Comparative leaf structure and its functional significance in Phyllospadix iwatensis Makino and Phyllospadix japonicus makino (Zosteraceae)

Aquatic Botany, 30 (1988) 169-187

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Elsevier Science Publishers B.V., Amsterd,m - - Printed in The Netherlands

COMPARATIVE LEAF S T R U C T U R E A N D ITS FUNCTIONAL SIGNIFICANCE IN P H Y L L O S P A D I X IWATENSIS MAKINO A N D P H Y L L O S P A D I X J A P O N I C U S MAKINO (ZOSTERACEAE)

JOHN KUO 1, KEIKO AIOI2 and HITOSHI IIZUMI~

1Electron Microscopy Centre, University of Western Australia, Nedlands, WesternAustralia 6009 (Australia) 2Ocean Research Institute, The University of Tokyo, Tokyo 164 (Japan) 30tsuchi Marine Research Centre, Ocean researchInstitute, The University of Tokyo, Akaharna, Otsuchi, Iwate 028-11 (Japan) (Accepted for publication 16 November 1987)

ABSTRACT

Kuo, J., Aioi, K. and Iizumi, H., 1988. Comparative leaf structure and its functional significance in PhyUospadix iwatensis Makino and Phyllospadixjaponicus Makino (Zosteraceae). Aquat. Bot., 30: 169-187. The leaf anatomy and ultrastructure of the Japanese Phyllospadix species have been studied. Subcuticular cavities, which sometimes contain fibrillar substances, are present in the leaf blades but are absent in the leaf sheaths. Blade and sheath epidermal cells have many chloroplasts, mitochondria, lipid droplets and much endoplasmic reticulum but no starch grains. Wall ingrowths are pronounced in the inner tangential wall of epidermal cells and are closely associated with mitochondria. Plasmodesmata are present between adjacent epidermal cells and between epidermal and mesophyll cells, suggesting both symplastic and apoplastic pathways for solute transport. Vascular bundles in both blade and sheath are represented by a single xylem lacuna and phloem tissue containing several nacreous-walled sieve elements. Fibre bundles are associated with the epidermis, vascular bundles and the leaf margin. The walls of fibre cells are thickened, but not lignified, suggesting they provide mechanical support and flexibility for leaves against wave action. The two Japanese PhyUospadixspecies can be distinguished by anatomical features including the leaf surface, shape of the epidermal cells, and distribution and size of fibre bundles.

INTRODUCTION

The genus Phyllospadix is ecologically distinct amongst seagrasses because the plants are normally attached directly to intertidal or subtidal rock substratum to form mat-like colonies. They are distinguished from the other Zosteraceae by being dioecious, and having congested rhizome internodes and short roots (den Hartog, 1970; Tomlinson, 1982 ). There are five species of this genus occurring only in the Northern Pacific, from America to Japan and adjacent 0304-3770/88/$03.50

© 1988 Elsevier Science Publishers B.V.

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regions. According to den Hartog (1970), the Northern American species (P. torreyi Watson, P. scouleri Hook. ) are distinguished from the Alaskan (P. serrulatus Ruprecht ex Aschers. ) and the Japanese species (P. iwatensis Makino, P. japonicus Makino ) by differences in the degree of branching of the reproductive shoots. There are also some differences in the number of roots and leaf veins among the Northern American species ( Sauvageau, 1890, 1891; Phillips, 1979) and the Japanese species (Miki, 1933). The Japanese PhyUospadix species were not included in the monograph on comparative anatomy of the Helobiae by Tomlinson (1982). He stressed that PhyUospadix species are still insufficiently known anatomically to decide whether they can be separated by the anatomical features of their vegetative parts. Furthermore, no ultrastructural investigations on Phyllospadix have been carried out until this study. This paper describes the comparative anatomy and ultrastructure of P. iwatensis and P. japonicus leaves in relation to their adaptation to the marine environment, as well as the usage of anatomical features for separating the species. M A T E R I A L S AND M E T H O D S

Plants of P. japonicus Makino were collected from Kominato and Choshi (Chiba Prefecture) and Ota ( Shimane Prefecture), at 1-3 m depth, in March 1985 and March-May 1986. Plants ofP. iwatensis Makino were obtained from Otsuchi (Iwate Prefecture) and Choshi, at 1-5 m depth, in May 1984 and April-May 1986. Plants were dug up intact with their substratum and transferred to plastic bags containing 2.5-5% glutaraldehyde in seawater. Small segments of vegetative tissues from the mid-point portions of the third leaf blade and from the second leaf sheath were placed in vials containing fixatives. Fixed plants and tissue segments were air-freighted to Perth, Western Australia. For gross anatomical study, free-hand sections were stained with toluidine blue O (pH 4.4) or whole tissues were mounted in distilled water. For histochemical studies the fixed tissues were dehydrated in ethanol, propanol and butanol and subsequently embedded in glycol methacrylate (O'Brien and McCully, 1981 ). The 2.5-/~m-thick sections were stained with toluidine blue 0 (pH 4.4), periodic acid-Schiff's reagent (PAS reaction), amido black 10B and Sudan black B (see Kuo, 1978). Some unstained sections were examined unFigs. 1-6. Scanning electron micrographs. Fig. 1. The surface of the leaf blade of PhyUospadix iwatensis. Scale = 10 ~m. Fig. 2. The surface of the leaf blade of P. japonicus. Scale = 10 #m. Fig. 3. A higher magnification of Fig. 2. Scale = 3/~m. Fig. 4. Transverse sectional view of the leaf blade of P. iwatensis. A = aerenchyma, V = vascular bundles. Scale = 100 ~trn. Fig. 5. The surface of the leaf sheath of P. iwatensis. Scale = 8/~m. Fig. 6. The surface of the leaf sheath of P. japonicus. Scale = 8 ~m.

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der autofluorescent or polarizing light. For scanning electron microscopy, fixed tissues were dehydrated in graded acetone and dried in a critical-point drier with carbon dioxide. Dried specimens were coated with a layer of gold-palladium and examined with a Philips scanning electron microscope 505 at 10-15 kV. For transmission electron microscopy, fixed tissues were post-fixed with 2% osmium tetroxide in 0.025 M phosphate buffer (pH 7), for 2 h at room temperature, then hydrated in graded acetone and embedded in Spurr's resin ( Spurr, 1969). Ultrathin sections were cut with a diamond or glass knife and mounted on 75 X 300-mesh naked grids. Grids were stained with saturated aqueous uranylate acetate and lead citrate (Reynolds, 1963 ) and then examined in a Siemens electron microscope 102 at 80 kV. The 0.5-/~m-thick sections were stained with toluidine blue O (pH 9) at 60°C and examined using an optical microscope. RESULTS

Gross anatomy and histochemistry Each shoot of both P. iwatensis and P. japonicus has three to five leaves, the outermost leaf being the oldest. Blade widths are uniform throughout the leaf length and are about 4.0 and 2.5 m m for P. iwatensis and P. japonicus, respectively. Leaf blades are up to 80 cm long in P. iwatensis and 60 cm in P. japonicus. 'Fin'-like projections occur on the margin of the young leaves but become less distinct on the older leaves. The appearance and density of these projections are similar in both species. Gross anatomy of the leaf blade is similar in both species: a transverse section through the mid-point of the leaf blade revealed a single layer of epidermal Figs. 7-18. Optical micrographs of the leaf blades of P. iwatensis(Figs. 7-12) and P. japonicus {Figs. 13-18). Fig. 7. Fibre bundles (arrowheads) are associated with the epidermis (E) and vascular bundles ( V ). Large aerenchyma (A) is present among the mesophyll tissue (M). Toluidine-blue stained. Scale = 100/~m. Fig. 8. The walls of both epidermal (E) and fibre cells (F) are uniformly stained with toluidine blue. Scale = 30 #m. Fig. 9. The walls of the epidermis (E) and the mesophyll (M) are strongly PAS-reaction positive, but those of the fibre cells (F) are only moderate positive. Scale = 30/tin. Fig. 10. A thin cuticle (-~) is covering the epidermis (E). Sudan-black stained. F = f i b r e cells. Scale = 30 ~tm. Fig. 11. Both the cuticle ( -~ ) and contents of the epidermal cell (E) are positively stained with amido black 10B. Vascular bundles consist of a single xylem element (X) and phloem tissue (P). Fibre bundles (F) are also associated with vascular bundles. Scale=80 #m. Fig. 12. A vascular bundle is surrounded by fibre cells (F) and consists of the phloem tissue (P) containing several nacreous-walled sieve elements (arrowheads) and a single xylem element (X) surrounded by a layer of xylem parenchyma cells. Toluidine-blue stained. Scale = 25 #m.

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cells covering mesophyll tissues, fibre bundles, vascular bundles and air-lacunae (Figs. 4, 7, 13 and 14). The upper and the lower epidermal cells have a similar structure. However, there are some detailed anatomical differences between the two Japanese Phyllospadix species. The most obvious difference is the appearance of the outer surface of the blade epidermal cells which is smooth in P. iwatensis (Figs. 1,7-11 ) but shows regular undulations in P. japonicus owing to the presence of ridge-like projections in each epidermal cell (Figs. 2, 14-18). At a higher magnification (e.g. Fig. 3), each of these ridges appears to be situated about the mid-point of each epidermal cell and running along the longitudinal axis of the cell. The surface of the ridges has some cracks which might be the result of specimen preparation. The non-ridge region of the leaf-blade surface contains numerous small oval-shaped depressions with a diameter of 0.2-0.3/~m (Fig. 3 ). Similar depressions also occur on the leaf blade surface of P. iwatensis, but are absent on the surface of the leaf sheath in both species. The outer tangential wall of each epidermal cell is thicker than the basal wall, while the lateral wall tapers in thickness from the apical to the basal end of each cell (Figs. 8-11). The entire wall of the epidermal cells shows the following histochemical reactions: stains pink-purple with toluidine blue (Fig. 8); has a strongly positive PAS reaction (Fig. 9) ; weakly stained with amido black (Fig. 11 ); strongly birefringent under polarizing light; negatively stained with Sudan black (Fig. 10), indicating that the wall contains hemiceUulose, cellulose and protein but no lignin. The fibre bundles with various cell numbers are normally associated with the epidermis, the leaf margins and vascular bundles. The distribution and size of these bundles are different in the two species. In P. iwatensis, a bundle usually consists of 6-12 cells and is always associated with each epidermal cell (Fig. 7). Whereas in P. japonicus, bundles consist normally of 2-5 cells and are less frequent, occurring every 2-3 epidermal cells (Fig. 14). The walls of the fibre cells are evenly thickened; histochemical investigation suggests that they consist of polysaccharides and cellulose, but do not contain lignin. Fig. 13. A leaf blade of P. japonicus containing three vascular bundles (V). Scale = 200/~m. Fig. 14. Regular ridges occur on the surface of the epidermis (E). Fibre cells (F) are associated with the epidermis (E) and vascular bundles (V). Aerenchyma (A) are present among the mesophyll tissue (M). Scale = 30/~m. Fig. 15. A large group of fibre cells (F) concentrates in the leaf margin. A small 'fin-like' structure (arrow) is also present. E = epidermis. Scale = 30/~m. Fig. 16. Contents of the epidermis (E) and vascular tissue (V) are stained positively with amido black 10B. )<510. Scale=25/~m. Fig. 17. The walls of the epidermis ( E ) and the fibre cells (F) are uniformly stained with toluidine blue. Scale = 10/~m. Fig. 18. The walls of the epidermis (E) and the fibre cells (F) are strongly positive, stained with PAS reaction, but the 'ridge' ( -, ) on the epidermis and the middle lamellae of the fibre cells are only moderately positive with PAS reaction. Scale= 10/nn.

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176 There are five and three vascular bundles in each leaf of P. iwatensis and P. japonicus (Fig. 13 ), respectively, but the structure of the vascular bundles is similar in both species. Unlike Posidonia or Thalassodendron (Kuo, 1978, 1983a), bundle sheath cells can not be recognized easily in Phyllospadix, except by their location and cell size. However, fibre cells are always located in the periphery of vascular bundles (Figs. 11, 14). Phloem tissue comprises several nacreous-walled sieve elements and their adjacent phloem parenchyma cells (Fig. 12 ). The irregular outline of the cell lumen in nacreous-walled sieve tubes reflects variable wall thickness along the length of the sieve tube. Sometimes the cell lumen became occluded. Xylem elements are represented in each vascular bundle by a single large lacuna surrounded by a layer of xylem parenchyma cells (Fig. 12). The 'wall' of the xylem lacuna is thickened but not lignified.

Ultrastructure The cuticle in the leaf blade is thin and has a low electron contrast appearance (Figs. 19 and 20), and is sometimes difficult to recognise (Figs. 24 and 25). There are numerous cavities, of various size, on the surface of the outer tangential wall of the epidermal cells (Figs. 19, 20, 24 and 25 ) and these structures have been termed subcuticular cavities (Barnabas et al., 1977 ). In Phyllospadix, the cavities contain electron-opaque fibrillar substances which appear to be similar to those associated with microorganisms (Figs. 19 and 20). Cavities which occur deep in the wall layers are enlarged in size and also contained fibrillar substances (Fig. 25 ). The bulk of the epidermal cell wall has moderate electron-opaque layers of cross-hatched appearance (Figs. 23 and 25). There is a thin, but electron-dense, inner-wall layer covering the plasmalemma. This inner wall material projects into the cell lumen to form wall ingrowths in the inner tangential wall and the lower portion of the lateral wall (Figs. 22, 23 and 26). The walls of the fibre cells have uniform low electron density with no detectable ultrastructure, however, the middle lamellae of both fibre and epidermal cells are extremely electron-dense (Fig. 21 ). Figs. 19-29 Transmission electron micrographs of the leaf blades of P. iwatensis (Figs. 19-23, 28 and 29) and P. japonicus (Figs. 24-27). Figs. 19 and 20. The cuticle ( ~ ) has low electron density. Several oval-shaped subcuticular cavities are present on the surface of the outermost portion of the outer tangential walls (C) of the epidermal cell.Some cavities contain substances similar to those associated with the microorganisms which lie on the leaf surface (*). Scale for both = 0.5/zm. Figs. 21-23. The bulk of the lateral and the inner tangential walls in the epidermal cell have a cross-hatched appearance. The innermost layer of the wall is thin but electron opaque, and has fine wall ingrowths (1) projecting into the cell lumen. Both chloroplasts (C) and mitochondria (M) are closely associated with wall ingrowths and plasmodesmata (arrowheads). The wall of the fibre cell (F) has low uniform electron density, whereas the middle lamellae (* ) and the walls of the epidermal and fibre cells have high electron density appearance. Scale = Figs. 21 and 22--2

/~m;Fig. 23= 0.5/lm.

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Blade epidermal cells are characterized by the presence of large nuclei and are rich in cytoplasmic contents, including numerous chloroplasts, mitochondria and endoplasmic reticulum in association with ribosomes (Figs. 21 and 24). Chloroplasts have well-developed grana and lipid droplets but lack starch grains (Figs. 21, 24 and 26). Mitochondria with well-developed cristae are more abundant in the basal than the distal portion of the cell (Figs. 21, 23, 24 and 26). Plasmodesmata are present, either singly or as a group between the adjacent epidermal cells (Figs. 21-24) or between the epidermal and mesophyll cells (Fig. 26). They appear as a simple form and are not branched (Figs. 22, 23 and 26). The mature sieve elements have a distinct plasmalemma, stacks of endoplasmic reticulum, mitochondria and plastids which contain protein crystals but lack a nucleus, ribosomes and dictyosomes. The nacreous thickening in sieve elements (Fig. 27) has a low electron-dense appearance and contains m a n y parallel microfibrils, arranged approximately at right angles to the longitudinal axis of the cells. In contrast, the middle lamellae and the walls of adjacent phloem parenchyma cells (Figs. 27 and 28) have an extremely electron-dense appearance. These sieve elements had m a n y symplastic connections to phloem parenchyma cells, via sieve areas along their c o m m o n walls (Fig. 28). In each sieve area the branched plasmodesmata occur in enlarged wall areas of the parenchyma cells.These cells also possess wall ingrowths with coarser projections (Fig. 28). Ultrastructurally, the original xylem wall appears to be hydrolyzed completely and the 'xylem wall' in fact, consists of the middle lamellae and the wall of xylem parenchyma cells. T h e middle lamellae appear as an electron-dense layer and the wall of xylem parenchyma cells that abutted the xylem is thickened (Fig. 29). The xylem parenchyma cells have a conspicuous central vacuole with a thin peripheral cytoplasm, containing chloroplasts and mitochondria (Fig. 29). As in most other seagrasses, the leaf sheath of the Japanese PhyUospadix encloses young developing leaf blades (Figs. 30 and 31 ). The general anatomy of the leaf sheath and structural composition of the sheath fibre and vascular bundles (Figs. 30-35) are similar to those of the blade. However, unlike the blade, the upper (adaxial) sheath epidermal cells contain more chloroplasts Fig. 24. The epidermal cellhas a distinctnucleus (N), many chloroplasts(C) lackingstarch grains;wall ingrowths (*) are more pronounced in the inner tangentialwall.Plasmodesmata (arrowheads) occurbetween the epidermalcells.Epiphyticmicroorganisms (E) are presenton the leafsurface.Scale= 2/~rn. Fig. 25. Small subcuticularcavities (-~ ) occur on the wall surface,whereas the largercavities (arrowheads) are locatedin the middle portionof the wall (W) and contain fibrillarmaterials. Endoplasmic reticulum (E) ispresentin the cytoplasm of the epidermalcell.Scale= 1 #m. Fig.26. Very intensivewall ingrowths (-.) occur in the innertangentialwall.Both chloroplasts (C) and mitochondria (M) are closelyassociatedwith wall ingrowths.Plasmodesmata (arrowheads) alsooccurbetween the epidermalcelland the mesophyllcell.Scale= 1/~m.

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Fig. 27. Anacreous-waUed sieve element (N) has reduced celllumen and the irregularinner wall surface but contains microfibrils in the thickened wall which has low electron density. Walls of phloem parenchyma cells (P) and the middle lamellae of the nacreous-walled sieve elements have an electron opaque appearance. C = chloroplast. Scale = 2/Ira. Fig. 28. Sieve area (arrowhead) connects the sieve tube (N) and phloem parenchyma cell (P) which has wall ingrowths (*). Scale = 0.5/ira. Fig. 29. The original xylem wall completely hydrolyzed and the electron-opaque middle lamellae ( -. ) are directlyabutting the xylem (X). Only the portion of the wall (W) of the xylem parenchyma cell (P) abutting the xylem, is thickened. Scale = 2 #m.

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Figs. 30-35. Optical micrographs of the leaf sheath of P. iwatensis (Figs. 30, 32, 34 and 35) and P. japonicus {Figs. 31 and 33). Figs. 30 and 31. Young leaves ( LE ) are protected by leaf sheaths (SH). A distinct 'fin-like' structure occurs in the margin of the second leaf (LE~) of P. iwatensis (Fig. 30). Scale = 200 #m.

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than the lower epidermis. Anatomical features of the leaf sheath are also different between the two Japanese species. For example, the surface of the upper leaf sheath is relatively smooth in P. iwatensis (Figs. 5 and 32 ) but has regular undulations or ridges in P. japonicus (Figs. 6 and 33). In contrast, the surface of the lower sheath epidermal cells is smooth in both species. DISCUSSION

The anatomical and structural characters of Phyllospadix leaves appear to be in agreement with the uniformity of the anatomical features in marine angiosperm leaves (Tomlinson, 1982). The ultrastructural organization in Japanese Phyllospadix leaves does not differ significantly from those described for other members of the family Zosteraceae (Barnabas et al., 1977, 1980; Barnabas and Guillard, 1979; Jagels, 1983; Kuo, 1983a,b; Kuo et al., 1988). The presence of subcuticular cavities in the blade epidermis of Phyllospadix indicates that the cavities are probably unique for the family Zosteraceae, as they have also been observed in other species in the family such as Zostera capensis Setchell (Barnabas et al., 1977, 1980), Z. marina L. (Jagels, 1983), Z. muelleri Irmisch ex Aschers. (Kuo et al., 1988) and Heterozostera tasmanica (Martens ex Aschers.) den Hartog (Kuo, 1983a) but have not been seen in seagrasses belonging to other families (Kuo, 1983a). Barnabas et al. (1977) suggested that this type of structure might represent a system of absorption or secretion or storage of ions. In a later paper (1980) they further speculated that certain materials might be excreted from the epidermal cells of the leaf through these cavities. However, a critical experimental study on the subcuticular cavities in seagrasses is needed, to determine the precise function of these structures. Histochemical and ultrastructural investigations reveal that the cell walls of Phyllospadix epidermis, as in other seagrasses, contain pectin, protein, cellulose and polysaccharide components (Madea et al., 1966; Ducker et al., 1977; Kuo, 1978; Cambridge and Kuo, 1982). The chemical nature of the cell walls

Figs. 32 and 33. Fibre cells (F) are associated with the epidermis (E) and vascular bundles (P). Note the outer surface of the sheath is smooth in P. iwatensis (Fig. 32 ) but has a wavy appearance in P. japonicus (Fig. 33). The inner surface of the sheath is smooth in both species. PAS/toluidine-blue stained. Scale = 50/~m. Fig. 34. The wall of nacreous-walled sieve elements (-*) is only weakly stained with toluidine blue but the middle larnellae,and the walls of phloem parenchyma cells (P) and of fibre cells (F) are strongly stained. Scale = 30 #m. Fig. 35. T h e walls of nacreous-walled sieve elements are negatively stained with amido black 10B, b u t they are visible owing to t h e cell lumens a n d t h e middle lamellae which are positively stained. F = fibre cells; P = phloem parenchyma cells; -~ = wall of nacreous-walled sieve elements. Scale = 30 /tm.

Fo

184 in seagrasses appears to be more closely related to other marine plants, such as seaweeds, rather than to their evolutionary cousins, i.e. terrestrial angiosperms. These findings suggest that a specialized process of ion exchange between the epidermal cell walls of seagrass leaves and their marine medium may exist. The leaf-blade epidermis in seagrasses is the main site of photosynthesis owing to the high concentration of chloroplasts. The fact that chloroplasts in the epidermis and mesophyll of Phyllospadix do not accumulate starches suggests that the photosynthates may be transferred from the blade epidermis to other parts of the plant as soon as they are produced. The presence of the high density of mitochondria with well-developed cristae in the basal portion of the epidermal cells and the occurrence of wall ingrowths in the inner tangential walls of these cells support this view. As in Z. muelleri (Kuo et ah, 1988), the leaf-blade epidermal cells in both P. iwatensis and P. japonicus are interconnected to each other and to the mesophyll cells through plasmodesmata. Thus, intercellular transport may well involve both symplastic and apoplastic pathways. In contrast, cytoplasmic connections have been reported as absent at the maturity of the blade epidermal cells of Thalassia testudinum Banks ex K~nig, Z. marina (Jagels, 1983), Z. capensis (Barnabas et al., 1977), and Cymodocea serrulata ( R. Br. ) Aschers. ex Magnus, C. rotundata Ehrenb. ex Hempr. ex Aschers. and T. hemprichii (Ehrenb.) Aschers. ( Doohan and Newcomb, 1976), it has been suggested that solute transport only takes place in these species by an apoplastic pathway through wall ingrowths. It is important to note that wall ingrowths associated with the blade epidermis and with the phloem parenchyma cells in PhyUospadix are different in their structural appearance and possibly also in chemical composition. In the epidermal cells, wall ingrowths occur as intense but very fine ingrowths with high electron density (Figs. 21-23, 24 and 26), whereas in phloem parenchyma cells, they appear as sparse and coarse finger projections with a moderate electron-opaque appearance (Figs. 27 and 28). Thus the amplification factor for the plasmalemma would be greater in the blade epidermis than in the phloem parenchyma cells. Whether these structural and possible chemical differences in wall ingrowths play any significant physiological role in relation to solute transport in the marine angiosperms remains to be determined. Wall ingrowths in phloem parenchyma cells have also been reported for Z. capensis (Barnabas and Guillard, 1979) and Z. muelleri (Kuo et al., 1988). The occurrence of nacreous-walled sieve elements in both Japanese Phyllospadix species confirms that the presence of these structures appears to be an unique feature in the Zosteraceae (Kuo, 1983b). Ultrastructural evidence indicates that these sieve elements are functional, but the apparent reduction in cell lumen in certain portions of nacreous-walled sieve elements suggests that the translocation rate in these sieve elements may be reduced. However, this

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possible reduction of translocation rate in these sieve elements can probably be compensated for by active involvement of phloem parenchyma cells in solute translocation. The presence of wall ingrowths in phloem parenchyma cells suggests that active transfer of solute may take place between these two tissues, and that both tissues may operate in long-distance transport of solutes in leaves of the Zosteraceae. On the other hand, in all other seagrasses, with the exception of Halodule, leaves possess normal thin-walled sieve elements and phloem parenchyma cellslack wall ingrowths (Kuo, 1978, 1983a,b). Thus, long-distance transport of solutes may be confined to the sieve elements alone. As in other members of Zosteraceae, e.g. Zostera muelleri (Kuo et al., 1988), xylem elements in Phyllospadix leaves are represented by a single large lumen. Because the original xylem wall is hydrolyzed completely at maturity, the xylem 'wall' in the Zosteraceae in fact contains the walls of xylem parenchyma cells and the middle lamellae which abut the xylem lumen directly. In contrast, in Posidonia leaves, xylem elements usually occur as several small vessels, and portions of their wall are thickened and lignified (Kuo, 1978; Cambridge and Kuo, 1982 ). Despite structural and chemical differences, xylem elements may serve the same function as a pathway for transpiration streams in marine angiosperms. Most seagrasses normally have at least the base of their sheaths covered by sediment and usually lack chloroplasts in the sheath epidermis (Kuo, 1983a). The leaf sheath of Posidonia australis Hook. [ differs from the blade by having a non-porous cuticle (Kuo, 1978 ). In contrast, the plants of Phyllospadix normally grow on the rock substratum and have both their leaf blade and sheath directly exposed to the medium. The presence of numerous chloroplasts in the sheath epidermal cells indicates that the sheath of PhyUospadix, at least in the upper epidermal layer, may also be involved in photosynthesis. The walls of both epidermal and fibre cells in the leaf of PhyUospadix are thickened but not lignified, which may also provide stronger mechanical resistance as well as flexibility - important features for the marine plants which are often subjected to vigorous wave action. Choshi (Chiba Prefecture) on the east coast of Honshu Island, is the southern limit for P. iwatensis and the northern limit for P. japonicus. The former species, which possesses more frequent and larger leaf-fibre bundles, normally occurs in high-energy areas. On the other hand, the latter species, which has less frequent and smaller leaffibre bundles usually occupies low-energy areas. The genus PhyUospadix consists of five species and occurs only in the Northern Pacific. There are some differences in the degree of branching of the reproductive shoots between North American species {P. torreyi; P. scouleri), the Alaskan ( P. serulatus ) and the Japanese species ( P. iwatensis; P. japonicus) (see Introduction ). The present study indicates that anatomical and structural features of the leaves are useful in identifying the two Japanese Phyllospadix species. Besides the number of vascular bundles (Miki, 1933),

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features such as the surface appearance in epidermal cells of the blade and sheath; shape of the epidermal cellin transverse-sectionalview and distribution and size of fibre bundles in the leafblade can be used for identification. Furthermore, leaf gross morphology (den Hartog, 1970; Phillips, 1979) and anatomical features such as distributionof fibrebundles and shape of the epidermal cells (Kuo et al.,in preparation) differbetween the three North American and the Alaskan species. Thus, as predicted by Tomlinson (1982) leaf anatomy can be used in separating species in the genus PhyUospadix. ACKNOWLEDGEMENTS

Our thanks are due to the University of Western Australia,The Australian Academy of Science and the Japan Society for the Promotion of Science who made itpossible for one of us (JK) to visitJapan in 1985 when the work was begun and in 1987 to finish it.W e are indebted to B. Nilsen for technical assistance,to D. Walker and K. Hillman for reading the manuscript, and to Prof. C. den Hartog and an anonymous reviewer for their criticalcomments on the manuscript.

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