Occurrence and Fruit and Seed Biology of Halophila tricostata Greenway (Hydrocharitaceae)

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1 Aust. J. Mar. Freshwater Res., 1993, 44, Occurrence and Fruit and Seed Biology of Halophila tricostata Greenway (Hydrocharitaceae) J. KUO~, W. Lee ~ o n and g ~ R. G. colesb A Centre for Microscopy and Microanalysis, The University of Western Australia, Nedlands, WA 6009, Australia. Northern Fisheries Centre, Queensland Department of Primary Industries, Box 5396, Cairns, Qld 4871, Australia. Abstract Halophila tricostata Greenway appears to be endemic to eastern Queensland, Australia, and occurs between 14O11'S and 23'45's. It was found at depths from 1.4 to 30 m in well sheltered habitats, including in shallow coastal sites near mangrove-lined estuaries, on the lee side of continental and coralreef islands, and on some commercial prawn-trawling grounds within the Great Barrier Reef lagoon. It grows on predominantly fine mud substratum in small monospecific meadows or mixed with other tropical seagrasses, mostly other Halophila species. Field observations indicate that H. tricostata is an annual angiosperm and produces an estimated seeds m-2 year-'. Halophila tricostata is dioecious. The plant has a horizontal rhizome bearing an erect shoot with eight to twelve nodes and a root at each rhizome node. Except at the first two or three nodes, the mature plants produce a reproductive organ at each node of their rarely branched erect shoot. The reproductive organs and fruits develop and mature acropetally along the erect shoot. There are seeds, with a mean of 41 seeds, per fruit. The seed has a coiled embryo protected by a cotyledon, and an enlarged hypocotyl. The hypocotyl acts as a nutrient store and contains starch, protein and lipid. The seed covering consists of pericarp remains and two thin cuticular layers of seed coat. The surface of the seed covering has numerous fine protrusions. The seed covering becomes loose and is discarded during germination, exposing the hypocotyl. The surface of the hypocotyl develops hair-like unicellular structures during seedling development. The majority of the seeds begin to germinate at 26-2g0C after two weeks of culturing, but germination is not synchronized. The culturing of H. tricostata seedlings beyond the three-leaf stage was not successful. Introduction The distribution and reproductive biology of Halophila tricostata Greenway are among the least known for the Halophila species. Since the first description of H. tricostata from sledge samples near Lizard Island, north-eastern Queensland (Greenway 1979), its presence has been recorded over a much wider distributional range (Coles et al. 1987a, 1987b, 1992; Lee Long et al. 1989). Observations during these surveys indicated that the plant may be much more ephemeral than most other seagrasses, and a monitoring study was implemented to examine this aspect of its biology. Morphological descriptions of fruits and seeds in Halophila are few (Balfour 1878; den Hartog 1970). This has been attributed to the inconspicuous fruits and seeds, which have probably been overlooked (den Hartog 1970). However, recent studies of fruit and seed morphology have been carried out on H. engelmannii Aschers. (McMillan 1986, 1987a, 1988a), H. decipiens Ostenfeld (McMillan 1986~; Parthasarathy et al. 1988b; McMillan and Soong 1989), H. beccarii Aschers. (Parthasarathy et al. 1988a) and H. ovalis (R. Br.) Hook. f. (Kuo and Kirkman 1992). The mature fruits and seeds of H. tricostata have

2 J. Kuo et al. not been previously described (Greenway 1979). The anatomy of fruits and seeds of the Halophila species, in particular the embryo and seed reserves, is not well understood and has been investigated only in H. ovalis (Kuo and Kirkman 1992). The morphology of germination and the establishment of seedlings in Halophila have been described for H. spinulosa (R. Br.) Aschers. (Birch 1981), H. engelmannii (McMillan 1987b; McMillan and Jewett-Smith 1988; Jewett-Smith and McMillan 1990), H. decipiens (McMillan 1988b) and H. ovalis (Kuo and Kirkman 1992). This paper reports on the distribution and habitats of Halophila tricostata and describes the morphology and anatomy of the mature fruits, seeds and seedlings of this species. Materials and Methods Information on the occurrence and distribution of H. tricostata was based mainly on Queensland Department of Primary Industries surveys of Queensland coastal and island areas conducted from 1984 to 1989 (see Coles et al. 1987a, 1987b, 1992; Lee Long et al. 1989, 1992). A further monthly or bimonthly survey was conducted at a permanent site, 16 m below mean sea level on the north-western lee side of Fitzroy Island (a small continental island in the Great Barrier Reef lagoon, 6 km from the mainland, 16"56'S), from October 1987 to February Flowering and fruiting plants of H. tricostata were collected from Fitzroy Island on 23 November Mature fruits and seeds were separated and stored in the dark in aerated recirculating sea water (salinity 35) at 28T for 35 days. Seed counts were carried out for 30 fruits, and seed diameter was measured on 70 seeds. For the germination trials, seeds were sterilized in 90% ethanol for 20 min, then rinsed twice in autoclaved sea water. Seeds were transferred to axenic conditions in 200-mL flasks containing 150 ml of sea water and kept at 26 to 28OC on a 12-h light/dark cycle. Ambient illumination on the experimental jars was pe m-2 s-' (1 pe= 1 pmol), monitored with a Li-Cor Model LI-188B quantum radiometer-photometer. Seed germination and seedling development were recorded and photographed periodically and were also examined by scanning electron microscopy (SEM). For SEM, seeds and seedlings were fixed in 2.5% glutaraldehyde in sea water and dehydrated with graded acetone, then critical-point-dried with C02. Specimens were examined with a Philips Model 505 scanning electron microscope. For anatomical and histochmeical studies, mature fruits with maturing seeds were fixed in 2.5% glutaraldehyde in sea water and embedded in glycol and methacrylate (O'Brien and McCully 1981). Serial sections (2.5 pm thick) were stained with saturated Sudan black B in 70% ethanol for lipids and cuticle, 1% amido black 10B in 7% acetic acid for protein, periodic acid-schiff's reaction (PAS) for starch and other polysaccharides, and 0.05% toluidine blue (ph 4.4) for general cell organization and phenols (see Kuo et al. 1990, 1991; Kuo and Kirkman 1992). Results Habitats and Distribution of H. tricostata Field observations of Halophila tricostata appear as occasional records in a series of reports on the distribution of seagrasses on the eastern Queensland coast and are compiled here (Table 1). The known latitudinal distribution of H. tricostata is from 14'11'S to 23'45's (Fig. 1). The species was found during surveys at depths between 1.4 and 30 m, always in sheltered waters. Sites included shallow-water (<5 m) coastal habitats near mangrove wetlands and estuaries, and deep waters of the Great Barrier Reef lagoon. Additional records have come from benthic otter-trawls engaged in commercial prawn-fishing operations at 20 m depth 6 nautical miles east of Cape Flattery (15's) in November and December 1989 and 1990 and south-east of Fitzroy Island (about 17"s) in December Habitats where H. tricostata were observed and collected all include a substratum of predominantly fine mud, with some carbonate sand and shell grit. Halophila tricostata occurred in small patches of monospecific meadows but was more commonly found mixed with H. decipiens and sometimes with H. ovata Gaud. and H. spinulosa of the Hydrocharitaceae, Halodule uninveris (Forsk.) Aschers., Syringodium isoetifolium (Aschers.)

3 Table 1. Records of occurrence of Halophila tricostata and associated seagrass species in surveys of Queensland coastal and island areasa Date ~ocality~ Latitude Depth Vegetation cover H. tricostata Other speciesc ("3 (m) (all species) (96) (shoots m-') A Based on Queensland Department of Primary Industries surveys (Coles et al. 1987u, 1987b, 1992; Lee Long et al. 1989, 1992). Locality types: *, continental island; #, coral island; +, mangrove-lined estuary and bay. Other seagrasses occurring with Halophila tricostata: CS, Cymodocea serrulata; HD, Halophila decipiens; HO, Halophila ovalis; HS, Halophila spinulosa; HT, Halophila ovata; HU, Halodule uninervk (wide-leafed); SI, Syringodium isoetifolium; ZC, Zostera cupricorni. Hinders Islands Group* Howick Islands Group* Low Islets# Fitzroy Island* Hutchison Island* Dunk Island* Hinchinbrook Island Channelt Magnetic Island* Upstart Bay+ Hook Island* Gladstone Harbour+ HD, HO, HS, HT CS, HD, HO, HS HD HD HD, HT HD, HS, HU, SI HD, HO, HU HD, HO, HU HO, HS, HU None HD, HO, HU, ZC

4 J. Kuo et al. Dandy and Cymodocea serrulata (R. Br.) Aschers. of the Cymodoceaceae, and Zostera capricorni Aschers. of the Zosteraceae (Table 1). Halophila tricostata abundance at the Fitzoy Island site was greatest in October and November, and plants were almost absent from the site between February and September (autumn and winter) each year (Fig. 2). However, the abundance of H. tricostata at the site also varied between years, and the bottom vegetation cover was sometimes less than that at adjacent sites (Fig. 2; Table 1). A density of about 284 erect shoots m-2 was counted at Fitzroy Island in December 1989, of which only about 5-10% were male shoots. From the seed production rate (see below) this equates to an estimated supply of about seeds m-2. Plant Morphology in H. tricostata Halophila tricostata is dioecious, although the morphology and appearance of the mature plant are similar in both male and female plants. Mature plants have a horizontal rhizome that produces an unbranched root and an erect shoot at each rhizome node (Fig. 3). Each erect shoot has eight to twelve unbranched or rarely branched nodes. Each of the first AUSTRALIA '6 - LOW ISLglS - PITZROY ISLAND - HINCHINBROOK ISLAND UPSTART BAY QUEENSLAND Fig. 1. Area map, showing major localities for collections of Halophila tricostata during seagrass surveys and monitoring ( ).

5 Occurrence and Fruits and Seeds of H. tricostata I I 1 I I I I 1, I I I I I l I I I, I /, I I , Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun Aug Oct Dec I Fig. 2. Changes in bottom vegetation cover of Halophila tricostata at Fitzroy Island from October 1987 to January two to three basal nodes normally bear two leaf blades representing 'junior leaves', but these leaves, particularly those from the first node, are usually abscissed in mature reproductive plants (Fig. 3). From the third or fourth node upwards, there is both a rosette of three leaf blades representing 'adult leaves' and a reproductive organ at each node (Fig. 3). The vegetative and reproductive organs develop and mature acropetally along the erect shoot (Figs 4-6). Each male or female flower is covered by two bracts and supported by the rosetted leaves (Figs 4 and 6). The male flower has a short pedicel, which becomes elongated during pollination, and three pale-yellow fused anthers (Fig. 4). The female flower has an ovoid ovary with a hypanthium bearing four to six styles up to 1 cm long (Fig. 5). After fertilization, these styles are detached, leaving the hypanthium on the apical end of the developing fruit (Fig. 6). Fruit and Seed Morphology in H. tricostata Five to eight (average 6.8) fruits are produced per erect shoot of H. tricostata at the site near Fitzroy Island. The maturing fruit has a globose to subglobose shape and is protected by a pair of bracts. A short (- 3 mm) stylar beak or hypanthium drops off after the fruit is detached from the parental erect shoot. The surface of the fruit is smooth and yellowish to brown in colour. From 30 fruits, seed counts vary from 24 to 60 (mean 41) mature seeds per fruit. There are also, on average, five immature or undeveloped seeds per fruit. The seed of H. tricostata is spherical, usually bears short fruit-stalk remains at the apical end, and is slightly apiculated at the basal end (Figs 7, 8 and 21). Seeds average 0.38k0.003 mm (range mm) in diameter. The surface of the seed covering in H. tricostata has numerous fine protrusions, each protrusion being about 2-3 pm in height and some of them being joined to each other (Figs 9 and 10). Fruit and Seed Anatomy in H. tricostata The fruit wall of H. tricostata consists of enlarged epidermal cells and three to four layers of parenchymatous cells (Figs 11 and 12). The outer wall of each epidermal cell is

6 J. Kuo et al. Figs : Portion of a female reproductive plant of Halophila tricostata, showing a horizontal rhizome (Rh) with a root (R) and an erect shoot (ES) bearing a developing fruit (F) at each node from the third or fourth node upwards. x 1. Scale marked in millimetres. 4: Erect shoot with portions of the bracts removed to show the developing male flowers (A) containing fused anthers. x 8. Scale bar, 1 mm. 5: Erect shoot bearing a female flower with four styles (St) and a developing fruit (F). x 1.4. Scale marked in millimetres. 6: A maturing fruit (F) has a distinct hypanthium (H) and is protected by two bracts (B). x6. Scale bar, 2 mm. thickened but not lignified, without a detectable cuticular covering. The inner parenchymatous cells are smaller in size but contain more starch grains (Fig. 12). The seed covering of H. tricostata is a complex structure and consists of pericarp remains and the seed coat (Figs 13-16). Pericarp remains are represented by several cell layers that are flattened and lie parallel to the seed surface. The walls of the outermost cell layer and the outer tangential walls of the second cell layer are thin, but the radial and particularly

7 Occurrence and Fruits and Seeds of H. tricostata Figs , 8: SEM photomicrographs show that a mature seed of Halophila tricostata has a distinct seed stalk (Sk). x 135 (Fig. 7) and x 117 (Fig. 8). Scale bars, 100 pm. 9, 10: The seed surface has numerous fine protrusions, some of which may be fused together. The seed covering also has some debris (asterisk), and it splits to expose the surface of the hypocotyl (Hc). ~477 (Fig. 9) and x 990 (Fig. 10). Scale bars, 20 am (Fig. 9) and 10 pm (Fig. 10). the inner tangential walls of the second-layer cells are thickened (Figs 13 and 14). These thickened walls contain mainly polysaccharides and are not lignified (Figs 13 and 14). In addition, the basal portion of the thickened inner tangential walls possesses numerous vertical fine lines (Figs 13-16) that are rich in protein (Figs 14 and 15) and lipid (Fig. 16). The seed coat is represented by two thin cuticular layers that stain positively with Sudan black B (Fig. 16), indicating that they consist of cuticular and/or fatty material. The inner cuticular layer tightly covers the surface of the hypocotyl epidermal cells (Figs 15 and 16). The seed has a large hypocotyl and a coiled embryo protected by a cotyledon situated near the apical end of the seed (Figs 17-20). The hypocotyl has numerous large uniform cells containing a large amount of starch (Figs 9, 10, 13 and 14), with little protein (Fig. 15) or lipid (Fig. 16).

8 J. Kuo et al. Figs 11 and : Longitudinal section through a maturing fruit of Halophila tricostata, showing the fruit wall (FW) and several developing starch-rich (darkly stained) seeds (S) that have been sectioned in different planes. Each seed is attached to a seed stalk (Sk). Toluidine blue counterstained with PAS. x50. Scale bar, 250 gm. 12: The fruit wall consists of several cell layers containing enlarged epidermal cells (E) and starch (Sh)-rich inner parenchymatous cells covering the developing seeds (S). Toluidine blue counterstained with PAS. x 126. Scale bar, 100 pm. Seed Germination and Seedling Morphology in H. tricostata None of the seeds of H. tricostata germinated during dark storage in the seven weeks after seed collection, but the seeds began to germinate one to two weeks after transfer to illuminated experimental jars. Germination was not synchronized; after two months of culturing, about 30% of seeds had germinated and were at various stages of seedling

9 Occurrence and Fruits and Seeds of H. tricostata 51 Figs The seed covering in Halophila tricostata consists of pericarp remains and the two layers of the seed coat (arrows and arrowheads). The pericarp remains have epidermal cells (E), and the third parenchyma cell layer has thickened inner tangential walls (asterisks) with numerous fine vertical lines. Sh, starch-rich cells. Toluidine blue counterstained with PAS (Fig. 13), amido black 10B counterstained with PAS (Fig. 14), amido black 10B (Fig. 15), and Sudan black B (Fig. 16). All x 435. Scale bars, all 15 pm : Serial sections of a mature seed, showing the relationship between the hypocotyl (Hc), the embryo (Eb) and a cotyledon (C). All toluidine blue. All x 110. Scale bars, all 100 pm.

10 Figs : Germinating seeds (S) and a young seedling of Halophila tricostata in which a cotyledon (C) has emerged from the hypocotyl (Hc) and the first leaf (L) has emerged from the cotyledonary pocket. Note that the seed covering (Sc) has already separated from the hypocotyl. x 2. Scale bar, 5 mm. 22: SEM photomicrograph of a young seedling at the one-leaf stage, showing a cotyledon (C) emerging from the apical end of the hypocotyl (Hc) and a long radicle (Ra) emerging from the basal end. Several hair-like structures are growing from the surface of the hypocotyl, and a discarded seed coat (Sc) is nearby. The first leaf (L) has emerged from the cotyledonary pocket. x 16. Scale bar, 5 mm. 23, 24: SEM photomicrographs of seeds with the seed covering (Sc) removed to show the apical end (asterisk) and basal end (arrowhead) of the hypocotyl as well as the surface of the hypocotyl epidermal cells. x 88 (Fig. 23) and x 112 (Fig. 24). Scale bars, 100 pm. 25: Young seedling at the two-leaf stage. The elongated first leaf (Ll) and second leaf (L2) and the emerging lateral root (R) are growing from the cotyledonary pocket. Note that the cotyledon (C) extends from the apical end of the hypocotyl (Hc) and a long radicle (Ra) extends from the basal end. The hypocotyl is covered with long hair-like structures. x 14. Scale bar, 1 mm.

11 Occurrence and Fruits and Seeds of H. tricostata development. Unfortunately, none of the seedlings developed beyond the three-leaf stage to produce a horizontal rhizome for further development. The initial sign of germination in H. tricostata was a swelling of the seeds, followed by splitting and finally discarding of the outer seed covering (Figs 21 and 22) to expose the rather smooth surface of the hypocotyl (Fig. 23). After the protrusion of the chalaza1 end of the seed, the cotyledon and then the coiled embryo emerged through the split seed coat (Fig. 24). The radicle then developed from the base of the hypocotyl, and there was enlargement of the cotyledonary pocket (Figs 21 and 22). The first true leaf, with only a single midrib, emerged from the cotyledonary pocket (Figs 21 and 22). By this stage, long unicellular hairs were usually growing from the surface of the hypocotyl (Fig. 22). The second and third leaves emerged successively from the sheath of the preceding leaf, and a lateral root developed at the base of the first leaf on the enlarged cotyledonary pocket (Fig. 25). Discussion Greenway (1979) did not find mature fruits of Halophila tricostata, but described the species as having numerous ovules with reticulate testa. The present study found the surface of the seed covering in H. tricostata to have numerous fine protrusions up to 2-3 pm in height. These protrusions appear to resemble what has been described as peg-like projections (about pm in height) in H. spinulosa (Birch 1981) but are different from the reticulate surface of H. engelmannii (McMillan 1987b) and H. ovalis (Birch 1981; Kuo and Kirkman 1992). Whether the appearance of the seed surface can be used for taxonomic and phylogenetic purposes in the genus Halophila remains to be determined. It is still not certain whether the surface sculpturing of the seed covering in Halophila species has any functional significance. The peg-like projections may provide surface friction against the substratum to loosen the seed covering (Birch 1981). On the other hand, the reticulate surface may facilitate positive buoyancy, by the entrapment of air bubbles, for further dispersal (McMillan 1987b). Despite the morphology and appearance of the mature plants and the differing appearance of the seed surface among the Halophila species, the seed anatomy and the morphology and initial development of seedlings are similar in the species that have been studied, including H. spinulosa (Birch 1981), H. engelmannii (McMillan 1987b; McMillan and Jewett-Smith 1988; Jewett-Smith and McMillan 1990), H. decipiens (McMillan 1988b) and H. ovalis (Kuo and Kirkman 1992). Seeds of H. tricostata have a complex seed covering consisting of pericarp remains and a two-layered seed coat, and each seed consists of both a coiled embryo protected by a cotyledon and a large hypocotyl. This type of seed organization appears to be typical of the genus Halophila (Lakshmanan 1963; den Hartog 1970; McMillan 19876; Kuo and Kirkman 1992), although Birch (1981) claimed that only a single integument is present in H. spinulosa. Both layers of the seed coat derive from inner and outer integuments during seed development in H. ovata (Lakshmanan 1963). Probably the most interesting feature during seedling development in the Halophila species is that the discarding of the seed covering ensures the early development of unicellular hair-like structures from the hypocotyl epidermal cells. Similar hairs, known as 'hypocotylar hairs' or the ring of 'anchoring hairs' on the 'hypocotylar collar', are apparently typical of freshwater monocotyledonous seedlings and also occur in some aquatic dicotyledonous seedlings and in a few terrestrial plants (see Arber 1925; Kaul 1978). Seeds of the tropical seagrass Thalassia species have an enlarged pyriform hypocotyl with a flattened base (Maiden and Betche 1909; Orpurt and Boral 1964; Kuo et al. 1991), and apparently only from this flattened portion of the hypocotyl do the anchoring hairs develop (Orpurt and Boral 1964). These anchoring hairs have been shown to be efficient anchors of seeds before the radicle emerges (Birch 1981). The ontogeny and the development of the seed covering

12 J. Kuo et al. in Halophila, and that of the hair-like structure on the hypocotyl during seedling development, deserve further microscopical investigation. A study on nutrient utilization in seed reserves during seedling development in H. ovalis indicates that starch and protein in the hypocotyl are used for the germination and initial development of the seedling until it reaches about the three-leaf stage (Kuo and Kirkman 1992). The nutrients required for further development and growth of the Halophila seedling appear to be obtained from the substratum and the water surrounding the seedling. This phenomenon could explain why, under normal laboratory conditions, successful germination and early development of H. tricostata seedlings are rather easily achieved but are rarely followed by continued growth, as is also the case for H. spinulosa (Birch 1981) and H. ovalis (Kuo and Kirkman 1992). Successful culturing of young seedlings has led to rosettes of six leaves and beyond in H. decipiens (McMillan 1988b) and H. engelmannii (McMillan 1987b; McMillan and Jewett-Smith 1988; Jewett-Smith and McMillan 1990). Field observations at Fitzroy Island indicated that seeds of H. tricostata have a winter dormancy requirement and that the timing of germination is controlled primarily by the temperature and light patterns in the area. It is impossible to determine whether the plants observed at Fitzroy Island in 1989 and 1990 were germinated from seeds produced in previous years (i.e and 1989, respectively). Germination in the laboratory at 26-28OC suggests that germination in H. tricostata is least likely in the low temperatures of autumn and winter and most likely when water temperatures rise with spring conditions. The wide variation in the timing of germination at 26-28OC assures continuous germination during summer. Furthermore, seeds of H. tricostata remain dormant if buried in the sediment or kept in darkness and germinate only when exposed to light. Seeds of H. engelmannii are similar in this respect (McMillan 1987b, 1988a; Jewett-Smith and McMillan 1990), but the mechanisms of both intrinsic and extrinsic factors in seagrass seed germination, as discussed by Birch (1981), are still little understood. Although the phenology of H. tricostata in the Great Barrier Reef is not fully recorded, the continuous field observations at Fitzroy Island indicate that, unlike most other Halophila species, which are perennial, H. tricostata is an annual species. Halophila tricostata germinates from seed, grows, and produces flowers and fruits all within a period of a few months, from about September-October to December-January each year. At the time of seed collection in November-December 1989 and 1990, all plants were reproductive. However, living plants were almost absent from the Fitzroy Island site during the other periods of the year. Late austral spring blooms of H. tricostata appear to germinate from presumably dense beds of seeds, which remain dormant in the fine mud and carbonate substratum during the austral autumn to early spring. This would indicate that there is only a brief period in which conditions are suitable for seed germination, seedling establishment, plant growth and development, and reproduction in H. tricostata. Plants of H. tricostata from Fitzroy Island had, on average, 41 seeds per fruit, higher than any recorded in the literature for the Halophila species. High numbers of seeds per fruit have also been observed in the annual H. decipiens from Panama (McMillan and Soong 1989) and south-western Australia (Kuo and Kirkman 1992 and unpublished observation). The mean number of H. decipiens fruits per square metre reported varies with depth at St Croix in the US Virgin Islands: 10.4 fruits m-2 at 15 m, 9.3 fruits m-2 at 21 m and 7.1 fruits m-2 at 27 m, with the mean number of seeds per fruit being 35.3 (Josselyn et al. 1986). McMillan (1988~) recorded (mean 36.8) seeds per fruit from 20 fruits of H. decipiens from shallow water in Panama. In Halophila species, the lowest average numbers of seeds per fruit reported were 8.6 for H. ovalis from south-western Australia (Kuo and Kirkman 1992), 7.1 for H. engelmannii from Redfish Bay, Texas (McMillan 1987a), and 1-4 for H. beccarii from southern India (Parthasarathy et al. 1988~). The mean seed production for H. tricostata at Fitzroy Island, in the Great Barrier Reef, is calculated as seeds m-2, which far exceeds the reported seeds m-2 for H. decipiens at Toro Point, Panama (McMillan 1988b), and 74 seeds m-2 for H. engelmannii in

13 Occurrence and Fruits and Seeds of H. tricostata Redfish Bay, Texas (McMillan 1986, 1987a, 1988~). The high number of seeds per fruit in H. tricostata further supports this species as being annual and its only means of survival as being seed germination. Keddy (1987) found that the seed production of the annual eelgrass Zostera marina L. was seven times that of perennial shoots. Most other seagrasses are perennial and can therefore be propagated both through sexual reproduction by seed production and through asexual reproduction by rhizome extension or vegetative propagule formation (see Kuo and McComb 1989). The fruits of most Halophila species normally develop on the rhizome, which is buried in sediment, resulting in narrow seed dispersal. In contrast, the fruits of H. tricostata are borne on erect shoots at the centre of a rosette of leaves, and the fruits float and are dispersed by water currents after detachment from the erect shoot, which would result in the wide dispersal of H. tricostata. The negatively buoyant seeds would settle to the bottom after the fruit dehisces. Ongoing collection surveys are required to augment the list of known sites for H. tricostata. Extensive surveys to date have failed to find evidence of this species outside the latitudinal range reported here (Lee Long et al. 1993). However, collections from trawler operators indicate that, within the known latitudinal range, H. tricostata may be a widely distributed annual species. Evidence of deep-water (up to 23 m) feeding by dugong (Dugong dugon) on seagrasses in the Great Barrier Reef lagoon (Lee Long et al. 1989), and the well documented association between seagrasses and commercially important penaeid prawns (Staples 1984; Coles and Lee Long 1985), warrant further investigations of the ecology of H. tricostata and the role of this species in the overall ecology of the Great Barrier Reef lagoon. Acknowledgments We acknowledge the assistance of G. Chisholm, who was vessel skipper and boatman during the surveys and monitoring and who also provided valuable assistance in diving-based studies. We also thank R. Christian, J. Coffey and M. Stevens for technical assistance in microscopical investigations and B. Bright for reading an early draft of this paper. This project was supported by the Australian Research Council, the Great Barrier Reef Marine Park Authority and the Fishing Industry Research and Development Council. References Arber, A. (1925). 'Monocotyledons.' (Cambridge University Press: London.) Balfour, I. B. (1878). On the genus Halophila. Botanical Society of Edinburgh Transactions 13, Birch, W. R. (1981). Morphology of germinating seeds of the seagrass Halophila spinulosa (R. Br.) Aschers. (Hydrocharitaceae). Aquatic Botany 11, Coles, R. G., and Lee Long, W. J. (1985). Juvenile prawn biology and the distribution of seagrass prawn nursery grounds in the south-eastern Gulf of Carpentaria, Queensland. In 'Second Australian National Prawn Seminar'. (Eds P. C. Rothlisberg, B. J. Hill and D. J. Staples.) pp (NPS2: Cleveland, Australia.) Coles, R. G., Lee Long, W. J., Squire, B. A., Squire, L. C., and Bibby, J. M. (1987a). Distribution of seagrasses and associated juvenile commercial penaeid prawns in north-eastern Queensland waters. Australian Journal of Marine and Freshwater Research 38, Coles, R. G., ~ellors, J. E., Bibby, J. M., and Squire, B. A. (1987b). Seagrass beds and juvenile prawn nursery grounds between Bowen and Water Park Point. Queensland Department of Primary Industries Information Series No. QI Coles, R. G., Lee Long, W. J., Helmke, S. E., Bennett, R. E., and Derbyshire, D. J. (1992). Seagrass beds and juvenile prawn and fish nursery grounds, Cairns to Bowen. Queensland Department of Primary Industries Information Series No. QI den Hartog, C. (1970). 'The Sea-grasses of the World.' (North-Holland Publishing: Amsterdam.)

14 J. Kuo et al. Greenway, M. (1979). Halophila tricostata (Hydrocharitaceae), a new species of seagrass from the Great Barrier Reef region. Aquatic Botany 7, Jewett-Smith, J., and McMillan, C. (1990). Germination and seedling development of Halophila engelmannii Aschers. (Hydrocharitaceae) under axenic conditions. Aquatic Botany 36, Josselyn, M., Fonseca, M., Niesen, T., and Larson, R. (1986). Biomass, production and decomposition of a deep water seagrass, Halophila decipiens Ostenf. Aquatic Botany 25, Kaul, R. B. (1978). Morphology of germination and establishment of aquatic seedling in Alismataceae and Hydrocharitaceae. Aquatic Botany 5, Keddy, C. J. (1987). Reproduction of annual eelgrass: variation among habitats and comparison with perennial eelgrass (Zostera marina L.). Aquatic Botany 27, Kuo, J., and Kirkman, H. (1992). Fruits, seeds and germination in the seagrass Halophila ovalis (Hydrocharitaceae). Botanica Marina 35, Kuo, J., and McComb, A. J. (1989). Seagrass taxonomy, structure and development. In 'Biology of Seagrasses: A Treatise on the Biology of Seagrasses with Special Reference to the Australian Region'. (Eds A. W. D. Larkum, A. J. McComb and S. A. Shepherd.) pp (Elsevier: Amsterdam.) Kuo, J., Iizumi, H., Nilsen, B. E., and Aioi, K. (1990). Fruit anatomy, seed germination and seedling development in the Japanese seagrass Phyllospadix (Zosteraceae). Aquatic Botany 37, Kuo, J., Coles, R. G., Lee Long, W. J., and Mellors, J. (1991). Fruits and seeds of Thalassia hemprichii (Hydrocharitaceae) from Queensland, Australia. Aquatic Botany 40, Lakshmanan, K. K. (1963). Embryological studies in the Hydrocharitaceae. 11. Halophila ovata Gaudich. Journal of the Indian Botanical Society 42, Lee Long, W. J., Coles, R. G., Helmke, S. A., and Bennett, R. E. (1989). Seagrass habitats in coastal, mid shelf and reef waters from Lookout Point to Barrow Point in north-eastern Queensland. Queensland Department of Primary Industries Information Series (in press). Lee Long, W. J., Coles, R. G., Derbyshire, K. J., Miller, K. J., and Vidler, K. P. (1992). Seagrass beds and juvenile prawn and fish nursery grounds, Water Park Point to Hervey Bay, Queensland. Queensland Department of Primary Industries Information Series No. QI Lee Long, W. J., Mellors, J. E., and Coles, R. G. (1993). Seagrasses between Cape York and Hervey Bay, Queensland, Australia. Australian Journal of Marine and Freshwater Research 44, Maiden, J. H., and Betche, E. (1909). Notes from the Botanical Gardens, No. 15. On a plant, in fruit, doubtfully referred to Cymodocea. Proceedings of Linnean Society of New South Wales 34, McMillan, C. (1986). Fruits and seeds of Halophila engelmannii (Hydrocharitaceae) in Texas. Contributions in Marine Science 19, 1-8. McMillan, C. (1987~). Recurrence of fruiting of Halophila engelmannii (Hydrocharitaceae) in Redfish Bay, Texas. Contributions in Marine Science 30, McMillan, C. (19876). Seed germination and seedling morphology of the seagrass, Halophila engelmannii (Hydrocharitaceae). Aquatic Botany 28, McMillan, C. (1988~). The seed reserve of Halophila engelmannii (Hydrocharitaceae) in Redfish Bay, Texas. Aquatic Botany 30, McMillan, C. (19886). Seed germination and seedling development of Halophila decipiens Ostenfeld (Hydrocharitaceae) from Panama. Aquatic Botany 31, McMillan, C. (1988~). The seed reserve of Halophila decipiens Ostenfeld (Hydrocharitaceae) in Panama. Aquatic Botany 31, McMillan, C., and Jewett-Smith, J. (1988). The sex ratio and fruit production of laboratory germinated seedlings of Halophila engelmannii Aschers. (Hydrocharitaceae) from Redfish Bay, Texas. Aquatic Botany 32, McMillan, C., and Soong, K. (1989). An annual cycle of flowering, fruiting and seed reserve for Halophila decipiens Ostenfeld (Hydrocharitaceae) in Panama. Aquatic Botany 34, O'Brien, T. P., and McCully, M. E. (1981). 'The Study of Plant Structure. Principles and Selected Methods.' (Termarcarphi: Melbourne.) Orpurt, D. A., and Boral, L. L. (1964). The flowers, fruits and seeds of Thalassia testudinum Konig. Bulletin of Marine Science, the Gulf and Caribbean 14, Parthasarathy, N., Ravikumar, K., and Ramamurthy, K. (1988~). Floral biology and ecology of Halophila beccarii Aschers. (Hydrocharitaceae). Aquatic Botany 31,

15 Occurrence and Fruits and Seeds of H. tricostata Parthasarathy, N., Ravikumar, K., and Ramamurthy, K. (1988b). Halophila decipiens Ostenf. in southern India. Aquatic Botany 32, Staples, D. J. (1984). Habitat requirements of juvenile prawns. In 'The Potential of Aquaculture in Queensland'. (Eds B. R. Pollock and R. H. Quinn.) pp (Queensland Department of Primary Industries Conference Workshop Series No. QC83012.) Manuscript received 14 May 1991; revised and accepted 18 March 1992