Marine Biology (2002) 141: 1029–1034 DOI 10.1007/s00227-002-0951-1

W-Q. Wang Æ L. Ke Æ N.F.Y. Tam Æ Y-S. Wong Changes in the main osmotica during the development of candel hypocotyls and after mature hypocotyls were transplanted in solutions with different salinities

Received: 13 May 2001 / Accepted: 30 November 2001 / Published online: 4 October 2002 Ó Springer-Verlag 2002

Abstract No direct correlation was found between salt 30& salinity treatments, and declined or remained tolerance and vivipary. Ion (Mg2+,Ca2+,Na+,K+ and constant when salinity was zero. If corrected for water Cl–) concentrations on a dry weight basis (mg/g) and on content, it was found that when roots formed there was a milli-molar basis per mass of water in fresh tissue a leakage of ions in K. candel hypocotyl during the zero (mM) were followed during the development of Kandelia salinity treatment. There was a faster ion uptake at sa- candel hypocotyls and after mature hypocotyls were linities of 15& and 30&. This showed that once the root transplanted under different treatments (salinities of 0, system is formed, exchange with the environment 15 and 30&). During hypocotyl development, ion con- becomes more rapid. centrations on a dry weight basis declined especially for Mg2+ and Ca2+. The decrease could not be explained by the decrease in water content. However, the con- centrations on a milli-molar basis did not decrease, but Introduction increased slightly at a later stage. Mass balance studies showed that the hypocotyl development was a salt ac- + + – Vivipary is the condition found in a few seed in cumulation process, especially for Na ,K and Cl . which the sexually produced embryo of the seed con- propagules adapt themselves to hyposaline tinues its development without dormancy into a seed- environments by accumulating salt, especially before ling, while still attached to the parent (Elmqvist leaving their parent plants. Substrate salinity showed no 2+ 2+ and Cox 1996). Vivipary in flowering plants occurs most modification of trends in Ca and Mg after trans- commonly in plants that occupy shallow intertidal plantation, increasing rapidly in the beginning, then re- habitats, e.g. seagrass and (Elmqvist and maining at a high level and declining rapidly when roots Cox 1996). It is known in the four genera , formed. There were no significant differences in Mg2+ 2+ Kandelia, Rhizophora and Ceriops of and Ca concentration among treatments. The signif- (Tomlinson 1986). The seedling develops without dor- icance of salt level changes during hypocotyl develop- mancy largely by elongation of the hypocotyl to produce ment to salt tolerance remains to be clarified. After a cigar-shaped seedling, which remains conspicuously hypocotyl transplantation, concentrations of Na+,K+ – pendulous on the parent tree for several months. The and Cl increased gradually after insignificantly early structure is eventually abscised (Tomlinson and Cox changes. However, once roots formed, K+,Na+ and – 2000). The agent of dispersal (propagule) is neither fruit Cl concentrations increased rapidly under 15& and nor seed, but the seedling itself (Tomlinson and Cox 2000). In general, the seedling is termed a hypocotyl. Communicated by G.F. Humphrey, Sydney Seedling development is critical for mangroves because of the salt regime, water movement, stressful W-Q. Wang (&) intertidal environment, and the need for long-term dis- School of Life Sciences, Xiamen University, persal by ocean currents (Dawes 1998). The viviparous Xiamen 361005, Fujian, People’s Republic of China condition is so strongly associated with mangroves that E-mail: [email protected] it is suggested to have adaptive significance in the Tel.: +86-592-2180937 intertidal environment (Tomlinson 1986). It was W-Q. Wang Æ L. Ke Æ N.F.Y. Tam Æ Y-S. Wong assumed that mangroves originated from fresh-water Department of Biology and Chemistry, Centre of Coastal Pollution Conservation, habitats (Jin and Fang 1958; Dawes 1998) and that vi- City University of Hong Kong, vipary in mangroves may be an adaptive characteristic Tat Chee Avenue, Kowloon, Hong Kong permitting avoidance of high salinity at germination 1030

(Joshi 1933; Henkel 1979). Some evidence has proved Table 1 Length, fresh weight, dry weight and water content that developing viviparous seedlings (i.e. hypocotyls) in of hypocotyls at different developing stages. The hypocotyls were collected from Mai Po, Hong Kong, China. Rhizophoraceae retain lower salt concentrations than Hypocotyls at Stage 8 are mature. Values are means±SD (n=3) the parent shoot and especially the leaves (Lo¨ tschert and Liemann 1967; Hogarth 1999; Zheng et al. 1999). This Stage Length (cm) Fresh Dry Water lowered salt concentration, whatever its origin, is inter- weight (g) weight (g) content (%) preted as a mechanism to ‘‘protect’’ the embryo from the deleterious effects of high salt concentrations until ma- 1 8.2±0.5 1.8±0.3 0.5±0.1 70.1±0.9 turity (Tomlinson 1986; Hogarth 1999). Nevertheless, a 2 10.7±0.4 3.0±0.3 0.9±0.1 70.5±1.6 few authors have argued that the adaptation of hy- 3 12.4±0.4 3.7±0.2 1.1±0.1 70.3±0.5 4 13.9±0.5 4.9±0.5 1.6±0.2 67.9±2.1 pocotyl to salt originates when it is still attached to the 5 16.2±0.3 5.5±0.7 1.8±0.4 67.6±2.2 maternal tree by continuously absorbing salt from the 6 18.1±0.6 7.7±1.1 2.5±0.4 67.0±1.3 tree (Jin and Fang 1958; Joshi et al. 1972; Lin 1988). 7 19.8±0.3 9.4±0.8 3.2±0.3 66.4±1.2 After following the element concentrations on a dry 8 21.7±0.6 14.7±0.3 5.3±0.2 63.7±1.1 weight basis, Zheng et al. (1999) concluded that the development of hypocotyls was not a salt accumulation process, but a desalinating process, because salt con- Salt solutions were prepared using tap water and Instant Ocean Synthetic Sea Salt (Aquarium Systems, Mentor, Ohio, USA). The centration on a dry weight basis declined with hypocotyl fresh weight of each hypocotyl was measured before transplanta- development. If it were truly a desalinating process, how tion and each hypocotyl had a fixed position. Tap water was added can the hypocotyl keep the osmotic balance and by what daily to keep the salinity constant. The salt solutions were changed means can the hypocotyl cope with the salt stress and once a week. At 0, 5, 10, 15, 20, 25 and 30 days after transplan- osmotic stress after the hypocotyl settles down in the tation, four hypocotyls for each treatment were collected randomly. At day 30, white roots occurred in all hypocotyls, but the leaves hyposaline environment? Current studies have focused were still not open. The fresh and dry weights (105°C, 24 h) of each on the development of the hypocotyl, and little research hypocotyl were measured. has dealt with the changes after a hypocotyl leaves the parent plant (Tomlinson and Cox 2000). Thus, no fully Measurements acceptable explanation for the correction has been offered. All samples were cleaned carefully with distilled water and blotted K. candel is the dominant mangrove species and the dry, weighed and dried at 80°C for 48 h. The dry samples were most important species used for mangrove rehabilitation weighed and water contents of the samples were calculated. Dry samples were ground, passed through a 1-mm sieve and stored for in China (Wang et al. 1999). Integrative physiological analysis. Samples were ashed in a muffle furnace at 500°C for 4 h. and ecological studies of vivipary are critical to devising Ashes were dissolved in hot 5 N HNO3 until dry, re-dissolved in + + 2+ seed-handling strategies, predicting seedling perfor- 0.1 N HNO3, filtered and stored for analysis. Na ,K ,Ca and 2+ mance, and understanding the evolution of reproductive Mg were analyzed by atomic absorption spectrophotometry (Perkin-Elmer Atomic Absorption Spectrophotometry, Plasma traits in general (Farnsworth and Farrant 1998). 1000 Emission Spectrometer, USA). Cl– extracts were prepared by + + 2+ 2+ – – K ,Na ,Ca ,Mg and Cl are the main inor- hot-water extraction and Cl determined by AgNO3 titration. ganic osmotica in mangroves. In K. candel leaves, these ions were found to account for 87% of the osmotica (Zhao et al. 1999). To study the salt-tolerant mechanism, Results we quantified levels and dynamics of these ions in K. candel hypocotyls at different stages of development During development of K. candel hypocotyls, the from hypocotyl formation to maturity and we studied element concentrations on a dry weight basis de- temporal changes after transplantation into different clined (Fig. 1). Compared to stage 1 hypocotyls, the salinities.

Materials and methods

K. candel hypocotyls were collected from Mai Po, Hong Kong. To minimize the genetic differences between individual hypocotyls, hypocotyls at different developing stages were collected from the upper canopy of the same tree. The development of hypocotyls was divided into eight stages by the weight and length of hypocotyls. Table 1 shows the ranges of weight and length. Hypocotyls at stage 8 meant the mature hypocotyls, i. e. falling from branches when shaken gently. For studies on maintenance of osmotic equilibrium and salt balance after hypocotyl transplantation, mature K. candel hy- pocotyls with similar lengths and fresh weights were cultured hy- droponically in sand beds at salinities of 0, 15 and 30& under Fig. 1 Changes in element content (mg/individual) during Kandelia natural photoperiods and mean day/night temperatures of 26/20°C. candel hypocotyl development. Values are means±SD (n=3) 1031 concentrations of Mg2+,Ca2+,Na+,K+ and Cl– in After hypocotyl transplantation, water content de- mature hypocotyls had declined by 53.7%, 50.2%, creased gradually (Fig. 3). This process was also ac- 14.4%, 7.6% and 20.6%, respectively. The concentra- companied by a loss of mass of about 10%. The ion tions of Ca2+ and Mg2+ changed more significantly changes after hypocotyl transplantation occurred in two than K+,Na+ and Cl–. Nevertheless, the development stages. From hypocotyl transplantation to day 25 is stage of K. candel hypocotyls was not a desalinating process, 1, and from day 25 to day 30 is stage 2. During stage 1, but a process of element accumulation. Compared to concentrations of Na+,K+ and Cl– increased gradually stage 1 hypocotyl, the contents of Mg2+,Ca2+,Na+, and slightly, while concentrations of Ca2+ and Mg2+ K+ and Cl– per mature hypocotyl increased 3.7-, 4.0-, increased rapidly at the beginning of the stage. Substrate 7.3-, 7.9- and 7.0-fold. The accumulation of Mg2+ and salinity showed no modification of ion concentrations Ca2+ was not as significant as K+,Na+ and Cl–. With between treatments. The ion concentrations were similar hypocotyl development, the rate of element accumula- to each other between treatments (Fig. 4). The slight tion accelerated. From stage 7 to stage 8, there was a increase in K+,Na+ and Cl– concentration was not the sharp increase in K+,Na+ and Cl– contents. result of uptake from the environment, but resulted from The changes in element concentrations of tissue sap the decline in water content and the loss of mass. (mM) were not the same as the changes in element At 25 days after transplantation, roots appeared. At concentrations on a dry weight basis (mg/g) nor as the this stage, the concentrations in Ca2+ and Mg2+ declined changes in mg/individual (Fig. 2). Although there was a sharply, becoming similar to those on day 0. No differ- decline in the concentrations of Ca2+ and Mg2+ in the ences in Ca2+ and Mg2+ concentrations between treat- tissue sap at the early stage of development, almost no ments could be found. However, K+,Na+ and Cl– changes in Na+,K+, and Cl– concentrations occurred behaved differently. The concentrations of Na+,K+ and during the period studied. From stage 7 to hypocotyl Cl– at 0& treatment declined or remained constant. maturity, the concentrations of K+ increased from 290 Taking the decline of water and mass into account, it to 395 mM. The total concentration of the five elements showed that ion leakage occurred. However, the con- increased by 141.4 mM during the course of develop- centrations of Na+,K+ and Cl– increased during 15& ment. and 30& treatments. At day 25, the K+/Na+ ratio of 0,

Fig. 2 Changes in element concentration (mM) of tissue sap during K. candel hypocotyl development. Total The total concentrations of K+,Na+, Cl–,Ca2+ and Mg2+). Values are means±SD (n=3) 1032 Henkel 1979; Hogarth 1999). These authors assumed that salinity imposes severe constraints on seed germi- nation, and that vivipary would thus allow the propa- gule to escape this vulnerable phase. Salt concentrations are lower in the cotyledons than in the pedicel, lower again in hypocotyls, and lower still towards the tip of the hypocotyls, reaching levels of less than one-third of those found in pedicels (Lo¨ tschert and Liemann 1967). Tissues of the seedling are thus preserved from prema- ture exposure to high salt levels; Zheng et al. (1999) suggested that the adaptation of the seedlings to saline environments is caused by breeding seedlings in highly Fig. 3 Temporal changes in water contents after K. candel concentrated salt environments. They called this process hypocotyls were transplanted to a different salinity. Values are a sign of atavism. However, for halophytes the salt level means±SD (n=3) in propagules is generally lower than in shoots and leaves (Ungar 1991). For example, seedlings of the non- 15 and 30& treatments was 6.6, 7.3 and 5.9, respectively. viviparous mangrove Acanthus also show lower Na+ Once roots formed, the K+/Na+ ratio increased rapidly. and Cl– concentrations than adult plants (Hogarth At day 30, the ratios were 16.4, 15.3 and 12.7, respectively. 1999). Therefore preserving seedlings from premature exposure to high salt levels is not necessary in viviparous mangroves. Discussion After following the element concentrations on a dry weight basis, Zheng et al. (1999) concluded that the Ion dynamics during hypocotyl development development of hypocotyls was not a salt accumulation process, but a desalinating process, because salt con- Some authors have suggested that vivipary among centrations on a dry weight basis (mg/g) declined with mangroves is an adaptation to salinity (Joshi 1933; hypocotyl development. The adaptation of the seedlings

Fig. 4 Temporal changes in ion concentration (mM) after K. candel hypocotyls were trans- planted to a different salinity. Values are means±SD (n=3) 1033 to saline environments is caused not by increasing salt content. This confirms that the epidermis of K. candel levels in the hypocotyl, but by breeding seedlings in hypocotyls is effectively sealed. The slow exchange rate highly concentrated salt environments, e.g. fruits and favors the long-distance dispersal of K. candel propa- persistent pericarps (Zheng et al. 1999). A few authors, gules. The changes in Mg2+ and Ca2+ cannot be ex- however, argue that the adaptation of hypocotyls to salt plained by a simple process of passive water and mass starts when they are still attached to the maternal tree loss. There must be an increase in Ca2+ and Mg2+ and salt is absorbed from the tree continuously (Jin and content during days 0–10 and a subsequent decrease. Fang 1958; Joshi et al. 1972; Lin 1988). Our results There is an increase in the K+/Na+ ratio implying that contradict these suggestions. Generally speaking, there K. candel preferentially absorbs or retains K+. were no significant changes in element concentrations Rates of ion exchange increased once roots appeared. (mM) during K. candel hypocotyl development (Fig. 3). This agrees with the suggestion of Tomlinson and Cox The decline in ion concentrations on a dry weight basis (2000) that root emergence makes the seedling less (mg/g) is offset by the decline in water content (Table 2). impervious to ion exchange with the environments. The net result is the non-significant change in element concentrations (Fig. 3). However, the development of K. candel hypocotyls is a salt-accumulating process, Significance of vivipary to mangroves because the amounts of salt per hypocotyl increased significantly (Fig. 2). Anatomical experiments showed Because many viviparous mangrove species belong to a that the K. candel hypocotyl has a uniformly and heavily number of different families, whose other members are cutinized epidermis and no stomata (Tomlinson and not viviparous, vivipary must have arisen through con- Cox 2000). This inevitably results in a slow transpira- vergent evolution, on a number of separate occasions. tion. Juncosa and Tomlinson (1988) suggested that This suggests that there is some advantage to being vi- vivipary is a simple consequence of the amplification of viparous in a tropical, tidal, or shallow marine envi- the normal torpedo stage of embryo development by the ronment (Hogarth 1999). Seedling development is extended growth of the hypocotyl. In addition, the critical for mangroves because of the salt water, the viviparous species of the Avicennia are generally water movement, the stressful intertidal environment, considered to be the ones most tolerant of high salinity and the need for long-term dispersal by ocean currents (Elster et al. 1999). However, all species of Avicennia (Dawes 1998). Plants with true vivipary are largely show cryptovivipary. Therefore the significance of salt confined to shallow marine systems. In these marine level changes during hypocotyl development to salt tol- habitats, plants face a rather predictable and uniform erance is still open to question. environment within both time and space. The tempera- ture, salinity and chemistry of both the water and sub- strate are subject to minimal and seasonal fluctuations Ion dynamics after hypocotyl transplantation (Elmqvist and Cox 1996). Mangrove propagules are widely distributed and must become established in Anatomical results showed that the hypocotyl of slightly to extremely unstable substrate conditions K. candel has a thick, cutinized and stomatal-free epi- (Tomlinson 1986). It is widely accepted that vivipary in dermis. This effectively hermetically sealed epidermis mangroves has adaptive significance in saline intertidal makes the exchange with the ambient fluid impossible environments (Tomlinson 1986). Current research has or, at best, minimal (Tomlinson and Cox 2000). In our focused on the adaptive significance of vivipary to salt experiments, at stage 1, concentrations of Na+,K+ and tolerance. In our present research, we found no direct Cl– increased gradually and slightly, and substrate evidence of the correlation between vivipary and salt salinity showed no modification of ion concentrations tolerance. Tomlinson (1986) also argued that it was between treatments. The slight increase in Na+,K+ and difficult to assign an adaptation for viviparous seedling Cl– levels was mainly caused by the decrease in water to a single factor. The sizes of propagules of viviparous species (species of genera Rhizophora, Kandelia, Bruguiera and Ceriops) Table 2 Changes in element concentration on a dry weight basis are much larger than those of non-viviparous or cryp- (mg/g dw) during K. candel hypocotyl development. Values are toviviparous species (Tomlinson 1986). Elster et al. means±SD (n=3) (1999) showed that the larger size provides many ad- Stage Mg2+ Ca2+ Na+ K+ Cl– vantages in seedling establishment. Regeneration ex- periments showed that Avicennia germinans and 1 2.0±0.2 3.0±0.1 15.2±3.9 18.3±0.5 11.3±0.3 Laguncularia racemosa are more sensitive to changes in 2 1.7±0.2 2.9±0.5 15.2±1.3 16.3±0.6 10.5±0.9 3 1.4±0.2 2.1±0.6 15.3±1.2 17.4±1.4 10.4±0.6 water level, wind, waves, temperatures exceeding 45°C, 4 1.3±0.4 1.6±0.8 13.7±1.2 15.5±1.2 9.7±0.8 and dry soils than Rhizophora mangle, because the 5 1.2±0.0 1.6±0.2 15.6±0.2 17.5±1.5 10.1±0.4 propagules of A. germinans and L. racemosa are smaller 6 1.1±0.1 1.5±0.2 14.4±0.8 14.0±0.4 9.7±0.4 than those of the latter and Rhizophora mangle is much 7 0.9±0.1 1.3±0.2 13.9±1.7 14.0±0.7 9.4±0.3 more resistant to most detrimental factors observed with 8 0.9±0.0 1.5±0.4 13.0±0.6 16.9±2.3 9.0±0.5 the exception of high salinities (Elster et al. 1999). 1034 The propagules can be set with their lower end 3–4 cm Elster C, Perdomo L, Schnetter ML (1999) Impact of ecological deep into the ground and will neither be harmed by factors on the regeneration of mangroves in the Cie´naga Grande de Santa Marta, Colombia. 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Acta Bot Sin 2:51–58 minans survive for more than 3 weeks when totally Joshi AC (1933) A suggested explanation of the prevalence of vivipary on the sea-shore. J Ecol 21:209–212 submersed (Elster 1997). However, waterlogging exper- Juncosa AM, Tomlinson PB (1988) A historical and taxonomic iments showed that K. candel hypocotyls can germinate synopsis of Rhizophoraceae and Anisophyllaceae. Ann Mo Bot and root even under the same conditions (personal Gard 75:1278–1295 data). Therefore we agree with Dawes (1998) that Lin P (1988) Mangrove vegetation. China Ocean Press, Beijing Lo¨ tschert W, Liemann F (1967) Die salzspeicherung im Keimling vivipary allows a rapid establishment of seedlings in von Rhizophora mangle L. wa¨ hrend der Entwicklung auf der unstable regions. Mutterpflanze. Planta 77:142–156 Tomlinson PB (1986) The botany of mangroves. Cambridge Uni- Acknowledgements Thanks to Dr. N.C. Chen and technicians of versity Press, Cambridge BCH for help in sample analyses. 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