sv-lncs RUHUNA JOURNAL OF SCIENCE Vol 9: 13-31, June 2018 eISSN: 2536-8400  Faculty of Science http://doi.org/10.4038/rjs.v9i1.38 University of Ruhuna  Faculty of Science, University of Ruhuna 13 Sri Lanka Structural and functional responses of xylem in Rhizophora mucronata Lam. seedlings under drought and hypersaline conditions N.P. Dissanayake, K.A.S. Kodikara*, S. Premachandra and L.P. Jayatissa Department of Botany, Faculty of Science, University of Ruhuna, Matara, Sri Lanka Correspondence: * sunandaruh@gmail.com ; ORCID: 0000-0002-2493-3580 Received: 5th April 2018, Revised: 28th June 2018, Accepted: 30th June 2018 Abstract. Water translocation in mangrove seedlings is often affected by water stress conditions such as drought, hyper-salinities and their frequent variations. This study was therefore aimed at studying the wood anatomical responses of xylem tissue and hydraulic conductivity of Rhizophora mucronata Lam., a common species in mangrove planting, under different levels of drought [25%, ~50% and ~100% of water holding capacity (WHC)] and soil salinity [high salinity (35 psu), moderate salinity (15 psu) and freshwater (0 psu)]. As wood anatomical responses, significantly higher vessel density, vessel grouping (P<0.001) along with narrow vessel elements (P<0.001) were observed in plants grown in the 25% and 50% WHCs and high salinity treatments. All these anatomical responses are more directed towards avoidance of vessel cavitation which is commonly found under water deficit conditions. The results showed that R. mucronata plants failed to maintain efficient transportation of water when the field capacity was 50% of WHC or lower and the level of salinity was 35 psu or greater, as evident by the reduction of water conductive areas, vessel areas and hydraulic conductivity (P<0.05). Overall, water use efficiency of R. mucronata seedlings under the imposed water stress conditions has remarkably reduced and it further indicated that such imposed stress conditions directly affect the survival of planted seedlings as depicted by the significantly low survival in 25% and 50% of WHCs and high salinity. Therefore, in-depth study on lagoon hydrology including inundation levels, water depth, salinity and the selection of correct tidal positioning is highly recommended as prerequisites in mangrove planting. Keywords. Hydraulic architecture, hydraulic conductivity, mangroves, restoration, water stress. 1 Introduction Mangrove forests are unique plant communities that grow in extreme environmental conditions such as high and changing salinity, frequent mailto:sunandaruh@gmail.com N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 14 Vol 9: 13-31, June 2018 inundation with associated hypoxia, low air humidity and high temperatures. Mangrove ecosystems are restricted to intertidal areas of lagoons, estuaries, and sheltered bays in tropical and sub-tropical areas worldwide (Tomlinson 1986; Spalding et al. 2010; Mukherjee et al. 2015). However, the destruction of mangrove ecosystems is increased at an alarming rate and hence, attempts for mangrove replanting are taking place as a common activity in such areas. Mangrove planting in Sri Lanka has received a higher attention particularly after realizing the unprecedented mangrove loss in previous decades (Polidoro et al. 2010; Richards and Friess 2016) and safeguarding action of mangroves against the tsunami in 2004 (Dahdouh‐Guebas et al. 2005b; Lee et al. 2014; Jayatissa et al. 2016; Satyanarayana et al. 2017). However, majority of the planting attempts showed higher failure rates (Ahmad 2012; Kodikara et al. 2017a) as they were not supported by sound scientific knowledge. Among the relevant causes for the failures, improper site selection without studying site history and planting inappropriate species have been identified as two major causes for the failures (Primavera and Esteban 2008; Lewis 2009; Kathiresan 2011; Ahmad 2012; Kodikara et al. 2017a). In many cases, mangrove planting practitioners tend to establish mangrove plantations out of intertidal range (i.e. natural mangrove growing area) of lagoon which is commonly known as “inappropriate tidal positioning” in mangrove planting (Samson and Rollon 2008; Brown et al. 2014a). This often brings about inappropriate ecological conditions, for example hard soil, low water content, high salinity and hypoxia which are detrimental for the survival of mangrove plants (Brown et al. 2014a). Therefore, planted mangrove propagules/seedlings are vulnerable for drought stress (also known as physical water stress) and salt stress (also known as physiological water stress), in case of planting beyond intertidal zone (supra- littoral) where soil water content is low (particularly in dry zone and dry season) and more deeper areas in lagoon water (infra-littoral) where high salinity (during dry season) and hypoxic (prolonged submergence) conditions are applied, respectively (Hoppe-Speer et al. 2011). However, in general, mangrove plants have robust adaptions to overcome prevailing extreme conditions (Tomlinson 1994; Shi et al. 2005) and it is therefore important to study their plasticity of tolerance under these created extremities. Under the above stressed conditions, the water movement could be negatively affected which results in numerous variations in vessel anatomy as adaptations to ensure safe water movement (Hacke et al. 2006). In case of water limiting conditions, air bubbles could be formed inside vessel elements (cavitation) which breaks the continuity of water column (Cochard 2006; Robert et al. 2009) disturbing the movement of water. In response to these conditions, anatomical adaptations like high vessel density, high vessel grouping index, small and short vessels have been recorded for some N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 15 mesophytes (Baas et al. 1983). However, such studies for mangroves are scarce. Schmitz et al. (2006) and Robert et al. (2009) studied the anatomical traits like vessel density, vessel grouping index and vessel size of Avicennia marina and Rhizophora mucronata in relation to prevailing environmental conditions with particular attention on water relations. Several variations in size, density, and grouping of vessels have been observed. Nevertheless, the wood anatomical studies, water translocation behavior (functional response) and their plasticity under created abiotic stress conditions such as drought, hypersaline conditions and permanent submergence have not been adequately addressed. Further, in most of the studies, plant behavior under such stress conditions, are only discussed in term of vessel anatomy. However, we argue that hydraulic conductivity, i.e. the ease of water moving through a water transporting system, is a more reliable measure which reflects physiological response on the functional behavior of plants under water limiting conditions and it is more trustworthy, when accompanied with compatible anatomical data. Thus, the following aspects were addressed in this study, a) anatomical responses of Rhizophora mucronata plants under drought and hypersaline conditions, b) variations of hydraulic conductivity (Kh) of R. mucronata seedlings under above conditions and c) the plasticity of R. mucronata plants in modifying their vessel anatomy and water translocation behavior to secure their survival under water stress conditions. 2. Methodology The research was conducted in a plant-house, in the Department of Botany, University of Ruhuna, Matara, Sri Lanka. Rhizophora mucronata Lam. was selected for the study as the most commonly used mangrove species in planting programmes (~97%) in Sri Lanka (Kodikara et al. 2017a). 2.1 Experimental details In the plant-house experiment, mature propagules of Rhizophora mucronata collected from the natural mangrove forest in Pambala (07º31´N–79º49´E) in July, 2016 were kept floating in low saline (i.e. 2-3 psu) water for about one month. Later, the propagules were transferred from the water-filled containers to the plant-house conditions and maintained in a nursery to be used as the planting material. The soil mixture was prepared using garden top soil, mangrove top soil, sand and compost in the proportion of 1:1:1:1. The pots were prepared using black polythene of 23 cm x 25 cm and filled with about 3.5 kg of the prepared soil mixture. Thereafter, seedlings with the first two N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 16 Vol 9: 13-31, June 2018 unfurled leaves (i.e. same vigor) were selected for the experiment. For the two separated experiments (drought stress and salt stress), three treatment levels namely 25% WHC (Water Holding Capacity), 50% WHC and 100% WHC (control) were used for drought stress experiment (physical water stress) while another set of treatments for salt stress experiment (physiological water stress), high salinity (35 psu), moderate salinity (15 psu) and freshwater (0 psu) were used, keeping moderate salinity (15 psu) as the control since it is reported that R. mucronata performs its best under moderate salinity condition (Jayatissa et al. 2008). The water holding capacity was calculated based on the volume of the water held in the oven-dried, 100 g of soil sample when 100 ml water is added and the retained water volume was considered as the field capacity (100% WHC). Half and quarter of the field capacity volume were taken as 50% WHC and 25% WHC of the soil respectively. The individual pots were treated with the respective water volume daily to keep the imposed stress levels. In addition, several soil samples, taken randomly from the pots, were tested for the water level by using oven-dried method for further confirmation (NCRS, 2010). Three replicates for each treatment level were used with 12 seedlings per replicate. A total of 216 seedlings were subjected for the plant-house study. Low saline water (i.e. 5 psu) that was prepared separately by mixing sea water and aged tap water (i.e. water kept in open containers for few days before the use in order to remove excess chlorine), was added to maintain the respective WHCs and the same procedure was followed in preparation of moderate saline condition. Pots with seedlings were placed individually in well-separated tanks according to a completely randomized design (CRD) and this was done separately for the two experiments. Based on our preliminary measurements, the salinity of water in the tanks was checked once in every two days using a hand refractometer (ATAGO S/Mill-E, Japan) and adjusted the salinity when necessary (added aged tap water when the level of salinity was higher than the expected and seawater was added when the salinity was lower). Commercially available fertilizer was also applied (in the form of pellets) once a month by providing the same amount per pot (Adopted from Jayatissa et al. 2008; Dissanayake et al. 2014; Kodikara et al. 2017b). 2.2 Data collection Data on level of survival of Rhizophora mucronata seedlings in each treatment were recorded. As growth parameters, cumulative height (CH) (summation of the height of main stem and lengths of branches) of the R. mucronata seedlings, was measured once a month using a scaled ruler. Average number of leaves was also recorded and total average leaf area N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 17 (TALA) of the seedlings in all treatments were measured once in a 2-week period by using a millimeter graph sheet. Stomatal conductance (SC) was measured with the help of Steady State Porometer (SC-1; Decagon Devices, USA). Two measurements were taken on the same day as one at the late morning (10:00-11:00 hrs) and the other in early afternoon (13:00-14:00 hrs). The youngest, fully expanded leaf exposed to full sunlight was selected and abaxial conductance was recorded. The same leaf was subjected to measure the stomatal conductance of R. mucronata plants to avoid the variability among the different leaves. Fig.1 Solitary and grouped vessels in a cross section of Rhizophora mucronata stem (X 400). Conductive area of a cross section of R. mucronata stem (under low power), considered for the calculation of the hydraulic conductivity Vessel density (VD), vessel diameter (Vdia), vessel grouping index (VGI), number of solitary vessels (SV) and conductive area (CA) and vessel area (VA) were measured as anatomical parameters, by following the procedure given; the stems from the middle part of R. mucronata plants were collected and hand sectioned to obtain transverse sections of the stems. In case of hard stems, (about 4 months after planting), the collected samples were stored in a softening solution i.e. copenhague mixture: 70% ethanol; 28% de-ionised water and 2% glycerol in a 1:1:1 ratio) for about one month and then transverse sections were obtained using a microtome. Sections were stained using Safranin for 5 minutes and were then dehydrated by transferring through an ethanol series of 50%, 70%, 96% and 100% keeping 3 minutes in each. Sections were mounted on glass slides using Canada balsam. Microscopic images of the sections were taken at different magnifications (25x, 40x, 100x) with an Olympus microscope (BX60F-3, Tokyo, Japan) equipped with a digital camera (RUH-CAM, University of Ruhuna, Sri Lanka). Vessel dimensions were measured using IMAGEJ software, a public Solitary vessel Fiber cells Conductive area Grouped vessels Cortex region Pith region N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 18 Vol 9: 13-31, June 2018 domain, Java-based image processing program. Afterwards, Vdia was directly measured by IMAGE software while VD (number of vessels per mm2) and VGI (mean number of vessels per vessel group) and CA were calculated based on the vessel counts and area measurements. Percentage conductive area was calculated by taking percentage of total conductive area to the total area of the cross section whereas percentage vessel area was obtained by taking percentage of total vessel area to the total conductive area (Fig. 1). Hydraulic conductivity of the R. mucronata plants was measured at the 6th month after planting, using manually designed “Choatometer” [See Fig. 2 adopted from Choat et al. 2005]. Fig.2 Diagrammatic illustration of the manually designed Choatometer used in the experiment [adopted from Choat et al. 2005] (“h” = height of the pressure head). Segments of about 15 cm long and about 1 cm diameter were cut under water, from the stems (at the middle part), for hydraulic measurements according to Choat et al. (2005). A seawater solution (1%) filtered with a syringe filter of 0.22 μm, was used as perfusion solution for the experiment. The ends of the branch segments were cut smoothly with a razor blade and the mass of the branch segments i.e. mass start (m0), was recorded, before wrapping the segment ends with Parafilm. Thereafter, the branch segments were placed in the tubing system, connected at one end to a solution reservoir creating a pressure head (h) and to a pipette, placed on millimeter paper and filled with coloured liquid at the other end. The movement of the liquid in the pipette was followed by a camera taking pictures with a 3 seconds time interval (in total 30 seconds) and small video clips were also kept for further confirmation. After taking ten pictures, the mass of the branch segments was N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 19 weighed again i.e. mass end (m1). Furthermore, the length of the branch, the diameter of the branch, the number of nodes per branch segment, the air temperature and the relative humidity during the experiment were also recorded. The bark, pith and xylem area were measured on pictures of a transverse section of the branch segments, using ImageJ software. Hydraulic conductivity (Kh) was then calculated using the equation (1). ……………………………………….…… (1) where Kh is hydraulic conductivity, F is flow rate, L is branch length, and P is pressure drop. 2.3 Statistical analyses Cumulative height (CH), total average leaf area (TALA), stomatal conductance (SC), hydraulic conductivity (Kh), and vessel diameter (VDia) were measured and treated as the continuous variables while vessel density (VD), vessel grouping index (VGI), conductive area (CA), vessel area (VA) number of solitary vessels (SV) was taken as the count data (proportional and percentage). Mean and standard deviation were calculated as descriptive statistics. Three hypotheses, normality (Shapiro test; P>0.05), linearity (scatter-plot) and homogeneity of variance (Levene’s test; P>0.05) were tested and all conditions were met for the data. Parametric tests were, therefore, performed. The mean differences of the aforementioned continuous variables were checked using one-way ANOVA test (at a significance level of P<0.05), taken the “levels of stress” as the “factor” (for the same species Rhizophora mucronata) followed by the Tukey- Kramer multiple comparison test to check all pairwise differences. Chi-square test was conducted for the count data. All statistical analyses were performed using the R-3.2.2 statistical software. 3 Results The level of survival of Rhizophora mucronata seedlings decreased over time in all treatments except the control treatments. At the end of 18th week, 61% of R. mucronata plants survived in the 50% WHC level while all the seedlings died in the 25% WHC level by the end of 16th week (Fig. 3). In salt stress experiment, 21% of seedlings out of the initial number was able to survive in the high salinity level and only a few seedlings died in both moderate and P LF Kh   Pith region N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 20 Vol 9: 13-31, June 2018 freshwater treatments showing no significant difference in level of survival between two treatments (Fig. 3). Fig. 3 Percentage survival of Rhizophora mucronata seedlings over the study period. (Top) drought stress experiment and (bottom) salt stress experiment. Error bars indicate the standard deviation (±). In drought stress experiment, both CH and TALA of Rhizophora seedlings grown in the 50% WHC level was significantly lower (P<0.05) than that of the seedlings in the 100% WHC (Fig. 4 & Fig. 5). Interestingly, there was no significant difference in growth performances of the seedlings grown in freshwater and moderate saline condition whilst CH and TALA of the R. Time (weeks) P e rc e n ta g e l e v e l o f su rv iv a l (% ) 100% WHC 50% WHC 25% WHC Moderate Salinity Freshwater High salinity N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 21 mucronata seedlings in high salinity were remarkably lower (P<0.05) when compared with that in the freshwater and the moderate saline conditions (Fig. 4 & 5). The SC also showed noteworthy variation among the treatments. It was significantly lower (P<0.05) in the 50% WHC treatment level when compared with that of the control treatment (Fig. 6). However, there was no significant difference in SC between the moderate saline and freshwater conditions. It was significantly lower (P<0.001) in the high salinity treatment (Fig. 6). Fig. 4 Cumulative height of Rhizophora mucronata seedlings over the study period, in drought stress experiment (top) and salt stress experiment (bottom). Error bars indicate the standard deviation (±). 100% WHC 50% WHC 25% WHC Moderate Salinity Freshwater High salinity C u m u la ti v e h e ig h t (c m ) Time (weeks) N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 22 Vol 9: 13-31, June 2018 Fig.5 Total average leaf area (cm2) of Rhizophora mucronata seedlings at the end of the study period in (top) drought stress experiment and (bottom) salt stress experiment. Vertical bars indicate the standard deviation (±) while significant level by different letters at 95% confidence level. Anatomical responses and hydraulic conductivity Rhizophora mucronata seedlings showed remarkable variation in their anatomy and Kh under the given stress conditions which indicated that all the stress factors tested had a significant effect on vessel anatomy and Kh. T o ta l A v e ra g e L e a f A re a ( cm 2 ) X Y P P Q N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 23 Fig.6 Stomatal conductance (mmol m-2s-1) of Rhizophora mucronata seedlings over the study period in (top) drought stress experiment and (bottom) salt stress experiment. Vertical bars indicate the standard deviation (±) while significant level by different letters at 95% confidence level. According to the results, percentage cover of CA of the stems of Rhizophora mucronata in both 50% WHC and high salinity treatments were significantly lower (P<0.001) when compared with those of the stems, harvested from their respective controls i.e., 100% WHC and moderate saline condition (Table 1). However, no significant difference in the percentage cover of CA was observed among freshwater and moderate saline conditions. When considering the vessel characters, both VD and VGI were significantly higher X Q Y P P S to m a ta l co n d u ct a n ce ( m m o l m -2 s- 1 ) N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 24 Vol 9: 13-31, June 2018 (P<0.001) in the mangrove plants grown in the 50% WHC when compared with that of the respective control, 100% WHC (Table 1). Table 1. Anatomical responses of vessel elements of Rhizophora mucronata seedlings under drought and salt stress conditions (Tukey multiple comparison; 95% confidence level, P<0.05; significant differences among treatments within drought stress and salt stress experiments separately for the same parameter are shown by different superscript letters) (n: number of cross sections & n*: number of measurements). Parameter Drought stress experiment Salt stress experiment 100% WHC 50% WHC 25% WHC Moderate saline Fresh water High saline Conductive area (%) (n= 84) 11.7±2.2a 4.1±1.7b A ll p la n ts d ie d a ft e r 1 4 w e e k s 12.9±3.1a 13.1±2.4a 6.1±1.5b Vessel area (%) (n=84) 12.8±1.1a 5.2±0.9b 13.8±2.6a 14.4±1.9a 6.7±1.8b Vessel density (mm-2 xylem area) (n= X) (n= 84) 25.8±1.6a 66.2± 3.9b 24.5 ±2.9a 25.9± 1.7a 68.2 ±4.1b Vessel grouping index (n=84) 2.0 ±0.5a 5.1± 0.6b 2.4± 1.8a 1.8± 1.0a 6.1± 1.4b Solitary vessels (%) (n=84) 72.4±4.2a 33.4±3.1b 75.6±1.9a 83.2±2.2a 38.9±4.1b Vessel diameter (μm) (n*=1680) 82.6±13.6a 37.2±2.7b 88.7±11.8a 92.1±12.8a 34.8±3.8b In contrast, Vdia of R. mucronata plants in the 50% WHC showed significant decrease (P<0.05) when compared that of the 100% WHC treatment. In salt stress experiment, increased VD and VGI were found in the high salinity treatment (P<0.05) while the Vdia was significantly lower (P<0.05) as compared to that of the moderate saline condition. Interestingly, vessel anatomy of R. mucronata seedlings grown in the freshwater condition showed the pattern as same as the control plants i.e. low VD and VGI while having larger Vdia. Percentage of SV elements in conductive area of R. mucronata seedlings grown in the 50% WHC was significantly low (P<0.001) when compared with the control. In the same way, percentage of SV in R. mucronata stems grown under the high salinity condition was remarkably low (P<0.001) as compared to the moderate saline condition. N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 25 Fig.7 Boxplot graphs of hydraulic conductivity (mmol/ms MPa) of Rhizophora mucronata plants in (A) drought stress experiment and (B) salt stress experiment. Tukey-Kramer multiple comparison; 95% confidence level, P<0.05. Significance level shows by different letters and were used separately for two treatments. Further, we did not observe significant difference in the percentage of SV between the plants grown under the freshwater and moderate saline conditions. In spite of a high variability in vessel anatomy, Kh also varied among the treatments. The Kh of R. mucronata seedlings grown in the 50% WHC level and the high salinity treatment were significantly lower (P<0.05) as compared to their respective controls (Fig. 7) i.e. 100% WHC and moderate saline conditions respectively. The highest Kh was observed in the freshwater treatment which was not remarkably different from the control plants. 4 Discussion Selection of incorrect tidal positioning in mangrove planting due to ignorance of the accepted technical guidelines such as EMR (Ecological Mangrove Restoration) methodology (Lewis 2005; Lewis and Brown 2014) and modified protocols (Bosire et al. 2008), often leads to create stress conditions like drought (or negative soil water potential) and hypersaline conditions for planted seedlings (Kodikara et al. 2017a). Mangrove seedlings have therefore no choice rather than facing to such stress conditions. Although, mangrove 100%WHC 50%WHC 25%WHC Moderate sal. Freshwater High salinity H y d ra u li c co n d u ct iv it y ( m m o l /m s M P a ) X Y P P Treatments Q A B N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 26 Vol 9: 13-31, June 2018 plants are well-armed with various strategies to overcome extreme conditions such as high saline conditions and frequent inundation (Alongi 2002), the level of tolerance secondarily depends on the type and intensity of stress (Hsiao 1973; Larcher 2003). We further emphasize that created stress conditions may have relatively more deleterious effects on planted mangrove seedlings, particularly in the early life stage, than naturally prevailing extreme conditions. Under natural conditions, both level of stress and exposure time are relatively low since the effects of stresses are naturally moderated, for example, effect of mangrove soil salinity increase is often diluted with periodic inundation. However, according to the results, survival, growth performances, stomatal conductance, hydraulic traits and hydraulic conductivity of Rhizophora mucronata plants were affected in different ways and to different degrees under aforementioned two stress conditions. In this study, the stressed R. mucronata plants in both drought and salt stress conditions showed similar responses in many aspects. The decreased level of survival and growth performances of the mangrove plants grown in the 50% WHC, 25% WHC and high salinity conditions, could be due to low water potential and osmotic stress (Munns and Tester 2008). Reduced cell turgor pressure is expected under both drought and hypersaline conditions due to which cell expansion is hampered (Naidoo 2006). Also, accumulation of salt ions like NaCl in cell vacuoles further intensifies negative water potential under hypersaline condition (Werner and Stelzer 1990) where it needs prompt osmoregulation to cope up with such conditions (Naidoo 1985). Allocation of energy for osmoregulation may be prioritized over plant growth and development which may ultimately restrict energy allocation for growth, showing the reduction of growth parameters such as plant height, leaf production and total leaf area (Naidoo 2006; Hoppe-speer et al. 2011). When R. mucronata plants run out of metabolic energy in case of exposing to intense stress conditions for a longer period, the plant may end up with early mortality. In that sense, hypersaline condition has made more lethal effects on R. mucronata plants as the level of survival was reduced up to 26% whilst the survival was 61% under drought conditions (50%WHC). Several anatomical responses could also be observed under the imposed stress conditions, and those responses could be considered as modifications which may be contributory in securing their plant functionality and survival. However, decreased CA in both 50% WHC and high salinity treatments is disadvantageous as it reduces water transporting area inside the plants. Cell shrinkage which has resulted in due to the reduced cell turgor pressure under drought and hypersaline conditions may be the cause for the reduced CA (Akram et al. 2002). The common observations i.e. increased VD and VGI as well as narrow vessel elements of the mangrove plants with the enhanced q N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 27 drought (e.g., 50% WHC and 25% WHC) and salinity (>35 psu) can be explained in different ways. The formation of vessel conduits with different vessel diameters primarily depends on the differentiation rates of conduits from the cambium. It has showed that negative water potential under water stress conditions caused plants to form small cambial derivates resulting in narrow vessels (Arend and Fromm 2007). Moreover, Aloni (2004) has suggested that the formation of narrow vessels under water stress is a result of the elevated auxin concentrations in the plant body which induces the early secondary wall formation and lignification preventing further cell expansion. However, on the other hand, such anatomical modifications, have several associated functional significances. As reported by Robert et al. (2009), the described anatomical features like increased VD, VGI and decreased Vdia are known to be the adaptations which support the plant to overcome risk of cavitation as air bubbles are easily formed inside the water filled vessel elements under negative water potential obstructing the flow of water. Therefore, high VD and VGI under the water stress conditions help to serve more efficient bypass (being closer) of embolized vessels which minimizes the number of dysfunctional vessel elements due to cavitation (Schmitz et al. 2006). Probably this could be the reason why higher number of solitary vessels was favored in non-stress conditions, for example, 100% WHC, freshwater and moderate saline conditions, since risk of cavitation is comparatively low under sufficient water availability. However, the chance for cavitation under negative water potential cannot be totally avoided by the anatomical modifications in conductive tissues (Robert et al. 2009). This is clearly evident from the fact that hydraulic conductivity of R. mucronata seedlings grown in the 50% WHC and high salinity conditions, has remarkably reduced even when occurring the suitable anatomical modifications in conductive tissue. Therefore, we propose that this would happen either due to the presence of a higher number of dysfunctional vessels through which a resistance to water flow is created or any internal adjustment to water flow, done by the plants themselves in case of higher water demand. The reduced hydraulic conductivity could secondarily depend on SC of the plants as SC of the R plants grown in the 50% WHC and high salinity was significantly lower while the highest was recorded in the plants grown in the freshwater treatment. Several studies reported that high stomatal conductance in R. mucronata was recorded in freshwater condition and it was reduced with the increase of salinity and similar results were recorded for other mangrove species as well (Naidoo 1985; Aziz and Khan 2000; Kodikara et al. 2017b). Moreover smaller leaf area is also a strategy to reduce water loss from plants under water deficit conditions (Ball 1988; Brugnoli and Lauteri 1991). Interestingly, Parker and Pallardy (1985) have observed a strong positive correlation of leaf area with vessel diameter, stem conductive area and total xylem area for seedlings of several species. We also observed N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 28 Vol 9: 13-31, June 2018 the reduced CA in both experiments of our study. However, this indicates that water use efficiency (WUE) decreases with the increased negative water potential under substrate drought and hypersaline conditions. According to the observations, when the Rhizophora mucronata plants face water stress conditions (substrate drought or hypersaline condition), both anatomy of the xylem tissues and the hydraulic conductivity were modified as a response to stress. Further, the plants make anatomical modifications such as increased vessel density, vessel grouping index with narrow vessel elements which are more directed toward the avoidance of vessel cavitation. Parallel to these anatomical changes, stomatal conductance and leaf area also decreased, most probably as a measure of water conservation under water stress conditions. The trade-off between the energy allocation for the above tolerant mechanisms and plant growth is achieved through retarded growth of R. mucronata plants as evident by the plants grown in the 50% WHC, 25% WHC and high salinity treatments. At extreme level, R. mucronata plants ended up in early mortality (e.g. 25% WHC), more likely due to physiological imbalances that occur with increased water and energy demand under such prolonged water stress conditions. On the other hand, R. mucronata seedlings could build up some safety mechanisms inside the plants with anatomical modifications though, efficiency of water transportation was not well- maintained as reflected by decreased hydraulic conductivity of the plants under highly stressed conditions. Further, it clearly demonstrated that the water use efficiency of the R. mucronata plants also reduces under the stress conditions. The results of the study demonstrate that, under abiotic stress conditions, Rhizophora mucronata seedlings are able to survive for a certain period (up to some critical level) through some structural and physiological adjustments. Nevertheless, if these highly stressed conditions prevail for a longer period (prolonged exposure), the R. mucronata plants are unable to secure their survival. This clearly depicts that the impacts of stress conditions, created on mangrove plants particularly in early life stage due to the inappropriate planting practices, are more detrimental when compared with the natural fluctuations of salinity and availability of water in mangrove ecosystems. This emphasizes the need of understanding the lagoon history with special attention to behavior of lagoon hydrology including inundation levels, water depth, salinity and selection of suitable tidal position accordingly for mangrove planting. Acknowledgement We kindly acknowledge the Department of Botany, University of Ruhuna, Sri Lanka for giving a working place for the laboratory experiments. Also, the authors would like to thank Mrs. SK Madarasinghe, post-graduate student, University of Ruhuna for her assistance in taking anatomical measurements and N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 29 performing descriptive analyses. Two anonymous reviewers are acknowledged for their comments on the initial draft. References Ahmad IU. 2012. Status of mangrove plantations in the living delta: an overview of the coastal afforestation experience of Bangladesh. In Macintosh DJ, Mahindapala R, Markopoulos M. (Eds.), Sharing Lessons on Mangrove Restoration (pp. 81-93), Proceedings and a Call for Action from an MFF Regional Colloquium, Mamallapuram, India. Akram M, Akhtar S, Javed IH, Wahid A, Rasul E. 2002. Anatomical attributes of different wheat (Triticum aestivum) accessions/varieties to NaCl salinity. International Journal of Agriculture and Biology 4: 166–168. Alongi DM. 2002. Present state and future of the world’s mangrove forests. Environment Conservation 29: 331–349. Aloni R. 2004. The induction of vascular tissue by auxin. In Davies PJ. (Ed.), Plant hormones: biosynthesis, signal transduction, action (pp. 471- 492), Dordrecht: Kluwer. Arend M, Fromm J. 2007. Season change in the drought response of wood cell development in poplar. Tree Physiology 27: 985–992. Aziz I, Khan MA. 2001. Effect of seawater on the growth, ion content and water potential of Rhizophora mucronata Lam. Journal of Plant Research 114: 369–373. Ball MC. 1988. Ecophysiology of mangroves. Trees - Structure and Function 2: 129–142. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. 1983. Flow cytometry studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. Journal of Immunology 130(4): 1910–1917. Bosire JO, Dahdouh-Guebas F, Walton M, Crona BI, Lewis RR, Field C, Kairo JG, Koedam N. 2008. Functionality of restored mangroves: A review. Aquatic Botany 89: 251–259. Brown B, Yuniati W, Ahmad R, Soulsby I. 2014a. Observations of natural recruitment and human attempts at mangrove rehabilitation after seismic (tsunami and earthquake) events in Simulue Island and Singkil Lagoon, Acheh, Indonesia. In Santiago-Fandino V, Kontar YA, Kaneda Y. (Eds.), Post-Tsunami Hazard Reconstruction and Restoration (pp. 311-327), Springer. Brugnoli E, Lauteri M. 1991. Effects of salinity on stomatal conductance, photosynthetic capacity, and carbon isotope discrimination of salt-tolerant (Gossypium hirsutum L.) and salt-sensitive (Phaseolus vulgaris L.) C3 non-halophytes. Plant Physiology 95: 628-635. Choat B, Ball MC, Luly JG, Holtum JAM. 2005. Hydraulic architecture of deciduous and evergreen dry rainforest tree species from north-eastern Australia. Trees-Structure and Function 19: 305–311. Cochard H. 2006. Cavitation in trees. Comptes Rendus Physique 7: 1018–1026. Dahdouh-Guebas F, Jayatissa LP, Di Nitto D, Bosire J, Lo Seen, Koedam N. 2005b. How effective were mangrove as a defence against the recent tsunami? Current Biology 15: 443- 447. Dissanayake NP, Madarasinghe SK, Kodikara KAS, Jayatissa LP, Perera AJD, Koedam N, Dahdouh-Guebas F. 2014. Preliminary study on the propagule dependency of Rhizophora seedlings. Journal of Department of wildlife conservation 2: 141-151. Hacke UG, Sperry JS, Wheeler JK, Castro L. 2006. Scaling of angiosperm xylem structure with safety and efficiency. Tree Physiology 26: 689–701. Hoppe-Speer SCL, Adams JB, Rajkaran A, Bailey D. 2011. The response of the red mangrove Rhizophora mucronata Lam. to salinity and inundation in South Africa. Aquatic Botany 95: 71– 76. Hsiao TC. 1973. Plant responses to water stress. Annual Review of Plant Physiology 24: 519- 570. N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science 30 Vol 9: 13-31, June 2018 Lewis RR. 2005. Ecological engineering for successful management and restoration of mangrove forests. Ecological Engineering 24: 403-418. Lewis RR. 2009. Methods and criteria for successful mangrove forest restoration. In Perillo GME, Wolanski E, Cahoon DR, Brinson MM. (Eds.), Coastal wetlands: an integrated ecosystem approach (pp. 787–80), Elsevier Press, Amsterdam, The Netherlands. Lewis RR, Brown B. 2014. Ecological mangrove rehabilitation – a field manual for practitioners. Version 3. Mangrove Action Project Indonesia, Blue Forests, Canadian International Development Agency, and OXFAM. Jayatissa LP, Kodikara KAS, Dissanayaka NP, Satyanarayana B. 2016. Post-Tsunami assessment of coastal vegetation, with the view to protect coastal areas from ocean surges in Sri Lanka. In Santiago-Fandiño V, Tanaka H, Spiske M. (Eds.) Tsunamis and earthquakes in coastal environments, Coastal Research Library. Springer International Publishing, pp 47–64. Jayatissa LP, Wickramasinghe WAADL, Dahdouh-Guebas F, Huxham M. 2008. Interspecific variations in responses of mangrove seedlings to two contrasting salinities. International Review of Hydrobiology 93: 700-710. Kathiresan K. 2011. Conservation and Management Strategies: Restoration Technologies. In Kathiresan K, Ajmal Khan S. (Eds.), Coastal Biodiversity in Mangrove Ecosystems: UNU- INWEH-UNESCO International Training Course (pp. 609–632). Online at: http://ocw.unu.edu/international-network-on-water-environment-and-health/unu- inwehcourse-1-mangroves/Restoration-technologies.pdf. Kodikara KAS, Mukherjee N, Jayatissa LP, Dahdouh-Guebas F, Koedam N. 2017a. Have mangrove restoration projects worked? An in-depth study in Sri Lanka. Journal of Restoration Ecology 25(5): 705–716. Kodikara KAS, Jayatissa LP, Huxham M, Dahdouh-Guebas F, Koedam N. 2017b. The effects of salinity on growth and survival of mangrove seedlings changes with age. Journal of Acta Botanica Brasilica 32(1): 37-46. Larcher W. 2003. Physiological Plant Ecology: Ecophysiology and stress physiology of functional groups. 4th edn. Springer-Verlag, Berlin, pp. 513. Lee SY, Primavera JH, Dahdouh-Guebas F, McKee K, Bosire JO, Cannicci S, Diele K, Fromard F, Koedam N, Marchand C, Mendelssohn I, Mukherjee N, Record S. 2014. Ecological role and services of tropical mangrove ecosystems: A reassessment. Global Ecology and Biogeography 23: 726–743. Mukherjee N, Dahdouh-Guebas F, Koedam N, Shanker K. 2015. An interdisciplinary framework to evaluate bioshield plantations: Insights from peninsular India. Acta Oecologica 63: 91–100. Munns R, Tester M. 2008. Mechanisms of Salinity Tolerance. Annual Review of Plant Biology 59: 651-681. Naidoo G. 1985. Effects of water logging and salinity on plant–water relations and on the accumulation of solutes in three mangrove species. Aquatic Botany 22: 133–143. Naidoo G. 2006. Factors contributing to dwarfing in the mangrove Avicennia marina. Annals of Botany 97: 1095–1101. National Resources Conservation Service (NRCS). 2010. Soil quality kit: Guides for educators, United Sates Department of Agriculture (USDA), U.S.A. http://www.nrcs.usda.gov/wps/portal/nrcs/site/national/home/ (Accessed in 25 May 2018). Parker WC, Pallardy SG. 1985. Stem vascular anatomy and leaf area in seedlings of six black walnut (Juglans nigra) families. Canadian Journal of Botany 63(7): 1266- 1270. https://doi.org/10.1139/b85-175 Polidoro BA, Carpenter KE, Collins L, Duke NC, Ellison AM, Ellison JC, Farnsworth EJ, Fernando ES, Kathiresan K, Koedam N, Livingstone SR, Miyagi T, Moore GE, Nam VN, Ong JE, Primavera JH, Salmo SG, Sanciangco JC, Sukardjo S, Wang Y, Yong JWH. 2010. http://www.vub.ac.be/APNA/staff/FDG/pub/Jayatissaetal_2008_InternatRevHydrobiol.pdf http://www.vub.ac.be/APNA/staff/FDG/pub/Jayatissaetal_2008_InternatRevHydrobiol.pdf http://ocw.unu.edu/international-network-on-water-environment-and-health/unu-inwehcourse-1-mangroves/Restoration-technologies.pdf http://ocw.unu.edu/international-network-on-water-environment-and-health/unu-inwehcourse-1-mangroves/Restoration-technologies.pdf http://www.nrcs.usda.gov/wps/portal/nrcs/site/national/home/ https://doi.org/10.1139/b85-175 N.P. Dissanayaka et al. Responses of xylem in Rhizophora seedlings Ruhuna Journal of Science Vol 9: 13-31, June 2018 31 The loss of species: Mangrove extinction risk and geographic areas of global concern. PLoS One 5: e10095. Primavera JH, Esteban JMA. 2008. A review of mangrove rehabilitation in the Philippines: successes, failures and future prospects. Wetland Ecology and Management 16: 173–253. Richards DR, Friess DA. 2016. Rates and drivers of mangrove deforestation in Southeast Asia, 2000–2012. Proceedings of the National Academy of Sciences 113: 344–349. Robert EMR, Koedam N, Beeckman H, Schmitz N. 2009. A safe hydraulic architecture as wood anatomical explanation for the difference in distribution of the mangroves Avicennia and Rhizophora. Functional Ecology 23: 649–657. Samson MS, Rollon RN. 2008. Growth performance of planted red mangroves in the Philippines: revisiting forest management strategies. Ambio 37: 234-240. Satyanarayana B, Van der Stocken T, Rans G, Kodikara KAS, Ronsmans G, Jayatissa LP, Mohd-Lokman H, Koedam N, Dahdouh-Guebas F. 2017. Island-wide coastal vulnerability assessment of Sri Lanka reveals that sand dunes, planted trees and natural vegetation may play a role as potential barriers against ocean surges. Global Ecology and Conservation 12: 144-157. http://dx.doi.org/10.1016/j.gecco.2017.10.001 Schmitz N, Verheyden A, Beeckman H, Kairo JG, Koedam N. 2006. Influence of a salinity gradient on vessel characters of the mangrove species Rhizophora mucronata. Annals of Botany 98: 1321–1330. Shi SH, Huang YL, Zeng K, Tan FX, He HH, Huang JZ, Fu YX. 2005. Molecular phylogenetic analysis of mangroves: independent evolutionary origins of vivipary and salt secretion. Molecular Phylogenetic and Evolution 34: 159–166. Spalding MD, Kainuma M, Collins L. 2010. World atlas of mangroves. London, U.K. Earthscan. Tomlinson PB. 1986. The Botany of Mangroves. London, Cambridge University Press. pp. 419. Tomlinson PB. 1994. The Botany of Mangroves. Cambridge University Press, Cambridge. Werner A, Stelzer R. 1990. Physiological responses of the mangrove Rhizophora mangle grown in the absence and presence of NaCl. Plant, Cell & Environment 13: 243–255. http://dx.doi.org/10.1016/j.gecco.2017.10.001