paper filter discs and exposed to freshwater conditions.
For example, 41 % of seeds had germinated in the saline treatment solution after 8
days of exposure to 4 g L
-1
. Transfer of the seeds to freshwater conditions on filter
discs resulted in an additional ~20 % of seeds germinating within 1 day, and
eventually 80 % of seeds germinated despite their earlier exposure to salt at 4 g L
-1
for
over one week (Figure 5.2 d). Similarly, 17 % of seeds had germinated in the 8 g L
-1
treatment solution after 8 days, and germination increased after transfer to freshwater
conditions such that nearly 75 % of seeds had germinated the end of the experiment
(Figure 5.2 e). Even seeds exposed to the highest salt concentration (16 g L
-1
) showed
some germination in the treatment solution (5 %) and ultimately about 58 % of seeds
germinated after transfer to freshwater conditions despite this severe earlier salt
exposure.
113
Second, and a corollary of the first conclusion, seeds continued to germinate in
subsequent freshwater conditions even after lengthy earlier exposure to the highest
salinity. Nearly 15 % of seeds had germinated in the 16 g L
-1
treatment solution after
16 days, and this rate increased after their transfer to freshwater conditions such that
nearly 50 % of seeds had germinated after 6 days in fresh water (Figure 5.2 f).
Third, despite the ability of seeds to germinate in saline solutions and increase their
germination rate after transfer to freshwater conditions, salt exerted a strong inhibitory
effect on the ultimate rate of germination. All seeds germinated in freshwater
conditions and at a salinity of 1 g L
-1
(Figure 5.2 a, b); this progressively fell to about
95 % germination at 2 g L
-1
(Figure 5.2 c), 80 % at both 4 g L
-1
and 8 g L
-1
(Figure 5.2
d, e), and 50-75 % at 16 g L
-1
(Figure 5.2 f).
Fourth, there was some evidence that soaking seeds in freshwater increased the
germination speed (Figure 5.2 a). Seeds soaked in fresh waters for 1 day prior to
transfer to filter papers showed slower germination (initiated on Day 3) than did seeds
soaked for 2 days (initiated on Day 2). However, overall germination rates under both
treatments on Day 3 were similar.
114
Figure 5.2 Effects of prior exposure to saline conditions for up to 16 days, followed
by exposure to freshwater conditions, on the germination of M. ericifolia seeds. The
six initial salinities used for prior exposure were: a) fresh water (0 g L
-1
); b) 1 g L
-1
; c)
2 g L
-1
; d) 4 g L
-1
; e) 8 g L
-1
; and f) 16 g L
-1
. The six curves shown in each graph a) to
f) indicate germination after seeds had been transferred from the saline treatments to
fresh water. Soaking periods are represented by ( ) = 1 day, ( ) = 2 days, (
) = 4
days, (
) = 8 days, ( ) = 16 days.
115
5.3.4 Effects of seed burial and substrate type
There was a strong effect of deep burial in soil on the germination of M. ericifolia
seeds (data not shown). Seeds failed to germinate at all if buried to 6 mm, and
germination was decreased by nearly two orders of magnitude compared with surface-
sown condition if seeds were buried to 3 mm with peat moss (1.6 % germination). In
contrast, there was no significant difference ( P > 0.05) in germination between
surface-sown seeds (97 % + 10 %) and those buried by 1 mm of peat moss (83 % + 9
%).
Although burial had a highly significant effect on germination, substrate-type
had no effect ( P>0.05) on germination success. Regardless of soil type used for the
trials, the percentage germination rate was about 95 % in each of clay, sand and peat
moss.
116
5.4 Discussion
The results obtained in these experiments have major implications for understanding
historical patterns in the natural recruitment of M. ericifolia into coastal wetlands and
for attempts to rehabilitate these environments by manipulating their salt and water
regimes. The key findings are that M. ericifolia shows low seed viability and that
germination is affected strongly by salinity. Salinity effects, however, are moderated
in a complex set of interactions with light and temperature. Seeds can germinate
when exposed to high salt concentrations, even if there is a very marked depression in
the ultimate germination rate of seeds exposed to a salinity of about one-half
seawater. The inhibitory effects of short-term exposure to saline water, however,
can be overcome by subsequent exposure to fresh water. Finally, it seems that
germination is little affected by the type of sediment that the seeds finds themselves
deposited onto, but is influenced greatly by burial by even a few millimetres of soil.
5.4.1 Poor seed viability and its causes
The viability of seed from the Dowd Morass population of M. ericifolia was low, at
only 6 %. Low viability has been found in other populations of this species and other
species of Melaleuca. Ladiges et al. (1981), for example, reported viability rates of
23 % to 28 % (of potentially germinable seed) for various populations of M. ericifolia
across the eastern part of its range in Victoria. Meskimen (1962) found rates of
between 3 % and 28 % viability for Melaleuca quinquenervia in northern NSW and
southern Queensland. Recent viability testing of M. ericifolia (R. Robinson
unpublished data) indicated total sterility for two isolated populations in western
117
Victoria, and between 0.25 % and 32 % viability for a range of populations
throughout central and eastern Victoria.
Limited genetic diversity of the parent populations is widely recognised as a major
contributing factor to low viability (e.g., Young et al. 1999; Cunningham 2000;
Tretyakova and Bazhina 2000; Cochrane et al. 2001). Environmental factors,
particularly high salinity, may interfere with effective seed set and significantly
reduce viability (Redondo et al. 2004; Boscaiu et al. 2005). Human-induced
alteration of environmental variables or management of populations, particularly via
altered water and salinity regimes, changed vegetation structure, soil degradation and
habitat fragmentation, have marked impacts on seed set and viability (Kingsford
2000; Tretyakova and Bazhina 2000; Renison et al. 2004). Major changes to
environmental conditions at Dowd Morass over the past quarter of a century,
especially altered water regime, increased salinity and increased nutrient loadings
(Grayson 2003), are likely to have had considerable impacts on the overall health of
the swamp paperbark populations, potentially limiting flowering ability and seed set.
The strongly clonal growth form of M. ericifolia may induce an inverse trade-off
effect on seed set and viability. This response has been observed in other species
(e.g., Mimulus: Sutherland and Vickery 1988). The extensive clonally-derived
population of M. ericifolia presently found at Dowd Morass may have established
from a much smaller founder population, which developed under vastly different
salinity conditions, possibly even a largely freshwater environment (Grayson 2003).
There is a strong indication that the present population is many times larger than the
original population found in the area in 1957 (aerial photographs Run 14-1258 photos
118
85-91 and Run 15-1258 photos 98-104 (Department of Primary Industries,
Government of Victoria, Aerial Photograph Library, Werribee). Recent rapid
population expansion, coupled with low viability rates, is suggestive of limited
genetic diversity in the founding population. It seems likely that a combination of the
above factors has had, and continues to have, a limiting influence on seed set and
viability on the Dowd Morass population of M. ericifolia.
5.4.2 Effects of chronic and acute exposure to salt
Periodic saltwater intrusions of various durations and flushing by fresh water are well
documented in the Gippsland Lakes (Grayson 2003) and the resultant fluctuations in
salinity are likely to have significant impacts on vegetation dynamics and recruitment
in the fringing wetlands. There is some evidence that M. ericifolia seedlings are
tolerant of high salinity (e.g., Ladiges et al. 1981; J. Salter et al. 2006). Furthermore,
Ladiges et al. (1981) indicated that seed soaked at salinity of 20 g L
-1
for 28 days
recovered when placed in fresh water. Nevertheless, intrusions of water of a lower
salinity and for shorter durations than those investigated by Ladiges et al. (1981) are
frequent in the Gippsland Lakes (Grayson 2003) and are likely to have a direct impact
on the seed bank and germination. The various salinity concentrations and durations
of inundation used in this study (0 - 32 g L
-1
and 1 - 16 days: Figures 5.1 and 5.2)
show that there is indeed a direct effect on the overall germination of M. ericifolia
seed at salinity levels and for durations well below those investigated by Ladiges et al.
(1981).
119
The toxic effect of salinity on M. ericifolia seed reported in this study clearly indicates
that the seed is largely intolerant of high levels of salinity even over relatively short
durations. Ladiges et al. (1981) reported variation in salinity tolerance of individual
provenances of M. ericifolia, and similar responses are known to occur in a wide
range of species at provenance and individual-plant level in brackish wetland taxa
(Marcar et al. 2003). However, the general intolerance of salinity by M. ericifolia, at
least in the initial stages of germination, would indicate that sexual reproduction in
this species has not evolved the same level of salt tolerance as shown by established
adult plants of the same species. Indeed, many halophytes exhibit germination
patterns restricted to periods of decreased salinity in the water column or substrate
(Churchill 1983; Krauss et al. 1998; Barrett-Lennard 2003).
The ability of germinable seed to recover from saltwater intrusions by subsequent
flushing with fresh water indicates a preference for low salinity environments for the
successful germination of M. ericifolia seed. Regular freshwater flushing of the
wetlands of the Gippsland Lakes area, caused by flooding from the lowland river
systems that feed the lakes complex, is likely to be less frequent than was the case in
the past (Grayson 2003). Such flooding would moderate the salinity regime in the
wetlands to what would be considered ostensibly fresh or near fresh conditions of <
2g L
-1
. Periodic drying of the wetlands and lowering of the water table in a drought
year, or as part of the natural wetting and drying cycle, followed by moderate rainfall
may also create minor flushing of the surface sediments, in particular of the
hummocks that are a frequent feature of the wetlands surrounding the Gippsland
Lakes. This in turn may provide the ideal germination conditions through the creation
of a limited number of ‘safe sites’, which allow germination to occur in what would
120
otherwise be a hostile germination environment. Conversely, however, water-level
drawdown could well result in markedly increased salinities over much of the
remainder of the wetland, due to evaporative processes, and in these areas
germination would be precluded.
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