nvestigating the influences of tidal inundation and surface elevation on the establishment and early development of mangroves for application in understanding mangrove rehabilitation techniques 1 5

The objectives of this controlled mesocosm experiment was to determine if Avicennia alba and Rhizophora mucronata seedlings exhibited species-specific variation in i overall survival an

Chapter 4 – Interspecific variations in survival and growth responses of mangrove seedlings to three contrasting

inundation durations

4.1 Introduction

Mangroves generally exhibit multiple adaptations to anaerobic substrate conditions and periodic inundation, primarily through having modified roots that comprise mostly

of aerenchyma tissue with air spaces that allow rapid diffusion of oxygen through the root lenticels to the rest of the submerged root system (Lovelock et al., 2006, 2006b) Yet, studies have reported that there exist species-specific responses to duration of inundation (Luzhen et al., 2005) Experimental treatments simulating natural tidal amplitudes showed that under different treatments, species grew at different rates (Ellison & Farnsworth, 1997; Chen et al., 2005; He et al., 2007) He et al (2007) reported species-specific differences in biomass, morality and carbon allocation to roots, stems and leaves, and that experimentally determined inundation tolerance of mangrove species paralleled the pattern of species distribution for that location Kitaya

et al., (2002) similarly reported differing growth responses and mortality to elevation (i.e inundation duration) for seven species However, little variation in mortality, establishment and growth rates was found among five species planted at low- and high-elevation sites (Clark, 2004)

Focussing on life stage, mangrove seedlings are more vulnerable to prolonged inundation when compared to saplings and trees as they are physically smaller (i.e more prone to whole-plant submergence) and less established (i.e developmentally immature) The ability of each individual to respond to the stresses of prolonged inundation thus becomes crucial towards it survival and growth Hence, to achieve

ecological mangrove rehabilitation success, it is imperative to gain an understanding of species-specific responses of mangrove seedlings to prolonged inundation periods In Chapter 4, the field study examined the influence of surface elevations on the establishment of mangroves Given that surface elevation inherently controls inundation period, whereby lower elevations are inundated more frequently, for longer durations and to a greater depth, a second mesocosm experiment was designed to specifically examine the complementary knowledge of inundation durations on survival and early development of mangroves Moreover, the mesocosm experiment further serve as a control for potential confounding factors in the field study (Chapter 3) that may affect observed field results The objectives of this controlled mesocosm

experiment was to determine if Avicennia alba and Rhizophora mucronata seedlings

exhibited species-specific variation in i) overall survival and, ii) seedling growth in response to three different inundation durations

4.2 Materials and Methods

Mesocosm set-up – 12 fibreglass tanks with inner dimensions of 3 x 1 x 1 m were

housed at 82 Sungei Tengah Road, northwest Singapore There were six experimental and six reservoir tanks, where each experimental tank was elevated above a reservoir tank, and connected by a pump (Sobo WP 9200, 2400 L h-1) (Figures 4.1 and 4.2) This set-up was adapted from a larger study by Balke et al (2013) The reservoir tanks were filled with seawater with a salinity of 20 ppt and pH 11 A timer (SoundTech MDT-338) on the pump was used to fill and drain the experimental tanks automatically To simulate tidal inundation, activating the pump pumped water from the reservoir tank into the experimental tank (i.e high tide) while de-activating the pump resulted in complete drainage of the experimental tank back into the reservoir

(i.e low tide) Inside the experimental tank, an overflow at a height 0.9 m maintained the water depth when filled (Figure 4.1b)

Figure 4.1: (a) Aerial view of experimental set-up, (b) side and (c) aerial view of each pair of reservoir and experimental tank

Figure 4.2: Photographs of (a) the actual mesocosm set-up and the experimental pots with (b)

Rhizophora and (c) Avicennia seedlings

Materials and Experimental Design – Fresh R mucronata propagules

(Rhizophoraceae, R mucronata Lamk.) propagules were collected from the sediment

surface and trees at Sungei Buloh Wetland Reserve and Chek Jawa Wetlands,

northwest Singapore Rhizophora propagules exhibiting embryonic leaves or a whitish collar were considered fresh Similarly, fresh Avicennia alba propagules (Avicenniaceae, A alba Blume) were collected from the sediment surface at Sungei Buloh Wetland Reserve Avicennia propagules that had just shed or have an intact seed coat were considered fresh (Balke et al., 2013) Rhizophora and Avicennia propagules

were sown, as per Kitaya et al (2002) and Balke et al (2013) respectively, in polystyrene bags filled with mangrove mud collected from mangroves near Sungei

Buloh To allow for seedling anchoring, Rhizophora propagules were watered with freshwater for 35 days and Avicennia propagules, 10 days Anchored seedlings were

then randomly assigned to one of the three experimental treatments: (i) short inundation of 5 h (ii) medium inundation of 7 h and (iii) long inundation of 9 h, semi-diurnal tidal regime The short and medium inundation treatments were chosen as they were within the typical inundation durations in Southeast Asian mangroves (Van Loon

& Van Mensvoort, 2007), whereas the long inundation treatment served to test the upper inundation tolerances of seedlings

It is recognised that the planting of Rhizophora propagules may be a limitation of the

study design as Rhizophoraceae propagules may not always establish in an upright position under natural conditions (Tomlinson, 1986) Propagules may establish on their sides and later, develop lateral roots that exhibit differential elongation to then allow

the rooting and vertical establishment of Rhizophora seedlings (Tomlinson, 1986) Hence, the planting of Rhizophora seedlings will prematurely raise the propagule and

new plumule above the water level of 0.9 m, exposing it to shorter inundation periods over time This may favour photosynthesis and result in an over-representation of

survival rates of Rhizophora seedlings Nonetheless, given that mangrove propagules

are naturally buoyant, planting was necessary to facilitate the rooting of both

Rhizophora and Avicennia seedlings to ensure that their development and survival

could be followed across time This experimental design is similar to other studies investigating the effects of surface elevation on establishment and survival of mangrove seedlings under field conditions (Kitaya et al., 2002; Chen et al., 2005; Lu et al., 2013)

A total of 138 Rhizophora seedlings and 222 Avicennia seedlings were used The

seedlings were monitored throughout the experiment for survival and stem height

every week The mesocosm experiment lasted for 11 weeks, from 26 August 2014 – 2 November 2014 Salinity was monitored weekly and marine salt added when necessary

to maintain constant salinity Temperature and daylight hours in Singapore are generally constant throughout the year A separate one-time final measurement of root and stem length was conducted from 13 – 15th December 2014 for Rhizophora seedlings only as all Avicennia seedlings had died by then Mean root length was

derived from measuring four randomly chosen roots, across four cardinal points at the base of propagule,

Data analyses – Effects of species and inundation treatments on seedling survival were

analysed with a generalised linear model (GLM); binomial distribution, logit link function (logistic regression), using R 3.1.2 (R development core team, 2014) Using only data from the 11th week, the response variable was defined as the “Status” of each seedling, and assigned a binary code of either 1 (alive) or 0 (dead) The explanatory

variables were “Species” (Avicennia or Rhizophora) and “Treatment” (5, 7 or 9 hours

of inundation)

A separate analysis was conducted to model the relation between stem height and inundation duration and species using a mixed-effects linear model The software used

was the nlme library in R 3.1.2 (R development core team, 2014) A mixed-effects

model incorporates a mixture of fixed and random effects Fixed effects are associated with the average dynamic that may differ among treatment groups, specifically, the variability in stem height between different species and inundation treatments Random effects are associated with the variability of the dynamic among groups, specifically, the experimental tanks (Pinheiro & Bates, 2000) This approach allows the analysis to account for unknown differences in species (given they are of different genera) and temporal correlation (as repeated height measures were taken from the

same individual) The response variable was “Stem height” (log transformed)

“Species” (Avicennia or Rhizophora) and “Treatment” (5, 7 or 9 hours of inundation)

were included as fixed effects “Block” (where experimental tanks were assigned numbers ranging from 1 to 6) was modelled as a random effect

Both analyses started with fitting a global model that contained all explanatory variables as well as two-way interactions A final model was subsequently determined

by step-wise exclusion of the least significant terms, starting with non-significant two-way interactions (p > 0.05), and then non-significant main effects not included in the interactions The best fitting model was determined to be that with the lowest Akaike’s Information Criterion (AIC) value (Akaike, 1974) The AIC is a measure of the parsimony of models, and is based on a trade-off between deviance reduction and the number of parameters fitted in the model

Finally, for the remaining survivals (which were Rhizophora seedlings), Analysis of

Variance (ANOVA) was used to analyse if root length differed between inundation treatments The data was tested for normality and homogeneous variances before applying the ANOVA

4.3 Results

4.3.1 Seedling survival

Across all three inundation treatments, Rhizophora seedlings exhibited 100 % survival rates (Figure 4.3; R5, R7 and R9 = 1.0) For Avicennia seedlings, the proportion of

seedlings alive exhibited an inverse relationship to inundation period Treatment A9 with the longest inundation period of 9 hours exhibited the lowest percentage survival 46.1 % whereas Treatment A5, with the shortest inundation period of 5 hours exhibited

the highest percentage survival of 94.4 % Avicennia seedlings experiencing moderate

inundation period had a survival rate of 48.6 %

Figure 4.3: Proportion of seedlings alive per inundation treatment (A = Avicennia spp.; R =

Rhizophora spp.; 5, 7 and 9 represent the number of inundation hours)

A total of three GLMs were fitted and a comparison of the AIC values indicated that the minimum adequate model was that which included both “Species” and

“Treatment” as parameters, with the AIC value of 261.06 (Table 4.1) “Treatment” was identified as a significant and negative parameter in explaining for the variance in survival rates between seedlings, thus showing that longer inundation periods lowered seedling survival rates

Table 4.1: Performance matrix of the GLM models fitted

Model

Type

Global Status ~ Species + Treatment + Species:Treatment

Coefficient Estimate

P-value

Species:Treatment 0.57 0.99

263.06

Best

Fitting

Status ~ Species + Treatment

Coefficient Estimate

P-value

261.06

Status ~ Treatment

Coefficient Estimate

P-value

364.16

4.3.2 Seedling growth responses

Rhizophora seedlings exhibited similar cumulative stem height across inundation

treatments over 11 weeks At the 11th week, the mean Rhizophora stem height for treatments R5, R7 and R9 are 17.0 cm, 16.0 cm and 16.6 cm (Figure 4.4) Avicennia

seedlings exhibited more variation in mean cumulative stem height across time and inundation treatments The shorter the inundation duration, the longer the final stem length in the 11th week Inundation treatment A5 (with the shortest inundation period

of 5 hours day-1, semi-diurnal) had the longest stem (11.0 cm) The mean stem length for Treatments A7 and A9 were 9.2 cm and 8.4 cm respectively (Figure 4.4)

Figure 4.4: Cumulative stem height of both Rhizophora (top row) and Avicennia seedlings

(bottom row), segregated by inundation treatment, across weeks 1 to 11 Standard errors are

indicated by whiskers

A total of three models were fitted and a comparison of the AIC values (Table 4.2)

indicated that the best fitting model was that which included “Block” as a random

effect and “Species” as a fixed effect (Table 4.2, p-value < 0.05) An examination of

the fixed effect parameters showed that the stem height of Rhizophora seedlings were

on average, 0.528 cm less than that of Avicennia seedlings (Table 4.2)

Table 4.2: Performance matrix of the mixed-effects models fitted

Model

Type

Value

Global LogHt ~ Species + Treatment + Species:Treatment

Species: Treatment -0.09 0.29

8248.14

LogHt ~ Species + Treatment

8241.12

Best Fitting LogHt ~ Species

8233.35

4.3.3 Root length of Rhizophora seedlings

In the last week, stem length data was similar across inundation treatments (ANOVA p-value > 0.05) The mean stem length observed for each inundation treatments R5, R7 and R9 respectively are 22.6 cm, 21.7 cm and 23.9 cm (R9) Root length data was significantly different across inundation treatments (ANOVA p-value < 0.05) Inundation treatment R7 had the longest mean root length (21.2 cm), followed by R9 (18.9 cm) and R5 (17.9 cm) (Figure 4.5)

Figure 4.5: Barplot of (a) mean stem height and (b) mean root length of Rhizophora seedlings

across three Inundation Treatments (R5, R7 and R9) Standard errors are indicated by whiskers

4.4 Discussion

4.4.1 Impacts of prolonged inundation on seedling survival

Of the explanatory variables tested, inundation treatment was flagged as most important explanatory variable in the GLM that influences seedling survival rates (Table 4.1; p-value < 0.05) Longer inundation treatments lowered seedling survival

rates This was more applicable to Avicennia seedlings as Rhizophora seedlings exhibited 100 % survival across all three treatments (Figure 4.3) For Avicennia

seedlings, the lowest percentage survival (46.1 %) was observed in the 9 hour inundation treatment, with the highest percentage survival (94.4 %) in the 5 hours

inundation treatment Avicennia seedlings experiencing moderate inundation period

had 48.6 % survival