Aquatic Botany, 37 (1990) 229-245 229 Elsevier Science Publishers B.V., Amsterdam

Fruit anatomy, seed germination and seedling development in the Japanese seagrass

Phyllospadix ( Zosteraceae )

John Kuo 1, Hitoshi I izumi 2, Bjorg E. Nilsen I and Keiko Aioi 3 ~ Electron Microscopy Centre, The University of Western Australia, Nedlands,

W.A. 6009 (Australia) 20tsuchi Marine Research Centre, Ocean Research Institute, The University of Tokyo, Akahama,

OtsuchL ltwate 028-11 (Japan) 3Ocean Research Institute, The University of Tokyo, Nakano, Tokyo, 164 (Japan)

(Accepted for publication 5 January 1990)

ABSTRACT

Kuo, J., Iizumi, H., Nilsen, B.E. and Aioi, K., 1990. Fruit anatomy, seed germination and seedling development in the Japanese seagrass Phyllospadix (Zosteraceae). Aquat. Bot., 37: 229-245.

Phyllospadix iwatensis Makino and Phyllospadixjaponicus Makino have similar fruit morphology and anatomy. The rhomboid fruit of Japanese Phyllospadix is dark brown in colour and is character- ized by two arms bearing stiff inflected bristles which can act as an anchoring system. The fruit cov- eting consists of a thin cuticular seed coat and pericarp remains mainly fibrous endocarp. In the groove region of the fruit, the cuticular seed coat and endocarp are replaced by nucellus cells with wall in- growths and crushed pigment strands with lignified walls. These tissues appear to control the transfer of nutrients to developing seed. The seed is oval with a small embryo and a large hypocotyl.

The embryo is straight and simple, with the plumule containing three leaf primordia and a pair of root primordia surrounded by a cotyledon. The hypocotyl has a large ventral lobe containing central provascular tissue and two small dorsal lobes. The hypocotyl contains starch, lipid and protein, and acts as a nutrient store. The seed ofP. iwatensis has a dormancy period of ~ 6 weeks and germination eventually reaches ~ 65%, but is not synchronized. During germination the leaves emerge first, and then after at least three young leaves have formed and abscised, the roots emerge, usually > 6 months after the commencement of germination. Utilization of the nutrient reserves is initially from the pe- riphery of the hypocotyl and then progressively towards its centre.

INTRODUCTION

The embryos of seagrasses and freshwater plants have been studied exten- sively in relation to the phylogeny of the Helobiae and the relationship be-

0304-3770/90/$03.50 © 1990 - - Elsevier Science Publishers B.V.

230 J. KUO ET AL.

tween this group and its terrestrial relatives (Tomlinson, 1982). However there is little information about the functional biology of seagrass fruits and seeds.

Seed germination and seedling development have been investigated for various seagrass species under either laboratory conditions or in situ. Seed dormancy has been suspected in the seagrass genera Halophila, Cymodocea, Halodule, Syringodium, Zostera and Heterozostera, whereas seed germina- tion without dormancy has been reported for all other genera including Phil- lospadix (den Hartog, 1970; McMillan, 1983). On the other hand, Miki (1933) found ripe fruits of Phyllospadix iwatensis Makino from August to October, but did not observe germination to commence until May or June of the following year, indicating a long period of seed dormancy in this species.

This paper describes the anatomy of mature fruits and seeds ofP. iwatensis and Phyllospadixjaponicus Makino, and documents germination and the uti- lization of seed nutrient reserves in P. iwatensis.

MATERIALS A N D M E T H O D S

Spadices containing mature fruits of P. iwatensis were collected from sea- grass meadows around Horay Jima, just off Otsuchi Marine Research Centre, University of Tokyo, Otsuchi, Iwate Prefecture, Japan. Collections were made by SCUBA at a depth of 2 m in late July 1987. Fruits of P. japonicus were obtained from Chosi, Chiba Prefecture, at a depth of 1-5 m in July 1986 and 1987.

Mature fruits of P. iwatensis were separated from the spadices and imme- diately placed in culture tanks with continuously running filtered seawater. The culture was maintained at room temperature and under a 12-h photoper- iod provided by 4 × 18-W fluorescent tubes placed ~ 50 cm above the culture tanks. The water temperature varied with the seasons as shown in Table 1.

Seed germination and seedling development were observed, measured and

TABLE 1

Variation of water temperature of cultures throughout the experiment on P. iwatensis Makino

Year Month Water temperature Year Month Water temperature (°c) (°c)

1987 July 13-15 1988 January 8-9 August 16-19 February 6-7 September 19 March 4-6 October 15-18 April 5-6 November 13-14 May 7-10 December 10 June 11-12

July 13-15

JAPANESE SEAGRASS PHYLLOSPADIX 231

photographed at various developmental stages during a period of 12 months. Non-germinated fruits were discarded after 8 months in culture.

For morphological studies, mature fruits of P. iwatensis and P. japonicus were photographed and examined with a Philips scanning electron micro- scope 505 at 15 kV after fruits were dehydrated in an acetone series and a critical point dryer with carbon dioxide.

For anatomical and histochemical studies, mature fruits were fixed in 2.5% glutaraldehyde in seawater for at least 24 h and then dehydrated and embed- ded in glycol methacrylate (O'Brien and McCully, 1981 ). Serial sections (2.5 pm) were cut in sagittal, transverse and longitudinal planes to the axis of the embryo with a Sorval J.B-4 microtome. They were stained with 0.05% tolui- dine blue O (pH 4.4) for general cell organization and phenols; periodic acid- Schiff's (PAS) reaction for starch and other polysaccharides; 1% amido black 10B in 70% acetic acid for storage proteins; and saturated Sudan black B in 70% ethanol for lipids and cuticle (see Kuo, 1978; Kuo et al., 1988 ).

RESULTS

The fruits ofP. iwatensis and P. japonicus

The fruits of Phyllospadix are dark and rhomboid with two arms bearing stiff inflexible bristles (Figs. 1 and 2). The two Japanese Phyllospadix species have similar fruits, but those of P. japonicus are smaller and their arms are formed at right angles to the main fruit body; the arms in P. iwatensis curve inwards towards the main fruit body (Figs. 1 and 2). During seed develop- ment, the pericarp differentiates into a soft exocarp and mesocarp, and a hard fibrous endocarp. When a mature Phyllospadix fruit becomes detached from the spadix of the maternal plant (Fig. 2 ), a distinct scar remains in the groove region at the base of the fruit (Fig. 1 ). The thin exocarp and the outer portion of spongy mesocarp gradually decay and expose the remaining mesocarp and sometimes even the endocarp, particularly in the region of the arms (Figs. 1 and 2 ). This endocarp forms the main body of the two arms with stiff inflex- ible bristles that provide the fruit with an excellent "grappling apparatus". The fruits, by means of these bristles, become easily entangled with marine algae, coralline algae in particular, and other plants on the substratum.

Seed anatomy ofP. iwatensis and P. japonicus

Seeds of both Japanese Phyllospadix species are essentially oval and have a similar anatomy. Serial transverse (Figs. 3-5) and median sagittal sections (Figs. 6-9 ) through a mature fruit reveal that within the seed coat the mature embryo is situated toward the dorsal part of the seed and is provided with a plumule with three leaf primordia. The base of the plumule is the embryo axis

232 J. KUO ETAL.

Fig. 1. Scanning electron micrograph showing the ventral view of a mature fruit of P. japonicus Makino. G: the groove region. Scale: 1 mm.

Fig. 2. Scanning electron micrograph showing the ventral view of a mature fruit of P. iwatensis Makino. FS: fruit stalk. Scale: 1 mm.

which is supported by a pair of root primordia. The embryo is protected by a small cotyledon which appears as a steep triangular structure in sagittal sec- tions (Figs. 6-9). The cotyledon is connected to the hypocotyl which occu- pies the bulk of the seed (Figs. 3-9). Based on the reconstruction of serial sections of transverse, sagittal and longitudinal planes, it appears that the hy- pocotyl has three lobes, namely two smaller dorsal and a large ventral one. The embryo is located between the lobes. A central provascular tissue occurs in the ventral lobe of the hypocotyl and is fused with that of the embryo at the base of the latter (Figs. 5 and 6).

JAPANESE SEAGRASS PHYLLOSPADIX 233

Figs. 3-5. The serial transverse sections of a mature fruit ofP. japonicus at 2100, 1530 and 1160 /tm from the base of the seed, respectively, showing the relationship between the hypocotyl and the embryo. Note that within the fruit (F) , the seed is triangular in shape in transverse sections and the basal portion of the seed is entirely occupied by the hypocotyl (H, Fig. 5 ) which consists of three lobes (a large ventral lobe and two smaller dorsal lobes); the embryo is situated between the lobes (Figs. 3 and 4 ). A central provascular tissue (PV) in the ventral lobe is connected to the base of the embryo (arrow-head, Fig. 5). The embryo contains a plumule with three leaf primordia (P) and a pair of root primordia (R), and is protected by a cotyledon (Co). Tolui- dine blue stained. Scales: all 500/zm.

2 3 4 J. KUO ET AL.

Figs. 6-9. The median sagittal sections of the mature fruit ofP. japonicus (Fig. 6) and P. iwa- tensis (Figs. 7-9 ) showing the relationship between the embryo and the hypocotyl (H) within the fruit covering (F) . Scales: all 500/2m. Figures 6 and 7 show the general organization of the seed. The seed has a small triangular-shaped embryo containing a plumule with leaf primordia (P) and is protected by the cotyledon (Co). A provascular tissue (PV) is present in the hypo- cotyl and is connected to that of the embryo. The groove region of the seed (G) has a special structural arrangement. Toluidine blue stained. Figure 8 shows that starch grains are abundant in the hypocotyl (H) , but there are very few in the cotyledon (Co) and the plumule (P) of the embryo. PAS reaction. In Fig. 9 there is more protein in the cotyledon (Co) and the plumule (P) of the embryo than in the hypocotyl (H). Amido black stained.

JAPAN ESE SEAGRASS PHYLLOSPADIX 235

Figs. 10-13. General anatomy of pericarp remains and the outer hypocotyl of a mature P. iwa- tensis fruit. Note that the hypocotyl (H) stores a large number of starch grains ( S, Figs. 11-13 ), and small amounts of protein (Fig. 12) and lipids (small arrowheads, Fig. 13 ). The hypocotyl epidermal cells (E) are smaller and contain smaller starch grains (Fig. 11 ). The seed coat is represented by a thin cuticular layer (large arrowheads). The pericarp remains consist of tube cells (T) and cross cells (C) of the endocarp, the inner (IM) and the outer (OM) mesocarp. Figure 10, toluidine blue stained. Figure 11, PAS reaction. Figure 12, amido black 10 B stained. Figure 13. Sudan black B stained. Scales: all 50 #m.

2 3 6 J. KUO ET AL.

Histochemical tests indicate that the embryo with its leaf primordia and the cotyledon contain protein (Fig. 9), but little carbohydrate (Fig. 8), whereas the entire hypocotyl stores a large amount of starch grains (Figs. 8 and 11 ) and a small amount of protein (Figs. 9 and 12 ) and lipids (Fig. 13 ), usually located between the starch grains.

The outermost layer of the hypocotyl differs from the rest of the tissue by having smaller cells and containing smaller starch grains. The epidermis of the hypocotyl has no nuclei (Figs. 10-13 ).

Fruit covering in P. iwatensis and P. japonicus

The covering layers of the mature Phyllospadix fruits are composed of the seed coat and the pericarp (Figs. 10-13 ). The seed is covered by the seed coat which is represented by a thin cuticular layer (Figs. 10-13 ). This layer, de- rived from the integument, shows a positive reaction for polysaccharides (Fig. 11 ) and fatty material (Fig. 13 ), but is negatively stained to toluidine blue (Fig. 10 ) and amido black (Fig. 12 ). The pericarp is differentiated into the endocarp, mesocarp and exocarp. The endocarp consists of an inner tube and outer cross cells. Three to five layers of much compressed elongated tube cells run parallel with the longitudinal axis of the embryo (Figs. 10-13 ) and have thickened non-lignified walls with a lignified middle lamella, above which is a single layer of cross cells which have thickened non-lignified walls and a lignified middle lamella (Figs. 10-13 ). The inner mesocarp consists of a layer of four to eight compressed, elongated, rectangular cells with lignified and thickened walls (Figs. 10-13 ). Phenol material in the small lumens of these cells gives the Phyllospadix fruits their characteristic dark brown colour. The outer mesocarp has several layers of elongated cells with thickened and pitted, but not lignified walls (Figs. 10-12 ). Finally the exocarp consists of a single epidermal layer. This epidermis and the outer portion of the mesocarp grad- ually decay away and are not always present in mature fruits (Figs. 10-12 ).

The groove region of the fruit in P. iwatensis and P. japonicus

The composition of the fruit covering in the groove region is markedly dif- ferent from the rest of the fruit. Over the hypocotyl there is no cuticular seed coat or endocarp (tube and cross cells) (Fig. 14). Instead, these are replaced by a nucellus projection and a pigment strand which is connected to the fruit stalk (Fig. 14). This stalk contains vascular tissue to provide a pathway for the transport of essential nutrients from the parent body to the developing fruits. The nucellus projection consists of several layers of cells with thick- ened but not lignified walls (Figs. 15, 17 and 18) and normally becomes crushed in the mature fruit. Sometimes these nucellar cells become transfer cells with numerous very fine wall ingrowths in the inner wall surface (Fig.

JAPANESE SEAGRASS PHYLLOSPADIX 237

Fig. 14. General organization of the groove region in a P. iwatensis fruit. Note that the cuticular seed coat (arrowheads), and the tube (T) and cross (C) cells of the endocarp, are interrupted by the nucellus projection (NP), the pigment strands (PS) and then by the fruit stalk (FS). Amido black and Sudan black stains. Scale: 250/zm.

Figs. 15-18. Detailed anatomy of the groove region of a P. iwatensis fruit. Note that the epider- mal cells of the hypocotyl become cubic and contain small starch grains (Figs. 16 and 17 ). Most of the nucellus cells are crushed (Figs. 15, 17 and 18), but have fine wall ingrowths in their inner wall surfaces (arrows, Fig. 16). The pigment strands (PS) contain phenolics (Fig. 15 ), polysaccharides (Fig. 17) and proteins (Fig. 18). Figure 15, amido black and Sudan black stained. Figures 16 and t 7, PAS reaction. Figure 18, amido black stained. Scales: all 50 pm.

238 J. KUO ETAL.

16 ). T h e p i g m e n t s t r and r e s e m b l e s t ha t o f the te r res t r ia l cereals; it has mu l - t i layers o f c r u s h e d cells wi th l ignif ied walls a n d con t a in s a m o r p h o u s phenol ic , p r o t e i n e o u s a n d c a r b o h y d r a t e m a t e r i a l (Figs. 15, 17 a n d 18 ).

Figs. 19-23. Seed germination of P. iwatensis. Figure 19 shows seedlings, after 2 months of culture, showing different germination stages, with some seedlings having leaves just emerged from the apical end of the fruit. Figure 20 shows a seedling after 4 months of culture, showing the seedling 2-cm long leaves, but without root formation. Figure 21 shows an eight-month-old seedling showing a pair of just-emerged roots (R). Scale: 1 cm. Figure 22 shows a seedling after 10 months of culture showing leaves > 10 cm long. Figure 23 is a higher magnification of Fig. 22 showing a pair of developing roots (R) covered with numerous root hairs. By this stage, several seedling leaves have been shed leaving the sheaths (LS) on the seedling. Scale: 1 cm.

JAPANESE SEAGRASS PHYLLOSPADIX 239

TABLE2

Observations (numbers and percentage) on seed germination and seedling development in P. iwaten- sis Makino under laboratory conditions

Date Ungerminated Emergence Emergence Emergence Emergence of seeds of the of the I st of roots 2nd shoot and

plumule shoot root hairs

28 July 1987 700 0 0 0 0 100%

28 September 1987 650 50 0 0 0 93% 7%

10 November 1987 575 105 45 0 0 88.6% 15% 6.4%

28 January 1988 325 165 210 0 0 46.4% 23.6% 30%

27 April 1988 2451 25 190 230 10 35% 3.6% 27% 33% 1.4%

28 June 1988 - 0 0 0 450 65%

~Ungerminated seeds had been discarded by this date.

Seed germination in P. iwatensis

Seeds first germinated ~ 6 weeks after the fruits were collected and after 4 months only 150 out of 700 seeds (20%) had germinated. Among the young seedlings, various stages of development were present; from seeds which had just opened (Fig. 19 ), to seedlings which had young leaf blades 25 mm long (Fig. 20, Table 2 ). The first leaf ofP. iwatensis has a blade ~ 1 mm long and a sheath 3 mm long. The second leaf has a blade 4-6 mm long and a sheath

6-8 mm long. The first seedling shoot contains three alternating leaf blades up to 80 mm long. After 6 months, 375 seeds of the initial 700 seeds (53.5%) had germinated. The largest seedlings had a shoot consisting of three leaves up to 10 cm long (30%). However none of these seedlings had produced ad- ventitious roots. After 9 months, some seedlings had produced two roots (Fig. 21 ) and by 12 months all 455 seedlings (65%) had produced two short roots, ~ 2 mm long, covered with numerous root hairs (Figs. 22 and 23 ). By this stage, several seedling leaves had senesced and abscissed, leaving basal sheaths attached to the seedlings (Figs. 22 and 23, Table 2).

Utilization of reserves during seed germination in P. iwatensis

As the seed germinates, the fruit becomes slightly swollen and the cotyle- don, containing developing leaves, emerges from the apical tip of the fruit through a slit along the dorso-ventral junction (Fig. 19). Anatomically, the most obvious change takes place in the hypocotyl with the disappearance of

2 4 0 J. KUO ET AL.

Figs. 24-31. A median sagittal section through germinating fruits of P. iwatensis to show the utilization of storage reserves during seed germination. Scales: all 500 #m. Figures 24-26 show a seedling after 2 months of culture: the plumule (P) emerges from the apical end of the fruit (F) . Note that starch (Fig. 25 ) and protein (Fig. 26 ) are the first to disappear from the apical end (asterisks) of the hypocotyi (H) . Figure 24, toluidine blue stained. Figure 25, PAS reaction. Figure 26, amido black stained. Figures 27-29 show seedlings after 4 months of culture; the young seedling leaves (P) grow longer and the root primordia (RP) are further advanced. Note that both protein (Fig. 28) and starch (Figs. 28 and 29) are much reduced in the periphery of

JAPANESE SEAGRASS PHYLLOSPADIX 241

the hypocotyi (H). V: vascular system. Figure 27, toluidine blue stained. Figure 28, PAS reac- tion counterstained with amido black. Figure 29, PAS reaction counterstained with toluidine blue. Figures 30-31 show a seedling after 6 months of culture. Note that both protein (Fig. 30) and starch (Fig. 31 ) have completely disappeared from the hypocotyl (H). The first pair of root primordia (PRj) have not emerged whereas the second pair of root primordia (PR2) are already recognizable. V: vascular system. Figure 30, amido black stained. Figure 31, PAS reac- tion counterstained with toluidine blue.

242 J. KUO ET AL.

storage starch and protein from the apical end of the hypocotyl near the slit (Figs. 24-26). After 3 months in culture, the seedling leaves may be 2 cm long, and storage starch and protein will have gradually disappeared from the periphery of the hypocotyl (Fig. 28). After 4 months, when seedlings carry leaves up to 10 cm long and the first pair of roots is developing, the second pair of root primordia is recognizable (Figs. 27 and 29). At this stage, most of the storage starch and protein have been used up, leaving a small amount near the centre of the hypocotyl at the base of the developing seedling (Figs. 29 and 30). By 8 months, when several seedling leaves have formed, the first pair of roots is ready for emergence and the second pair is still in a less ad- vanced state. At this stage, no detectable carbohydrate or protein remain in the hypocotyl (Fig. 31 ).

D I S C U S S I O N

Phyllospadix is a typical member of the Helobiae, in having no endosperm in mature seeds. The embryo of Zostera has been described as a complex and curved structure (Taylor, 1957a, b) lacking a primary root (Yamashita, 1973 ). Miki (1933 ) reported that the embryo of the Japanese Phyllospadix has characteristics which resemble those of Zostera. The present study shows that the embryo of Phyllospadix is a simple straight structure, consisting of a plumule with three leaf primordia and a pair of root primordia, surrounded by a cotyledon. The epidermal cells of the hypocotyl in Phyllospadix, includ- ing cells in the groove region, are similar to aleurone cells in cereal. One dif- ference is that there are no nuclei in the epidermal cells of the Phyllospadix hypocotyl.

Gibbs ( 1902 ) described the testa in the American Phyllospadix as a single layer of cells with thick lignified cell walls. In fact these cells are part of the endocarp and do not belong to the seed coat. The seed coat of Phyllospadix is only represented by a thin cuticular layer derived from the integument. Sim- ilarly, there is always a membranous material covering the Australian Posi- donia seeds; however in contrast to Phyllospadix, the entire pericarp of Posi- donia is shed when the fruit matures (Kuo, 1983 ).

The pericarp in the mature Phyllospadix fruit is complex, consisting of the exocarp, mesocarp and endocarp, and is responsible for the hardness and col- our of the mature fruits. The stiffbristles on the arms of the Japanese Phyllos- padix fruits mainly consist of fibrous materials of the endocarp with thick- ened and lignified cell walls, as described for the American Phyllospadix species (Gibbs, 1902; den Hartog, 1970 ).

JAPANESE SEAGRASS PHYLLOSPADIX 243

One of many interesting features found in this study is the presence of transfer cells in the nucellus projections. Transfer cells have also been found at the interface between the developing viviparous seedling and its parent plant in the seagrass genera Amphibolis and Thalassodendron (Kuo and Kirk- man, 1990 ). It has been suggested in terrestrial plants that transfer cells play an important role in solute and nutrient transportation to the developing seeds (Pate and Gunning, 1972; Gunning, 1977). It is possible that a major func- tion of the developing nucellar projection and pigment strand in seagrasses, as in the cereals (Zee and O'Brien, 1970; Cochrane and Duffus, 1980; Oparka and Gates, 1982 ), is that they provide an efficient means of controlling, and eventually stopping, the physiological development of the seed. Zee and O'Brien (1970) further suggested that the crushed pigment strand at grain maturity may also provide a route by which water can enter the cereal grain during germination. Whether the pigment strand may have a similar function during seagrass germination remains to be determined.

The break of seed dormancy in Phyllospadix and the emergence of cotyle- don and plumule from the apical end of the fruit may involve both mechani- cal and enzymatic processes. In contrast to Miki's ( 1933 ) field observations, our laboratory study indicates that P. iwatensis can germinate after a short dormancy period of a few weeks, although the initiation of seedling develop- ment is far from synchronous, and finally 65% germination was achieved (Table 2 ). In comparison, up to 89% germination was obtained in situ for the American Phyliospadix scouleri Hook. (Turner, 1983 ). In the present study, some seeds ofP. iwatensis had not germinated after 8 months when the water temperature was < 10 o C. Presumably they would have germinated when the water temperature reached > l 0 °C in June-July. Whether the southern spe- cies P. japonicus would have a wider temperature range for its germination remains to be determined; Miki (1933) recorded that fruits ofP. japonicus ripen in early July and germinate from December to the following January.

Storage products of the Japanese Phyllospadix consist largely of starch, lip- ids and protein, providing nutrients for germination, as has been demon- strated for seeds of Posidonia spp. (Hocking et al., 1980, 1981 ). The present study clearly indicates that starch and protein in Phyllospadix seeds are uti- lized from the periphery of the hypocotyl first and then progressively towards the centre of the hypocotyl or the tissue close to the developing embryo. In cereals, hydrolytic enzymes such as amylases and proteinase have been lo- cated in aleurone and scutellar tissues, and are responsible for the mobiliza- tion of starch and protein stores in the endosperm during grain germination (Bernfeld, 1955; Ashton, 1976 ). Hydrolytic enzymes for the mobilization of seed reserves in seagrasses may probably derive from the embryo, the cotyle- don or even from the hypocotyl itself; a critical study of these aspects is highly recommended.

As in the American Phyllospadix (Gibbs, 1902), roots of the Japanese Phyllospadix emerge late in seedling development, after several seedling leaves

244 J. K U O ET AL.

have been produced and abscissed, and long after the exhaustion of storage materials in the hypocotyl. It is therefore likely that the reserves in the Phil- lospadix seed only help to establish the young shoot and are not directly in- volved in the production of roots; these needs are presumably provided by photosynthate from the young shoot. A preliminary study on nutrient uptake and photosynthesis of P. iwatensis seedlings supports this view (H. Iizumi, unpublished data, 1989). The delay in emergence of roots from the seedling may be closely related to the possession of the two arms or "grappling appa- ratus" in the fruit. The two arms are thought to serve as attachment organs to the substratum or other suitable surface, including plants of the parent mead- ows. A grappling apparatus which is modified from the pericarp for anchoring purposes also occurs in Amphibolis seedlings (Tepper, 1882a, b; Black, 1913; Ducker et al., 1977; McConchie et al., 1982 ). Amphibolis seeds germinate and grow on the parent plant, and the roots emerge after the seedling has detached and becomes anchored. The apparatus may remain for more than a year after seedling release from the parent plants (Kuo and Kirkman, 1990).

In many other seagrasses, including the Australian Posidonia species, roots are produced during germination, shortly after the young leaves emerge, sug- gesting that seed reserves in these species supply both shoot and root devel- opment (J. Kuo, unpublished data, 1989). Taylor (1957b) has described similar root development for Zostera marina L. seedlings. In seagrass species without a grappling apparatus, early production of a root system is essential for anchoring seedlings to the substratum.

ACKNOWLEDGEMENTS

We are grateful to the University of Western Australia, the Australian Academy of Science, the Ocean Research Institute, the University of Tokyo, the Japanese Ministry of Education, Culture and Science, and the Japan So- ciety for the Promotion of Science, for making this collaborative research pos- sible. This study was supported by grants from the Australian Research Coun- cil to the senior author who also wishes to express his sincere thanks to Professor K. Numachi, Professor K. Kawaguchi and the staff at the Otsuchi Marine Research Centre for their help, support and friendship during his stays at Otsuchi. We are indebted to Professor A.J. McComb and Dr. H. Kirkman for reading the manuscript.

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JAPANESE SEAGRASS PHYLLOSPADIX 245

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