Aquatic Botany, 40 ( 1991 ) 165-173 165 Elsevier Science Publishers B.V., Amste rdam

Fruits and seeds of Thalassia hemprichii (Hydrocharitaceae) from Queensland, Australia

John Kuo a, Robert G. Coles b' 1, Warren J. Lee Long b and Jane E. Mellors b aElectron Microscopy Centre, University of Western Australia, Nedlands, W.A. 6009, Australia

b Northern Fisheries Centre, Queensland Department of Primary Industries, Box" 5396, Cairns Mail Centre, Qld. 4871, Australia

(Accepted for publication 22 November 1990)

ABSTRACT

Kuo, J., Coles, R.G., Lee Long, W.J. and Mellors, J.E., 1991. Fruits and seeds of Thalassia hemprichii (Hydrocharitaceae) from Queensland, Australia. Aquat. Bot., 40:165-173.

Fruits of Thalassia hemprichii (Ehrenb.) Aschers. have a dome-shaped capsule and form on a short peduncle. Mature fruits slit open from the upper pan of the fruit and split into six to nine valves to release generally three, but occasionally one or two, pyriform seeds. The seed has an enlarged hypo- cotyl, and a laterally inserted embryo with leaf and root primordia protected by a cotyledon. A well- developed provascular bundle extends from the base of the embryo to the centre of the hypocotyl, where it branches and travels along the hypocotyl axis. The basal two-thirds of the hypocotyl store a large amount of starch with little protein, which is regarded as nutrient storage. A seed coat is absent. There are numerous tannin cells present in the hypocotoyl tissues and the remains of the soft pericarp. Seeds of Thalassia have no dormancy period and they probably begin to germinate before they are released. Nutrient storage is utilised initially from the apical end of the hypocotyl and around the provascular tissues.

INTRODUCTION

The seagrass genus Thalassia consists of two morphologically similar, but geographically disjunct, species in tropical waters. Thalassia testudinum Banks ex K/Snig exists in the Caribbean, whereas Thalassia hemprichii (Ehrenb.) Aschers. occurs from the western Pacific to the West Indian Ocean (East Africa ). These two species have been considered as twin species (den Hartog, 1970).

There are few studies on the fruits and seeds of T. testudinum (Rydberg, 1909; Orpurt and Boral, 1964) and of T. hemprichii (Maiden and Betche, 1909; Pascasio and Santos, 1930). Maiden and Betche present accurate ob-

1Present address: Austral ian Fisheries Service, P.O. Box 376, Thursday Island, Qld. 4875, Australia.

0304-3770 /91 /$03 .50 © 1991 - - Elsevier Science Publishers B.V.

166 J. KUO ET AL.

servations and illustrations, even though the specimen was identified as Cy- modocea (?) ciliata (Forssk. ) Ehrenb.

The anatomy of the seeds, particularly embryo and seed reserves, is totally unknown in these species. This paper reports the morphology and anatomy of fruits and seeds of T. hemprichii from Green Island, Queensland, Australia.

MATERIALS AND METHODS

Fruits with the parent plants of T. hemprichii were collected from the inter- tidal zone of Green Island offCairns, Queensland, Australia, in July 1989, by digging into the coral and sand substratum. Some detached fruits were also found on the shore. For anatomical and histochemical studies, seeds of T. hempriehii were fixed, embedded and serial sectioned as previously de- scribed (Kuo and Kirkman, 1990; Kuo et al., 1990). Sections were stained with 1% amido black 10B in 70% acetic acid for storage proteins; 0.05% to- luidine blue O (pH 4.4) for phenols and general cell organization; periodic acid-Schiff's (PAS) reaction for starch and other polysaccharides; and satu- rated Sudan black B in 70% ethanol for cuticle and lipids. Some PAS-stained sections were counter stained with either toluidine blue or amido black.

RESULTS AND DISCUSSION

The mature fruits of T. hemprichii are attached to the short peduncles (Fig. 1A-C), which are barely raised above the surface of the coral and sand sub- stratum. The fruit is a dome-shaped capsule about 20-25 mm in diameter at its widest, and 25-30 mm in height, with a stylar beak of 2-5 mm (Fig. 1A).

Fig. 1. Fruits and seeds of T. hemprichii (A) The dome-shaped fruit is attached to a short pe- duncle and the surface of the fruits is covered with numerous short trichomes. (B) The mature fruit splits open at the top. (C) The pericarp splits open into six to nine irregular valves to release generally three seeds. (D) The mature pyriform seeds. Scale: 1 cm.

Fig. 2. The median sagittat section of the mature seed of T. hemprichii showing the relationship between the embryo (E) and the hypocotyt (H). A provascular bundle (V) is present in the hypocotyl, and is connected to that of the embryo. Starch grains (dark stained material) are abundant in the basal portion (white H) but are not in the apical portion (black H) of the hypocotyl, there are also very few in the embryo. The remains of the pericarp (PR) do not cover the entire seed. Note that the positions marked as A, B, C and D, correspond to the positions shown in serial transverse sections of Fig. 4-7, respectively. PAS reaction counter-stained with toluidine blue O. Scale: 2 mm.

Fig. 3. The triangular-shaped embryo containing a plumule with leaf primordia (P) and a pair of root primordia (R), is protected by the cotyledon (C). Protein is rich in the embryo but not in the apical portion of the hypocotyl (H). V: provascular bundle. Amido black 10 B stained. Scale: 500 tim.

FRUITS AND SEEDS OF THALASSIA HEMPRICHll 167

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The fruit is bright yellow-green in colour, and the surface of the fruit has numerous short t r ichomes (Fig. 1 A). The mature fruits usually detach at the base of the peduncle, most likely through wave action. Free fruits may remain afloat and provide an excellent means of dispersal. Numerous dehiscent fruits were found on the shore. However, mature fruits may dehisce while still at-

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Figs. 4-7. The serial transverse sections of a mature seed of T. hemprichii correspond to the positions marked as A, B, C and D, respectively, in Fig. 2, showing the relationship between the remains of the pericarp (PR), the hypocotyl (H) containing the provascular bundle (V) and the embryo (E) with leaf primordia (P) protected by the cotyledon (C). Toluidine blue O stained. Scales: all 2 mm.

Figs. 8- l 1. The general anatomy of the remains of the pericarp and the outer hypocotyl of the mature T. hemprichii seed. Both the remains of the pericarp (PR) and 'seed coat' (asterisks) consist of several layers of the thin-walled parenchyma cells. A thin cuticular layer (arrows, Fig. 8), is not continuous and separates two tissues. Note that tannin cells (T) are present in the hypocotyl tissue and the remains of the pericarp. A large number of starch grains are present in the basal (Fig. 11 ), but are absent from the apical portion (Fig. 10) of the hypocotyl. Figure 8, Sudan black stained; Fig. 9, toluidine blue O stained; Fig. 10, PAS reaction; Fig. 11, PAS reac- tion. Scales: Figs. 8-9, 100#m; Figs. 10-11,500/~m.

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tached to a maternal plant. Seed dehiscence results from slitting at the top of the capsule or pericarp (Fig. 1B ). The pericarp splits into six to nine irregular valves, whereas the base of the pericarp is still joined (Fig. 1C). Within the fruit there are usually three, occasionally one or two, seeds (Fig. 1D). These observations confirm those of Maiden and Betche (1909) for T. hernprichii, and of Orpurt and Boral (1964) for T. testudinurn. However, Pascasio and Santos (1930) reported that the mature fruits of T. hernprichii from the Phil- ippines split into "many" valves, and contained three to nine seeds. Rydberg ( 1909 ) also mentioned that the fruits of T. testudinurn had "numerous" seeds.

The brownish pyriform seeds were on average 8 mm in width by 10 mm in height, with the flattened side down. The chalazal end of the embryo is pointed upward so that the plumule can emerge (Fig. 2). Because of the pyriform shape, the seed is always kept standing in an upright position in water, that is with the cone-shaped cotyledon upward.

A longitudinal median section through the axis of the seed reveals that the pyriform seed has a large hypocotyl fused with the cotyledon and an embryo laterally inserted about one-third from the base of the seed (Fig. 2; see also Figs. 5 and 6). The embryo contains a plumule with developing leaves and a pair of root primordia, and it is rich in protein with little starch storage (Figs. 2 and 3 ). The seed of T. hernprichii is bare and it does not have a distinct seed coat. Only a certain portion, particularly in the apical part of the seed, is cov- ered by the remains of the soft pericarp and these are easily removed (Figs. 2, 4-7 ). Histochemical stains indicate a discontinuous cuticular layer, and the remains of the integuments are occasionally present between the hypoco- tyl and the remains of the pericarp (Fig. 8). The epidermal and the outer hypocotyl tissues, as well as the remains of the pericarp, sometimes contain tannin cells (Figs. 8-11 ). These phenolic substances may protect seeds against the invasion of micro-organisms, but are probably not associated with seed dormancy and germination. The provascular bundle extends from the base of the embryo to the centre of the hypocotyl, where it branches into two. One branch continues upward to about one-third from the apical end of the hy- pocotyl where it ends blindly. The other branch extends downward to the base of the hypocotyl (Figs. 2, 4-7). The apical one-third of the hypocotyl does not store starch and proteins (Figs. 2, 4-7), but probably stored water or

Fig. 12. A median sagittal section through the seed of T. hemprichii immediately released from the fruit to show that the nutrient reserve, particularly starch (white H ) has been reduced slightly from the apical portion of the hypocotyl (black H), and the tissues around the provascular bundle (V). The embryo with leaf ( P ) and root ( R ) primordia is protected by a cotyledon (C). PAS reaction counter-stained with toluidine blue O. Scale: 2 mm.

Figs. 13-14. Higher magnification of longitudinal (Fig. 13) and transverse (Fig. 14) sections through the provascular bundle of the hypocotyl (H) to show it contains xylem vessels (x) and sieve tubes (s). Figure 13, PAS reaction; Fig. 14, toluidine blue O stained. Scales: both 50#m.

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172 J. KUO ET AL.

other soluble substances which were removed during the specimen prepara- tion for the anatomical investigation. On the other hand, the basal two-thirds of the hypocotyl contain a large amount of starch, with small amounts of lip- ids and proteins (Figs. 2, 4-7 ), and this is considered to be the nutrient stor- age for seed germination. Similar nutrient storage also occurs in the hypocotyl of Posidonia of the family Posidoniaceae (Kuo and McComb, 1989) and Phyllospadix of the family Zosteraceae (Kuo et al., 1990). In contrast, seeds of seagrasses which have viviparous seedlings, viz. Thalassodendron and Am- phibolis of the family Cymodoceaceae, apparently do not store starch and protein. Instead, numerous transfer cells are present at the interface between the germinating seedling and the parent plant, suggesting these seedlings ob- tain the nutrients required for growth directly from their parent plants (Kuo and Kirkman, 1990).

Seeds of Thalassia immediately released from the fruit show that the nu- trient storage, starch and protein in particular, has already been utilised from the apical end of the hypocotyl (Fig. 12) and from the tissues adjacent to the provascular bundle (Fig. 13). This vascular bundle contains several differ- entiating sieve tubes and xylem elements (Figs. 13 and 14). These observa- tions suggest that seeds of Thalassia probably have no dormancy period, and may begin to germinate well before they are released from the mature fruit. Seed germination without distinct dormancy has also been found in the sea- grass genera Enhalus, Posidonia, Thalassodendron and Amphibolis. Seed dor- mancy in seagrasses appears to be correlated well with the composition of the seed covering. In those genera without dormancy, the seed is usually covered by the remains of a soft pericarp and lacks a distinct seed coat, on the other hand, seeds with a dormancy period normally have a hard pericarp and a distinct seed coat (Kuo et al., 1990).

Nutrient storage of Thalassia is utilised initially from the apical end of the hypocotyl and around the provascular tissues. The pattern of nutrient utilis- ation from the hypocotyl of Thalassia appears to differ from that of Posidonia and Phyllospadix. In the latter genera, nutrients are initially consumed from the periphery, and then progressively toward the centre of the hypocotyl (Kuo et al., 1990).

ACKNOWLEDGEMENTS

The research was supported by a grant provided by Australian Research Council. The expert assistance of I. Tierney, M. Timmins, B. Bright and M. Stevens is gratefully acknowledged.

REFERENCES

Den Hartog, C., 1970. The Sea-Grasses of the World. North-Holland, Amsterdam, 275 pp. Kuo, J. and Kirkman, H., 1990. Anatomy of viviparous seagrass seedlings of Amphibolis and

Thalassodendron and their nutrient supply. Bot. Mar., 33:117-126.

FRUITS AND SEEDS OF THALASSIA HEMPRICHII 173

Kuo, J. and McComb, A.J., 1989. Seagrass taxonomy, structure and development. In: A.W.D. Larkum, A.J. McComb and S.A. Shepherd (Editors), Biology of Seagrasses. A Treatise on the Biology of Seagrasses with Special Reference to the Australasian Region. Elsevier, Am- sterdam, pp. 6-73.

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

Maiden, J.H. and Betche, E., 1909. Notes from the Botanical Gardens. No. 15. On a plant, in fruit, doubtfully referred to Cymodocea. Proc. Einn. Soc. N.S.W., 34: 585-587.

Orpurt, D.A. and Boral, L.L., 1964. The flowers, fruits and seeds of Thalassia testudinum Koe- nig. Bull. Mar. Sci. Gulf Caribb., 14: 296-302.

Pascasio, J.F. and Santos, J.K., 1930. A critical morphological study of Thalassia hemprichii (Ehrenb.) Aschers. from the Philippines. Nat. Appl. Sci. Bull., 1: 1-19.

Rydberg, P.A., 1909. The flowers and fruits of the turtle grass (Thalassia). J.N.Y. Bot. Gard., 10: 261-265.