Regeneration mechanisms in Swamp Paperbark (Melaleuca ericifolia Sm.) and their implications for wetland rehabilitation

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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.   

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
C and night temperatures of 10
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 
mol m
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 

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
) to over half seawater (19 g L
), 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
C in July to 
a daily maximum of 25
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
, 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 
mol m
); and iii) temperature 
(constant 10
C, 20
C or 30
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.  

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
) of reconstituted sea water as described above and soaked in 
the dark at 20
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. 

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;, verified September 2006) and Systat (version 5;, 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. 

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
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
, and only about 5 % of seeds germinated at 8 g L
this temperature.  In comparison, nearly 50 % germination was observed for seeds at 
C in fresh water.  Seeds at 30
C had consistently higher germination at all 
salinities in constant darkness than they did in a 12:12 hour light:dark cycle.  
At 20
C, seeds showed maximum germination success (40-50 %, depending on light 
conditions) at a salinity of 1 g L
 and germination fell at a roughly constant rate with 
increasing salinities.  Although about 10 % still germinated at a salt concentration of 
16 g L
, no seeds germinated at a salinity of 32 g L
.   Unlike the case at the highest 

incubation temperature, seeds at 20
C showed statistically significantly higher 
percentage germination in the light:dark cycle than in complete darkness. 
The percentage of seeds germinating at 10
C was about one half of that at 20
C at a 
given salinity.  The maximum germination at 10
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
Germination success fell regularly, but slowly, at higher salinities and, as before, no 
germination was observed at a salinity of 32 g L
.   Nevertheless, about 5 % of seeds 
still germinated at 16 g L
 at 10
C, a result in strong contrast to that found for seeds at 
C.   There was a very strong effect of light:dark on seeds incubated at 10
C, with 
seeds in the dark treatment showing about one-half the germination success of those 
kept in alternating light:dark cycles.  

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.  
 F-ratio P
Main effects 

658.658     < 0.001 
 Salinity  2.450 

Light regime   

76.477       < 0.001   
Interaction terms 
152.520     < 0.001 

75.543       < 0.001 

12.886       < 0.001 
25.317       < 0.001 
(error)  0.035 

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.  

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
 (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
.  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
over one week (Figure 5.2 d).  Similarly, 17 % of seeds had germinated in the 8 g L
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
) 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 

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
 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
(Figure 5.2 a, b); this progressively fell to about 
95 % germination at 2 g L
 (Figure 5.2 c), 80 % at both 4 g L
 and 8 g L
 (Figure 5.2 
d, e), and 50-75 % at 16 g L
 (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.   

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
); b) 1 g L
; c) 
2 g L
; d) 4 g L
; e) 8 g L
; and f) 16 g L
.  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. 

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 

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 

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 

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
 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
 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

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
.  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 

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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