Figure 1: Site map showing where reproductive shoots were collected. In
Lake Macquarie there is two thermally affected locations which are
illustrated by red markers and a power station image. Seagrass mapping
data sourced from NSW Department of Primary Industries.
Reproductive shoots were collected over two different reproductive
seasons, one during 21/22 spring/summer and one during the 22/23
spring/summer. At thermally affected sites (Myuna Bay and Mannering Park
in Lake Macquarie), reproductive shoots were collected late October,
while at ambient locations reproductive shoots were collected late
November. The difference in collection times was due to the likelihood
of temperature affecting seed maturation times (Smith et al. 2016). Reproductive shoots were transported to the laboratory in a 30 L
tub filled with seawater from the collection site. Shoots were then
placed outdoors in a shaded area in aerated 50 L containers with natural
seawater (34 ppt) sourced from Swansea Channel, the entrance to Lake
Macquarie. After maturation (~4 weeks after collection)
negatively buoyant seeds were siphoned from the bottom of the tanks and
stored in autoclaved seawater (sourced from Swansea Channel) at 4 °C for
a maximum of 2 days before being setup in an experiment. To simulate a
freshwater pulse and induce germination, seeds were moved from storage,
placed into a 30 ml sample jar of distilled water, stirred lightly for 5
minutes and then stored for 24 hours to assist in initiating germination
(Stafford-Bell et al., 2016; Cumming et al. ¸ 2017).
Viability
To determine viability, seeds (n = 100) from each site except Gwandalan
21/22 (n = 50) were subject to a cut test prior to being placed into the
germination experiment (Smith et al., 2016). The cut test is an
efficient (both time and economically) test which involves slicing the
seed in half and deems seeds which are firm and have a blue tinge to be
viable (Borza et al., 2007; Smith et al., 2016) highlighted
no difference between tetrazolium staining and a cut test in determining
seed viability. However, to ensure the cut test was being completed
correctly, tetrazolium staining was also performed on four sites (Myuna
Bay, Mannering Park, Murrays Beach and Yarrawonga Park; n = 100) and
compared by ANOVA which found no significant differences in viability
between the two tests. Consequently, in the second year only the cut
test was performed. Unfortunately, in Brisbane Water and Tuggerah Lakes
tanks, aerator failures resulted in usable seeds from only one site from
each estuary which were Saratoga and Pipeclay Point respectively. This
meant that the original design of n = 3 control sites in two separate
control estuaries was not accomplished, and because of no significant
differences between estuaries, ambient sites from Tuggerah Lakes and
Brisbane Water were pooled with ambient sites from Lake Macquarie.
Germination experiment
For each site, 100 seeds were randomly allocated to five petri dishes (n
= 20 per petri dish). However, insufficient seeds were collected at
Gwandalan in 2021, so three replicates (n = 14) were placed in the 16 °C
and 20 °C treatments only. Likewise, Saratoga Point only had sufficient
seeds for 5 replicates (n = 20) in the 16 °C treatment. Each petri dish
was filled with approximately 5-10 ml of sterilized seawater sourced
from the entrance of the lake on a single sampling occasion (34 ppt) and
fitted with an autoclaved sponge to reduce evaporation and
wet-strengthened filter paper to ensure the seeds did not move for the
duration of the experiment (Tol et al. 2021). Seeds were then
placed into plastic containers blacked out with aluminum foil and
randomly assigned to one of four (16 °C, 20 °C, 24 °C, 28 °C)
temperature control cabinets which allowed no ambient light. Excess
seeds from five of the sites were used in an additional five replicates
(n = 20) to be held at 8 ppt salinity in the 16 °C and 28 °C treatment
to investigate any effect of salinity. In the 16 °C treatment, this
included all sites except for Gwandalan and Saratoga and in the 28 °C
treatment it included Mannering Park, Myuna Bay and Pipeclay Point.
Seeds were checked for germination every 2-3 days and evaporative loss
topped up when required with saline solution (Cumming et al., 2017). Water was siphoned and changed fortnightly or earlier in the case
of unusual evaporation to ensure salinity did not fluctuate. Temperature
treatments and salinity were based on likely scenarios within the
estuaries where the seeds were sourced. On conclusion of the first year
of experiment, seeds were subjected to another freshwater pulse for 48 h
and results recorded for separate analysis.
Statistical analysis
To determine the effect of seed location (Lake Macquarie – ambient and
thermally affected; Brisbane Water – ambient, Tuggerah Lakes - ambient)
on mean time to germination (MTG), cox models were fit to seed
germination data using the R package ‘survival’ (Therneau, 2022) and
significant differences identified using estimated marginal means with
the ‘emmeans’ package with a Tukey adjustment (McNair et al., 2012; Tol et al. , 2022; Lenth, 2022). Because there were no
differences detected between ambient treatments regardless of estuary,
all seeds were grouped into either ‘ambient’ or ‘thermally affected’
treatments. Non-proportional hazards were checked using Kaplan-Meier
survivorship functions and the model with the lowest Akaike information
criterion (AIC) was used (McNair et al., 2012). Germination
analyses did not include the freshwater pulse in the first year which
concluded the experiment to avoid inflating the original germination
rate.
To determine the influence of temperature treatments and location of
seeds on seed germination, a multiple logistic regression model was fit
to individual seed data. To ensure the model fit the data and to
determine which covariates or interactions (Site, Location,
Treatment ) significantly influenced the model, deviance tables were
constructed on various models and compared using ANOVA with a Chi square
test. Multi-collinearity was tested by calculating the variance
inflation factors (Heiberger and Holland, 2015; Cumming et al., 2017). Factors which did not significantly influence the model or
suffered from multicollinearity were identified and removed from the
model (Site and the interaction of Location and Treatment). As in the
cox model, pairwise differences were identified using estimated marginal
means with the ‘emmeans’ package with no adjustment (Lenth et
al., 2022). The MASS package (Venables and Ripley, 2002) was then used
to calculate eβ and 95% confidence intervals to
quantify the influence of location on seed germination.
To compare means of dead seeds on experiment completion and maximum mean
germinations pre and pulse post and any interaction between location and
pulse, data was first analysed for homogeneity of variance which was
confirmed by plotting residual values vs fitted values and normality
using a Q-Q plot. ANOVA was then used with a Tukey’s HSD post hoc to
determine where significant differences occurred (Z ar, 2005).