Chapter – 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 47 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 x x 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 48 (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 49 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 50 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 h (ii) medium inundation of h and (iii) long inundation of h, semidiurnal 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 51 every week The mesocosm experiment lasted for 11 weeks, from 26 August 2014 – 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 (alive) or (dead) The explanatory variables were “Species” (Avicennia or Rhizophora) and “Treatment” (5, or 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 52 same individual) The response variable was “Stem height” (log transformed) “Species” (Avicennia or Rhizophora) and “Treatment” (5, or hours of inundation) were included as fixed effects “Block” (where experimental tanks were assigned numbers ranging from 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 twoway 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 hours exhibited the lowest percentage survival 46.1 % whereas Treatment A5, with the shortest inundation period of hours exhibited 53 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, and 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 54 Table 4.1: Performance matrix of the GLM models fitted Model Type Global Function AIC Value Status ~ Species + Treatment + Species:Treatment 263.06 Intercept Species Treatment Species:Treatment Best Fitting Coefficient Estimate 4.66 14.91 -0.57 0.57 P-value 0.00* 0.99 0.00* 0.99 Status ~ Species + Treatment Intercept Species Treatment 261.06 Coefficient Estimate 4.66 19.24 -0.57 P-value Coefficient Estimate 4.47 -0.44 P-value 0.00* 0.98 0.00* Status ~ Treatment Intercept Treatment 364.16 0.00* 0.00* 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 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) 55 Figure 4.4: Cumulative stem height of both Rhizophora (top row) and Avicennia seedlings (bottom row), segregated by inundation treatment, across weeks 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) 56 Table 4.2: Performance matrix of the mixed-effects models fitted Model Type Global Fixed Effects LogHt ~ Species + Treatment + Species:Treatment Intercept Species2 Treatment Species: Treatment Value -0.454 -0.370 0.00 -0.09 P-value 0.14 0.50 0.99 0.29 LogHt ~ Species + Treatment Intercept Species2 Treatment Value 0.590 -0.611 -0.022 8241.12 P-value 0.00* 0.00* 0.46 Best Fitting LogHt ~ Species Intercept Species2 AIC Value 8248.14 8233.35 Value 0.627 -0.528 P-value 0.00* 0.00* 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) 57 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 hour inundation treatment, with the highest percentage survival (94.4 %) in the hours inundation treatment Avicennia seedlings experiencing moderate inundation period had 48.6 % survival 58 The trend where prolonged inundation reduces seedling survivorship is expected as two direct mechanisms exist by which inundation acts to influence seedling survival First, prolonged inundation reduces oxygen available to roots, thereby reducing the rate of aerobic metabolism and water-use efficiency (Naidoo, 1985; McKee, 1996) Hovenden et al (1995) demonstrated that Avicennia marina seedlings had sufficient aerenchyma to supply the oxygen requirements of the root system for a period of approximately 1.5 – 3.5 hour per tide When inundation periods exceed this range, plants become anaerobic, consuming more energy in order to maintain their metabolisms Second, short-term inundation depresses photosynthetic capacity of mangrove seedlings When tidal inundation depth and duration exceeds the natural tolerance of mangroves, the leaf photosynthetic capacity becomes strongly limited (Ellison & Farnsworth, 1997; Chen et al., 2005) These natural tolerances for optimal inundation periods occur across a spectrum and are species-specific, eliciting differing responses across species (Kitaya et al., 2002) For example, Sonneratia caseolaris seedlings function best in the range of – hours with that for Sonneratia apetala to be – hours (Chen et al., 2013) This species-specific reason was attributed to the fact that compared to the exotic S apetala, S caseolaris was a local species and hence, have adapted best to local environmental conditions Kandelia obovata and Bruguiera gymnorrhiza functioned best in shorter optimal inundation periods of about and hours respectively (Chen et al., 2005; Wang et al., 2007) Seedling mortality was observed only in Avicennia but not Rhizophora seedlings Seedling mortality began in the 3rd week for the long inundation treatment R9, and was not observed until the 6th week in the short inundation treatment R5 (Figure 4.3) Effects of prolonged inundation elicit an immediate, extreme response of seedling mortality as unlike mature trees/saplings, mangrove seedlings have yet to develop 59 aerial roots and stem lenticels as adaptations to lowered soil oxygen Moreover, the short time span might prove insufficient for seedlings to adapt via increasing root porosity, an anatomical adaptive strategy employed to increase root oxygen reserves and reduce volume of respiring tissue (Youssef & Saenger, 1996) Throughout the experiment, all Avicennia seedlings were subjected to whole-plant submergence at the fixed inundation depth of 0.9 metre Seedlings thus faced reduced gaseous exchange and depressed light intensity, inhibiting respiration and photosynthetic assimilation (Lu et al., 2013) While all Rhizophora seedlings were subjected to whole-plant submergence at the start of the experiment, their taller seedling height resulting from an extended hypocotyl allowed some individuals to grow and extended their plumule beyond the water column This allows the distal portion of the seedling to assume aerobic metabolism, potentially conferring the advantage of overcoming complete inundation in a tidal environment In a field study by Kitaya et al (2002), similar results were observed that relates elevation (i.e inundation period) to seedling survival – R mucronata was concluded to have higher tolerance to prolonged inundation compared to Avicennia officinalis at an early growth stage At low elevations, R mucronata achieved 80% survival whereas all A officinalis seedlings died Likewise, all A marina seedlings died when fully submerged for 12 hours (Lu et al., 2013) Pezeshki et al (1987) argued that the survival and growth of any species exposed to underwater stress conditions is dependent on its ability to maintain a functional photochemical and biochemical system that enables maintenance of positive net photosynthesis In this sense, Avicennia seedlings possibly have a lower tolerance to underwater stress compared to Rhizophora seedlings A study by Mangora et al (2014) showed that ability of Avicennia marina to photosynthesis underwater was lower compared to B gymnorrhiza (family Rhizophoraceae) Seedling survival in early 60 development stages can also be influenced by the effect of propagule reserves (Smith & Snedaker, 2000) Seedlings likely survived fairly well until depletion of propagule reserves, at which point survival shifts to a higher dependence upon photosynthetic capacity of individual seedlings Smaller propagule sizes and lower cotyledon reserves in Avicennia may thus explain for the observed lower survival rates The critical amount of time in which growth responses begin to depend largely on photosynthesis over propagule reserves was probably crossed during the 11-week period – potentially around the 4th to 6th week where Avicennia seedling mortality rates were the highest (Figure 4.3; A7 & A9) This was probably not crossed during the 11-week period for Rhizophora seedlings since survival rates remained at 100 % However, this might just be attributed to larger propagule reserves as Rhizophora propagules are of a much larger size Once the reserves have been depleted, mortality rates might increase 4.4.2 Impacts of prolonged inundation on seedling growth This study also showed differential growth response of Rhizophora and Avicennia seedlings to inundation treatments as substantiated in the mixed-effects model wherein “Species” was a significant parameter (Table 4.2) Rhizophora seedlings did not exhibit significant variation in stem height across treatments in comparison to Avicennia seedlings (Figure 4.4) The data demonstrates that these treatments represent optimal inundation periods (5 – hours) for Rhizophora seedlings This concurs with findings by Hoppe-Speer et al (2011) where average weekly increase in height was similar across the 3, and hours semi-diurnal inundation treatments In those treatments, seedlings performed best as they exhibited maximum photosynthetic performance, high stomatal conductance and had the highest biomass accumulation and leaf production Yet, Rhizophora seedlings may be able to tolerate longer inundation periods as they have not been stressed enough to exhibit a stem elongation 61 response, a typical response of wetland plants to prolonged inundation (Jackson & Drew, 1984) Stem elongation represents plants adapting to prolonged inundation conditions through shifting biomass accumulation from roots to shoots, thereby allowing rapid initial growth to increase biomass above water surface Alternatively, the observed stem elongation responses in R apiculata (Kitaya et al., 2002) and R mangle (Ellison & Farnsworth, 1993) could in actuality be driven by a combination of environmental factors given that these were field studies in which conditions were not ideally kept constant across time and space Also, longer inundation periods suppressed Avicennia seedling growth and height In the hour inundation treatments, the greatest Avicennia seedling height documented was 44 cm compared to 26 cm and 24 cm in the and hours inundation treatments Potential reasons would be that inundation durations exceeding a threshold reduced respiration and photosynthetic abilities, thereby resulting in low- or non-functioning photochemical and biochemical systems in seedlings Also, the root length of seedlings was significantly longer in inundation treatments with prolonged inundation durations (R7 and R9; Figure 4.5) While higher frequency of inundation has been shown to maximise growth and aboveground productivity (Krauss et al., 2006), these results are coherent in suggesting that permanent flooding might function to stimulate root biomass allocation as an adaptation to more soil reducing conditions and sulphide accumulation (CastañedaMoya et al., 2011) In the long term, this translates into a response to changes in hydroperiod that is reflected as changes in aboveground and belowground biomass and productivity (Krauss et al., 2008) An examination of the fixed-effect parameters showed that the height of Rhizophora seedlings were on average, 0.528 cm less than that of Avicennia seedlings (Table 4.2) Vivipary is the condition in some seed plants in which the sexually produced embryo 62 of the seed continues its development without dormancy into a seedling, while still attached to the parent plant (Elmqvist & Cox, 1996) Inherent physiological differences exist in that Rhizophora exhibits vivipary whereas Avicennia provides a good example of cryptovivipary (Tomlinson & Cox, 2000) For Rhizophora, this occurs through elongation of the hypocotyl to produce a cigar-shaped seedling Thus, Avicennia being a good example of “cryptovivipary”, its growth strategy encompasses a lack of dormancy but rapid seedling growth after dispersal from the parent plant (Tomlinson, 1986) 4.4.3 Interactions between inundation and other physical factors that affect seedling growth Under field conditions, physical factors exert a combined influence on seedlings instead of in isolation as designed in this experiment Thus, observed responses to prolonged inundation may be confounded through interactions with other physical factors such as salinity (Ball, 2002) Experiments on Kandelia candel seedlings showed that while prolonged inundation stimulated stem elongation, this response remained unchanged in treatments that combined conditions of prolonged inundation with increased salinity (Ye et al., 2010) Stem diameter of K candel seedlings did not change as well A separate experiment involving Laguncularia racemosa and Rhizophora mangle seedlings showed inter-specific responses to combined effects of inundation and salinity All growth characteristics of L racemosa seedlings (e.g relative growth rate, leaf area, stem height etc.) were higher than that of R mangle across all treatments Under prolonged inundation, this species growth differentiation was also more pronounced at low salinity compared to high salinity (Cardona-Olarte et al., 2006), suggesting that under the same conditions of low to mild stress by 63 inundation and salinity, L racemosa exhibited the advantage of being competitively more dominant than R mangle Combined effects of inundation with salinity could also determine spatial distributions of certain species Field observations of mature K obovata trees highlighted that inundation tolerance thresholds were influenced by a salinity gradient – plants exhibited longer inundation duration tolerances as salinity decreased (Yang et al., 2013), and thus preferentially occupied areas with such conditions Hence, inundation as a factor in isolation, or in combination with other factors across varying ranges, will correspond to differences in responses across individual growth characteristics, species and life stages Faced with a change in such environmental conditions, certain species may be conferred a competitive advantage if better growth is encouraged This may manifest as some form of spatial restructuring of mangrove communities (i.e changes in species composition) and/or mangrove distributions, depending on spatial and temporal scales of these environmental changes In the context of mangrove rehabilitation, specifically the change in environmental conditions through the re-establishment of tidal connectivity and surface elevation changes, local inundation frequencies, duration and depth will be altered This may translate into landcover changes (e.g a shift from bare aquaculture ponds to reforested land) when conditions in degraded site prove favourable for mangrove colonisation 4.5 Summary This study highlights that survival and growth of mangrove seedlings vary across both inundation periods and is species-specific The threshold of Avicennia seedlings to prolonged inundation treatments is potentially constrained at hours, as prolonged inundation past this threshold lowered both the survival and growth rates of Avicennia 64 seedlings Rhizophora seedlings may have a higher threshold to prolonged inundation as both survival and growth rates remained unaffected These results provide information for use in rehabilitation projects should the restorer resort to planting mangroves seedlings (as a last resort) in areas prone to prolonged inundation periods Yet, results are only representative for early developmental stages and other environmental factors such as wave energy and salinity, factors contributing to seedling survival, must be considered before use in mangrove rehabilitation projects 65 ... 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... of h and (iii) long inundation of h, semidiurnal tidal regime The short and medium inundation treatments were chosen as they were within the typical inundation durations in Southeast Asian mangroves. .. duration, the longer the final stem length in the 11 th week Inundation treatment A5 (with the shortest inundation period of hours day -1, semi-diurnal) had the longest stem (11 .0 cm) The mean stem