Halophila beccarii Aschers (Hydrocharitaceae) Responses to Different Salinity Gradient

MATERIALS AND METHODS

Plant material and substrate: Halophila beccarii plant ramets consisting of 4 nodes intact with roots and shoots were collected from Pantai Sari (040°57.195’ N, 115°24.394’ E), Punang-Sari-Lawas River Estuary, Sarawak, Malaysia during low tide in late August 2008 and subsequently cleaned and, grown and maintained in a glass tank filled with artificial seawater of salinity 25 psu in shaded outdoor condition at the Research Centre, Universiti Putra Malaysia Bintulu Sarawak Campus from August 2008 to February 2010. Plant from the field were processed to grow them in the laboratory. Ramets were harvested using hand to avoid damage and ensure intact rhizome fragment, washed with seawater to remove any adherent debris before they were placed in a plastic box and immediately transported to a laboratory at a research centre located at Universiti Putra Malaysia Bintulu Sarawak Campus, Bintulu, Sarawak, Malaysia. Thirty individual ramets were measured for the number of leaves per shoot, length and width of longest leaves on the shoots and petiole length of leaves by using Mitutoyo Vernier Calliper and grown in two 120x45x45 cm glass tanks filled with 25 cm depth of 25 psu artificial seawater (prepared from commercial sea salt Marinemix, Marine Enterprises International Inc., Baltimore, USA i.e., a formulation containing all essential major, minor and trace elements) with the bottom spread of 5 cm thick loamy sand substrate collected from Tg. Batu Beach (3°13’ 46.37” N, 113°3’ 52.19” E), Bintulu, Sarawak. The culture system was fitted with an external filter and a submersible pump to provide filtration and circulation of water inside the tank. The Marinemix artificial seawater provides the nutrients to sustain H. beccarii growth and development while in culture. The culture system was kept under a shaded out-door natural light regimes that fluctuated from 160 μmol m^-2 sec^-1 (10% of the ambient light intensity outside) to ~240 μmol m^-2 sec^-1 (15% of the ambient light intensity outside) recorded between 12.00 noon to 2.00 pm. Water temperature and pH were recorded as 24-29°C and 7.5-8.4, respectively. Water level height of 25 cm (the water salinity maintained) in each of the tank was maintained by adding distilled water. The ramets propagated and colonized the substrate and they formed the stock plant propagules for experiments to examine H. beccarii tolerance and morphological changes when exposed to salinity gradient as performed below.

Setup of salinity experiment: Four individual ramets (Fig. 1a) of H. beccarii harvested from the stock plant propagules above were planted in a plastic tray (33x26x9 cm) filled with loamy sand substrate. The planted ramets occupied only half of the substrate surface to allow plants colonizing the remaining substrate surface. Four of such plastic trays were immersed in two separate 122x45x45 cm glass tanks filled with 25 cm depth of 25 psu artificial seawater (Marinemix) as shown in Fig. 1b. Plants in the plastic trays in the two tanks were allowed to grow and propagate for eight weeks. For each plastic tray, the planted and the initial bare area of the substrate colonized by new shoots of H. beccarii was marked, recorded and measured for following: the number of leaves per shoot, length and width of longest leaves on the shoots and petiole length of leaves. Leaf length, width and petiole length were measured using Mitutoyo Vernier Calliper.

Fig. 1(a-b):	(a) General morphology of a ramet consisting of 4 shoots with 7-8 leaves used as a planting material; number of leaves per shoot (NS), leaf length (LL), leaf width (LW) and petiole length (PL) and (b) A culture system for growing Halophila beccarii in different salinities

Fig. 2:	All shoots on the ramet were initially marked by placing small wooden pegs beside the shoots

This is the baseline conditions of H. beccarii plants at salinity of 25 psu before two separate experiments were performed to test how salinity affected H. beccarii performance where; (1) tank-1: salinity at 25 psu were consecutively increased to 30 and 45 psu (above the level reported in the literature) after two weeks intervals and, (2) tank-2: salinity at 25 psu were decreased consecutively 20, 15, 10, 6, 4, 2, 0.90, 0.52 psu at 2 weeks interval and 0 psu where the plants were left to continue to grow. All shoots on the ramets were initially marked by placing a small wooden peg beside them (Fig. 2). This is to ensure the newly produced shoot corresponded to the shoot without marks. The number of leaves per shoot, length and width of longest leaves on the shoots and petiole length of leaves from new established shoots were recorded and measured just prior to the target salinity treatment. The maintenance of the culture system basically followed the general procedure described above.

Analysis: The effects of salinity on the response variables i.e., number of leaves per shoot, length and width of longest leaves on the shoots and petiole length of leaves were subsequently analysed using One-Way ANOVA. Duncan’s new multiple range comparison test (DNMRT, p<0.05) was used to identify means that differed from each other. The original data from four response variables from each experiment were scatter plotted against the different salinities and Pearson correlation coefficients were obtained using SAS 9.0. Principle Component Analysis (PCA) was carried out using Primer 5, version 5.2.8 to obtain the relationship between the response variables and the treated salinities.

RESULTS AND DISCUSSION

Survival and production of shoot and leaves: There was no shoot mortality after immediate exposure till the end of the tested salinity. Irrespective of salinity gradient either increased or decreased salinities, planted ramets produced newly formed shoot with leaves. Young shoots were produced within 2-3 days, bearing 6-11 leaves after 5-7 days. Old leaves ceased to grow and detached from the shoot. The number of leaves per shoot varied markedly among replicate plastic trays within each salinity (Fig. 3a, b), tended to be high (13.0-18.3%) at low salinity (2-6 psu) and freshwater, respectively. At salinities above 25 psu the variations were (12.7-14.4%). The number of leaves per shoot, although variable, was not correlated with decreased (r = -0.061, p>0.05) or increased (r = -0.23, p>0.05) salinities.

Fig. 3(a-b):	Halophila beccarii responses, number of leaves per shoot after exposure to different levels in salinity at 2-week intervals, (a) Increased salinity from 25-45 psu and (b) Decreased salinity from 25-0 psu

Fig. 4:	Halophila beccarii grown in freshwater and continue to propagate via rhizome. Under this condition the plant propagated vegetatively

Under laboratory conditions, number of leaves per shoot were within the range values recorded in the field (6-11 leaves) and other locations e.g., 4 (Ascherson, 1871 cited from Den Hartog, 1970), 6-10 (Den Hartog 1957, 1970; Hodgkiss and Morton, 1978; Menez et al., 1983), 6-11 leaves (Parthasarathy et al.,1988), 4-12 leaves (Muta Harah et al., 2003). A study conducted by Hillman et al. (1995) on Halophila ovalis, showed plants only survived 7 days when expose to low salinity (10 psu), while Benjamina et al. (1999) reported H. ovalis leaves became smaller and shorter after exposed to low salinity before deceased. In contrast H. beccarii survived and continue to propagate at low salinity until the level of freshwater. The ability to grow under 0 psu more than 303 days is first reported under laboratory culture. To date the adapted plants is still being grown in our laboratory. Halophila beccarii demonstrated its tolerance to salinity gradient manipulation and continue to survive in freshwater (Fig. 4). The plant ability to survive indefinite to freshwater may involve combinations of dynamic cell wall elasticity and adjustment of cells cytoplasm osmotic gradient which was observed in other seagrass e.g., Ruppia maritima (Touchette, 2007).

Responses of H. beccarii to different salinity: The longest leaf length under increased salinities (Fig. 5a) and decreased (Fig. 5b) were both shorter and attained maximum values between 20 and 25 psu.

Fig. 5(a-f):	Halophila beccarii response variables after exposure to increased (from 25-45 psu) and increased (from 25-0 psu) salinity, (a, b) Leaf length, (c, d) Leaf width and (e, f) Petiole length

Leaves tend to be longer at freshwater level. There were discernible trends in leaf length with increased or decreased salinities. Leaf length became shorter and negatively correlated with increased (Fig. 5a) or decreased (p<0.05, r = -0.159, Fig. 5b) salinities. Similar responses were shown by leaf width under increased (Fig. 5c) and decreased (Fig. 5d) salinities. There were no obvious trends in leaf petiole length with salinity (Fig. 5e, f).

Factor analysis of the data from the increased salinity (from 25-45 psu) experiments resulted in three principal components with eigenvalues approximately one or greater than one that together explained about 93% of the total variance in the original response variables (Table 1). The loadings (correlations) of each response variable on the two components (Table 1) showed that leaf length and leaf width contributed almost equally to PC1, with all parameters being positively correlated with PC1. In contrast the number of leaves per shoot and petiole length had the greatest loading on the positive and negative site of PC2, respectively. PC1 varied significantly with salinity, leaf length (ANOVA, p<0.0001, F = 133.35) and leaf width (ANOVA, p<0.0001, F = 242.95).

Fig. 6:	Halophila beccarii responses and increased salinity: principal components 1 (PC1), 2 (PC2) and 3 (PC3) summarizing most of the original variation (ca. 93%) in the data set. All values are Means±SE (25 psu, n = 69; 30 psu, n = 38; 45 psu, n = 56)

Table 1:	The increased salinity (25-45 psu) experiments. Principal components (PC1, PC2 and PC3), their eigenvalues, the amount of variation explained by each component and loading of each response variable on the three principal components explaining more than 93% of the total variance in the data set

PC1 was significantly higher at 25 psu, than all other salinity and lowest of PC1 was observed at 45 psu (Fig. 6). The experiments showed increased salinity gave effects toward leaf length and leaf width where the leaves observed to be shorter and narrower at maximum salinity of 45 psu. PC2 was mainly contributed by number of leaves per shoot (ANOVA, p 0.96>0.05, F = 0.04) which was not affected by salinity. PC3 (petiole length) (ANOVA, p 0.12>0.05) also does not showed any effect toward increasing in salinity. PC2 and PC3 remain constants toward the increased salinity.

Factor analysis on the parameters observed from decreased salinity (25-0 psu) experiments resulted in three main principle components with eigenvalues approximately one or greater than one together explained about 85% of total variance in the original response variable (Table 2). The loadings (correlations) of each response variable on the three components (Table 2) showed leaf length and leaf width contributed almost equally with all parameters being positively correlated with PC1. PC1 varied significantly with salinity, leaf length (ANOVA, p<0.0001, F = 23.55) and leaf width (ANOVA, p<0.0001, F = 130.05). PC1 significantly higher at 25 and 20 psu the lowest of PC1 was observed within range of 4-0.52 psu (Fig. 7). There was a sudden decreased in the PC1 at the salinity from 20-15 psu. At 0 psu, PC1 showed good respond by having longer leaf length and wider leaf length, respectively. PC2 which was mainly contributed by number of leaves per shoot was not affected by salinity (ANOVA, p 0.83>0.05, F = 0.55). PC3 (petiole length) was also affected by salinity (ANOVA, p<0.0001, F = 5.65) being significantly shorter at 25 psu compared to other salinities (Fig. 7).

Fig. 7:	Halophila beccarii responses and decreased salinity: principal components 1 (PC1), 2 (PC2) and 3 (PC3) summarizing most of the original variation (ca. 85%) in the data set. All values are Means±SE (25 psu, n = 69; 20 psu n = 64; 15 psu, n = 54; 10 psu, n = 54; 6 psu, n = 56, 4 psu, n = 59; 2 psu, n = 35; 0.90 psu, n = 40; 0.52 psu, n = 31; 0 psu, n = 69)

Table 2:	The decreased salinity (25-0 psu) experiments. Principal components (PC1, PC2 and PC3), their eigenvalues, the amount of variation explained by each component and loading of each response variable on the three principal components explaining more than 85% of the total variance in the data set

Despite the hyposaline conditions, H. beccarii leaf dimensions remained at a relatively stable and within the range of those from the fields. Our results suggest that H. beccarii tolerated hyposaline conditions better than hypersaline conditions. According to Jagtap and Untawale (1981), gradual increase in salinity from 0-35 psu (when the monsoon ends) have no harmful effect on the growth of H. beccarii. It was also observed that new growth occurred when salinity was reduced to minimum by precipitation and flowering detected when salinity was 5.38 psu. Most seagrasses are thought to be more sensitive to increased salinity (Ogata and Matsui, 1965; Biebl and McRoy, 1971; Zieman, 1975; Adams and Bate, 1994; Kamermans et al., 1999; Van Katwijk et al., 1999). The cause of these effects may be due to toxicity by salt excess (Zhu, 2001), elevated metabolic cost to maintain internal ionic balance (Sibly and Calow, 1989), or negative alterations in the photosynthetic and respiratory rates (Ogata and Matsui, 1965; Biebl and McRoy, 1971; Kraemer et al., 1999). The species may therefore be similar to Ruppia, Zannichellia and Potamogeton pectinatus that can tolerate very sudden and very large fluctuations in salt content (Den Hartog, 1970). Although, H. beccarii has wide salt tolerance and can penetrate purely marine environment (salinity >30 psu, Muta Harah et al., 1999; Abu Hena et al., 2007; Abu Hena and Short, 2009), it is generally restricted to mangroves (Den Hartog, 1970; Jagtap and Untawale, 1981) and brackish water areas (Van Tien, 1998; Muta Harah et al., 2003).

CONCLUSION

Halophila beccarii survived and continue to propagate at high salinity (i.e., 45 psu beyond the high field salinity of 35 psu) and low salinity until the level of freshwater. The respond of above ground parts e.g., leaf dimensions remained at a relatively stable and within the range of those from the fields. The results of the culture experiments suggest H. beccarii tolerated hyposaline conditions better than hypersaline conditions. The ability to grow under 0 psu or freshwater condition for more than 303 days is first reported under laboratory culture.

ACKNOWLEDGMENTS

We would like to thank Vice-Chancellor, Universiti Putra Malaysia, for encouragement and facilities. This research is funded by the Ministry of Science, Technology and Environment Malaysia, under the Science Fund Priority Areas’ programme entitled “Indoor and outdoor culture of tropical spoongrass, Halophila ovalis R. Brown: Project number: 04-01-04-SF0867.