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Morphology of germinating seeds of the seagrass Halophila spinulosa (R.Br.) Aschers. (Hydrocharitaceae)
Aquatic Botany, 11 (1981) 79 # 0 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
79
MORPHOLOGY OF GERMINATING SEEDS OF THE SEAGRASS HALOPHILA SPINULOSA (R.Br.)ASCHERS. (HYDROCHARITACEAE)
W.R. BIRCH
Botany Department, James Cook University of North Queensland, Queensland 4811 (Australia) (Accepted 12 February 1981 )
ABSTRACT
Birch, W.R., 1981. The morphology of germinating seeds of the seagrass Halophila spinulosa (R.Br.) Aschers. (Hydrocharitaceae). Aquat. Bot., 11: 79--90. Laboratory germination of field harvested seeds of Halophila spinulosa (R.Br.) Aschers. showed a period of almost complete dormancy for about five weeks; thereafter the germination rate was high. All seedlings raised by various laboratory culture methods died at the second or third leaf stage. Halophila spinulosa (and probably H. ovalis (R.Br.) Hook. f.) has an unusual mechanism for early shedding of the testa from the seed from whose naked surface there then develops a profuse growth of 'seed-hairs'; these have an anchoring function before emergence of the radicle. This mechanism is likely to occur in all Halophila species because they have, so far as is known, coiled cotyledons.
INTRODUCTION
Halophila is a genus of some eight species (den Hartog, 1970) which grow in the tropics and subtropics. The sub-littoral species H. spinulosa (R.Br.) Aschers. is widely distributed in Malaysian and tropical Australian waters where it may grow alone or mixed with other seagrasses. Observations in Townsville waters indicate that H. spinulosa can often be found flourishing along tide-swept channels where the current is t o o fast for Cymodocea and Halodule. There are no published observations on the germination or seedling morphology of Halophila spinulosa, hence, a study of capsules and seeds of this species from the strand line on the beach at Cape Palarenda, Townsville, Queensland, was started in early October 1978. The main object was a morphological study, with incidental non-quantitative observations on the germination of the seeds. The release of the seeds and fruits and their subsequent deposition along the Palarenda strand must have followed a more or less massive synchronous flowering of the seagrass somewhere in the bay, followed b y an equally syn-
0304-3770/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
80 chronous abscission of the capsules, which float. The capsules were cast up in large numbers along at least a kilometre of beach during a two-or threeday period. The capsules dehisce easily on drying or with rough handling, and the released seeds, being denser than seawater, sink. They are then almost invisible among the sand grains. METHODS Capsules and seeds were collected and stored overnight in seawater. They were washed in seawater and all debris removed. The cleaned seed was stored in an open 3 1 beaker of aerated seawater on a b o t t o m substrate of clean sand and broken seashell at ambient laboratory temperatures (ca. 26 ° C) in fluorescent light (12 h photoperiod, 6 #E m -2 s-l). The seawater for the seed storage was taken from the School of Biology's seawater aquarium which draws its water from inshore waters close to Townsville. Experimental treatments used filtered oceanic seawater. Samples of seed were withdrawn from the large storage beaker and transferred to 50-ml vials or petri dishes, in order to try and increase the rate of germination. Treatments included the use of: (1) (2) (3) (4) (5) (6)
diluted seawater (1/1 or 2/1); darkness or low (6 g E m -2 s-1) or high light intensity (25/~E m -2 s -1); Schreiber's nutrient solution; seed scarification; 25 or 30°C constant temperatures; sandy shell-grit substrate or no substrate.
Scarification was attempted b y shaking the seed vigorously on an orbital shaker when mixed with coarse sand for 20 or 40 rain. In the first three weeks after collection only t w o seeds germinated in the storage beaker. Ungerminated samples were then taken from it and subjected to the various treatments. Samples of 50 seeds were sometimes used, but usually approximately equal volumes of seeds were taken b y spatula. Approximately 50 ml of culture solution was used for each treatment. Water lost from the vials b y evaporation was replenished as necessary during the period of the observations. Seeds and seedlings were photographed in situ, or using a Siemens SEM after specimens had been freeze-dried, and then coated in the usual way; figures are based on tracings made of live specimens using a Nikon profile projector. Most treatments were run for six weeks before being discarded, b u t the Schreiber's treatments under high light intensity were discarded after three weeks because of dense algal growth.
81 RESULTS
Germination No consistent germination trends were noted, regardless of treatment. Under high light intensity and Schreiber's solution germination was only ca. 1%. Sporadic mass germination occurred in localised patches in some vials, and in the storage beaker about five weeks after collection. This resulted in clumps of as many as 20 seedlings being tangled together while nearby other seeds remained ungerminated. A scan for ungerminated seeds, without counting or separation of them from the substrate suggested that most of the seeds in the storage beaker had germinated three months after collection, when observations ceased.
Seed morphology Standard seedling terminology is used even though there may be some doubt as to the homology of the cotyledon of Halophila spinulosa. The seeds
Fig. 1./-/. spinulosa seed with intact testa (bar scale 0.75 rnrn)
82
are white, oval and 0.75 m m long. The testa is characteristically sculptured with small peg-like projections (Figs. 1, 2 and 3). The following germination stages were recognised: (1) Splitting of the sculptured testa, which may be shed before the emergence of the cotyledon and radicle. This procedure does not seem to be as c o m m o n as stage 2; a smooth naked seed is left (Fig. 2). (2) Uncoiling of a coiled cotyledon from its pocket at the apex of the seed; the tip of the cotyledon usually carries with it the whole of the dehiscing testa (Fig. 4), unless shed previously as in stage 1. (3) The radicle starts to emerge. (4) Concomitant with stage 3, or even before it, long unicellular superficial hairs develop from the surface of the naked seed (Figs. 5 and 6). (5) Development of root hairs behind the apex of the radicle. (6) Extension of the cotyledon and enlargement of the cotyledonary pocket containing the first true leaf (Figs. 7 and 8). (7) Emergence of the first true leaf from the cotyledonary pocket; this leaf has only one vein, the midrib, and a smooth margin.
Fig. 2. H. spinulosa seeds, one (s) w i t h testa shed (bar scale 0.75 mm).
83
Fig. 3. Peg sculpturing of the testa of H. spinuiosa (bar scale 50 urn).
(8) Development of a lateral root primodium at the base of the first leaf at the node (Fig. 9). (9) Extension of the lateral root and the development of m a n y root hairs from it (Fig. 10). (10) Protrusion of the second leaf (three-veined) in the axil of the first leaf (Fig. 11). (11) Protrusion of the third leaf (three-veined) distichous to the second leaf. Under laboratory conditions it t o o k about 10 weeks for a seedling to reach this stage; all seedlings then died (or previously at stage 10) whatever culture treatments were used. These ranged from continuing in normal seawater or Schreiber's culture solution, to transplanting to sand and shell
84
Fig. 4. Germinating seeds of H. spinulosa in situ, showing the testas carried up on the elongated cotyledons (bar scale 10 mm).
mixtures, or even to loam or clay in seawater. Chloromycin was used in some cultures in an a t t e m p t to control algal growth. DISCUSSION The germinations showed that Halophila spinulosa seeds have a d o r m a n t period of at least one m o n t h before germination starts. This dormancy, coupled with the b u o y a n c y of the capsules m a y enhance propagule dispersal. However, that many capsules can be washed-up above the intertidal zone, can only result in many seeds reaching habitats inappropriate to the ecological range. Both Thalassia testudinum Banks ex KSnig and Zostera marina L. have been reported to be capable of immediate germination (Taylor, 1957 Thorhaug, 1974), though winter dormancy is more normal with Zostera. The tropical Thalassodendron ciliatum (Forsk.) den Hartog is viviparous (Isaac, 1969) as are b o t h the temperate-water species of Amphibolis (den Hartog, 1970). A spectrum of establishment strategies is thus exploited b y the seagrasses. The strategies so far investigated point to rapid, rather than to long delaying mechanisms for the control of germination. The short delay in the germination of Halophila spinulosa can be due to one, or more, of a number of intrinsic or extrinsic factors. Intrinsic factors could include the need for a ripening period of the e m b r y o , and the presence of germination inhibitors in the e m b r y o , endosperm, or testa, which need to be leached o u t before germination can occur. Of these, the ephemeral
85
Fig. 5. Naked seed of H. spinulosa with seed-hairs arising from the epidermis (bar scale
0.25 ram).
testa (see below) makes physical impermeability, and the presence of inhibitots in it, unlikely. This leaves after ripening, or internal inhibition as likely mechanisms for delaying germination. Extrinsic environmental factors as major controls in germination seem unlikely, unless in combinations of wider ranges of the treatments than were used in this exploratory work. It will be recalled that treatments involved combinations of three light levels, t w o temperature regimes, reduced salinity, absence or presence of substrate, scarification, and with or w i t h o u t nutrient solution. L o w oxygen concentration as a major factor can be eliminated because germination eventually occurred after five weeks in b o t h the seedstore beaker, which was always vigorously aerated with an aquarium aerator, and in the treatment vials, which were not artificially aerated. Intrinsic control of d o r m a n c y in H. spinulosa is indicated. The likelihood that extrinsic factors are less important than intrinsic factors in the control of seagrass germination is also suggested b y work on Zostera
86
C
- - S
I
c
Lt
I
R
5
--L
I
MM
!]"
Lt ~ \
C
I
LR
i
Figs. 6--11 (from t o p left to b o t t o m right). Stages in the germination of H. spinulosa from profile projection tracings (6, 8, 9, 10, 11 ) and showing emergence of first leaf from cotyledonary p o c k e t (Fig. 7, bar scale 0.25 mm): (C) = cotyledon; (CF) = cotyledonary pocket; (S) ffi seed; (R) ffi radicle; (L1) = first leaf; (L2) ffi second leaf; (LR) ffi lateral root.
87
marina L. and Z. japonica Aschers. and Graebn. The former germinated equally well in light or dark (Turin, 1938), and germination of neither species was affected by a wide range of chemical and organic substances (Arasaki, quoted in Taylor, 1957b). The complete seedling mortality in culture is unexplained. Lack of water movement is unlikely because the seed-storage beaker was aerated vigorously enough to keep the water moving. Nutritionally, starch was still present in the endosperm when the seedlings started to die, so a mineral or trace element imbalance could be implicated. Damage from microorganisms is also a possibility; seedlings of Thalassia testudinum Banks ex KSnig have been reported to be susceptible to fungal and bacterial attack in laboratory culture though these were not seen in field specimens (Thorhaug, 1974). Finally, the oxidation-reduction potential of the substrate is another possible factor, but seedlings died regardless of the substrate. The seeds of Halophila spinulosa, with their peg-like sculpturings, are smaller than those of H. ovalis (Fig. 12) which have a more or less rectangular sculpturing of the testa (Fig. 13). The coiled cotyledon was recorded first for H. ovalis (R.Br.) Hook. f. (Balfour, 1877), but not apparently until now for H. spinulosa. The uncoiling and expansion of the cotyledon during germination is, in H. spinulosa, a means of discarding the testa, so facilitating an early
Fig. 12. Comparison of seedlings of (A) H. ovalis and (B) H. spinulosa.
88
Fig. 13. H. ovalis seed; rectangular sculpturing of the testa (bar scale 0.25 mm). development of the hairs from the seed surface. In culturing on a sandy substrate these seed-hairs can be seen to be efficient anchors of the seed before the radicle emerges. The m e t h o d of discarding the testa, and the anchoring seed-hairs are t w o remarkable morphological attributes which are unique n o t only to H. spinulosa b u t probably to the genus Halophila as a whole. This can be predicted because coiled cotyledons are unlikely to have different functions in different species. It should be n o t e d that specimen A (Fig. 12) was observed growing among H. spinulosa seedlings; it was recognised by its greater c o t y l e d o n and seed size. Comparisons of locally harvested authentic H. ovalis seeds left no d o u b t that specimen A was H. ovalis even though the scultured testa was n o t attached; it was however, loose nearby in the vial. The nearest morphologically comparable plants would appear to be aquatic freshwater monocotyledons;Limnocharis flava (L.) Buch. has been shown to produce anchoring root hairs from the base of the h y p o c o t y l while Lophotocarpus, Alisma, and Echinodorus spp. rely on adventitious roots for early anchorage rather than the radicle (Kaul, 1978); in this respect they resemble Zostera marina, which does n o t develop its radicle properly (Taylor, 1957a). Zannichellia polycarpa Nolte. and Elatine hexandra D.C. also
89 produce hairs from the hypocotyl or 'collett' (Arber, 1920). By contrast, Thalassia testudinum KSnig (which has a loose seed-coat which slips off easily) apparently has a flattened basally-positioned cotyledon which does not uncoil, and from whose lower surface there develops a profuse growth of anchoring hairs (Orpurt and Bored, 1964). All produce roothair-like hairs, but none from the seed. Zostera is remarkable in its vascularless laterally developed tuber-like hairy hypocotyl (Taylor, 1957b); the lack of vasculation in this organ is suggestive of the vascularless, often hairy, epiblast in some Gramineae (Birch, 1963). The wall sculpturings may be of some functional significance in providing surface friction against the substrate whereby the testa is loosened around the seed before emergence of the coiled cotyledon. It may be significant here that at seed shedding there is, functionally, only a single integument which is only one cell thick in Halophila spinulosa. In H. ovalis the double integuments, each two-celled thick, which have been demonstrated in its early post-fertilisation stages later become compressed and only the single outer layer of cells of the outer integument remains intact; the other cells disintegrate to form a discontinuous papery layer (Lakshmanan, 1963), and so, as with H. spinulosa, finally provide a delicate loosely-fitting testa which can easily be ruptured by frictional forces. A similar disintegration of the inner integument and the freeing of it from the outer integument was reported in Zostera marina L; at maturity, however, the outer integument became lignified in this species (Taylor, 1957a). The morphological identity of the cotyledon in Halophila is of some interest. Under low light intensities the 'hypocotylar' region can elongate greatly (Fig. 12) carrying with it the node from which the plumule and leaves develop laterally. This agrees with Lakshmanan's (1962) figures for H. ovata Gaud. in which he clearly shows a recognisable lateral cotyledon. However the relative positions of cotyledon and epicotyl at the earlier proembryo stages were more terminally placed. Zostera marina also has this capability for hypocotylar extension; it helps overcome too deep burial of the seeds in silt (Taylor, 1957b). The same function is likely in Halophila. Finally, the seasonality of fruiting in Halophila spinulosa should be mentioned. Observations in 1979, the year after the present study, confirmed a fruiting period in October, but it was not nearly so marked as the year before. October has been noted as a fruiting period elsewhere (den Hartog, 1970), but some fertile specimens have also been collected in March, and October--December. The evidence, so far, suggests that H. spinulosa has a main fruiting period in spring, about three months after the tropical winter which occurs in June--July in southern latitudes. ACKNOWLEDGEMENTS
The author is grateful to J. Darley for his help with the electron microscopy and to his wife for her help with the collection of material.
90 REFERENCES Arber, A., 1920. Water Plants. A Study of Aquatic Angiosperms. Cambridge University Press, Cambridge, 436pp. Balfour, I.B., 1877--78. On the genus Halophila. Bot Soc. Edinburgh, 13: 290--343. Birch, W.R., 1963. Epiblast in Gramineae. Nature (London), 198 (4877): 304. Den Hartog, C., 1970. The Seagrasses of the World. North-Holland, Amsterdam, 275 pp. Isaac, F.M., 1969. Floral structure and germination in Cymodocea cil~ta. Phytomorphology, 19: 44--51. Kaul, R.B., 1978. Morphology of germination and establishment of aquatic seedlings in Alismataceae and Hydrocharitaceae. Aquat. Bot., 5: 139--147. Lakshmanan, K.K., 1962. The origin of epicotylary meristems and cotyledons in H. ovata Gaud. Ann. Bot., 26: 243--249. Lakshmanan, K.K., 1963. Embryological studies in the Hydrocharitaceae. II. H. ovata Gaudich. Indian Bot. Soc. J., 42: 16--18. Orpurt, P~A. and Baral, L.L., 1964. The flowers, fruit and seeds of Thalassia testudinum K~nig. Bull. Mar. Sci. Gulf Caribb., 14: 296--302. Taylor, A.R.A., 1957a. Studies on the development of Zostera marina L. I. Embryo and seed. Can. J. Bot., 35: 477--499. Taylor, A.R.A., 1957b. Studies on the development of Zostera marina L. II. Germination and seedling development. Can. J. Bot., 35: 681--695. Thorhaug, A., 1974. Transplantation of the seagrass Thalassia testudinum KSnig. Aquaculture, 4: 177--183. Tutin, T.G., 1938. The autecology of Zostera marina in relation to its wasting disease. New Phytol., 37 : 50--71.
79
MORPHOLOGY OF GERMINATING SEEDS OF THE SEAGRASS HALOPHILA SPINULOSA (R.Br.)ASCHERS. (HYDROCHARITACEAE)
W.R. BIRCH
Botany Department, James Cook University of North Queensland, Queensland 4811 (Australia) (Accepted 12 February 1981 )
ABSTRACT
Birch, W.R., 1981. The morphology of germinating seeds of the seagrass Halophila spinulosa (R.Br.) Aschers. (Hydrocharitaceae). Aquat. Bot., 11: 79--90. Laboratory germination of field harvested seeds of Halophila spinulosa (R.Br.) Aschers. showed a period of almost complete dormancy for about five weeks; thereafter the germination rate was high. All seedlings raised by various laboratory culture methods died at the second or third leaf stage. Halophila spinulosa (and probably H. ovalis (R.Br.) Hook. f.) has an unusual mechanism for early shedding of the testa from the seed from whose naked surface there then develops a profuse growth of 'seed-hairs'; these have an anchoring function before emergence of the radicle. This mechanism is likely to occur in all Halophila species because they have, so far as is known, coiled cotyledons.
INTRODUCTION
Halophila is a genus of some eight species (den Hartog, 1970) which grow in the tropics and subtropics. The sub-littoral species H. spinulosa (R.Br.) Aschers. is widely distributed in Malaysian and tropical Australian waters where it may grow alone or mixed with other seagrasses. Observations in Townsville waters indicate that H. spinulosa can often be found flourishing along tide-swept channels where the current is t o o fast for Cymodocea and Halodule. There are no published observations on the germination or seedling morphology of Halophila spinulosa, hence, a study of capsules and seeds of this species from the strand line on the beach at Cape Palarenda, Townsville, Queensland, was started in early October 1978. The main object was a morphological study, with incidental non-quantitative observations on the germination of the seeds. The release of the seeds and fruits and their subsequent deposition along the Palarenda strand must have followed a more or less massive synchronous flowering of the seagrass somewhere in the bay, followed b y an equally syn-
0304-3770/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
80 chronous abscission of the capsules, which float. The capsules were cast up in large numbers along at least a kilometre of beach during a two-or threeday period. The capsules dehisce easily on drying or with rough handling, and the released seeds, being denser than seawater, sink. They are then almost invisible among the sand grains. METHODS Capsules and seeds were collected and stored overnight in seawater. They were washed in seawater and all debris removed. The cleaned seed was stored in an open 3 1 beaker of aerated seawater on a b o t t o m substrate of clean sand and broken seashell at ambient laboratory temperatures (ca. 26 ° C) in fluorescent light (12 h photoperiod, 6 #E m -2 s-l). The seawater for the seed storage was taken from the School of Biology's seawater aquarium which draws its water from inshore waters close to Townsville. Experimental treatments used filtered oceanic seawater. Samples of seed were withdrawn from the large storage beaker and transferred to 50-ml vials or petri dishes, in order to try and increase the rate of germination. Treatments included the use of: (1) (2) (3) (4) (5) (6)
diluted seawater (1/1 or 2/1); darkness or low (6 g E m -2 s-1) or high light intensity (25/~E m -2 s -1); Schreiber's nutrient solution; seed scarification; 25 or 30°C constant temperatures; sandy shell-grit substrate or no substrate.
Scarification was attempted b y shaking the seed vigorously on an orbital shaker when mixed with coarse sand for 20 or 40 rain. In the first three weeks after collection only t w o seeds germinated in the storage beaker. Ungerminated samples were then taken from it and subjected to the various treatments. Samples of 50 seeds were sometimes used, but usually approximately equal volumes of seeds were taken b y spatula. Approximately 50 ml of culture solution was used for each treatment. Water lost from the vials b y evaporation was replenished as necessary during the period of the observations. Seeds and seedlings were photographed in situ, or using a Siemens SEM after specimens had been freeze-dried, and then coated in the usual way; figures are based on tracings made of live specimens using a Nikon profile projector. Most treatments were run for six weeks before being discarded, b u t the Schreiber's treatments under high light intensity were discarded after three weeks because of dense algal growth.
81 RESULTS
Germination No consistent germination trends were noted, regardless of treatment. Under high light intensity and Schreiber's solution germination was only ca. 1%. Sporadic mass germination occurred in localised patches in some vials, and in the storage beaker about five weeks after collection. This resulted in clumps of as many as 20 seedlings being tangled together while nearby other seeds remained ungerminated. A scan for ungerminated seeds, without counting or separation of them from the substrate suggested that most of the seeds in the storage beaker had germinated three months after collection, when observations ceased.
Seed morphology Standard seedling terminology is used even though there may be some doubt as to the homology of the cotyledon of Halophila spinulosa. The seeds
Fig. 1./-/. spinulosa seed with intact testa (bar scale 0.75 rnrn)
82
are white, oval and 0.75 m m long. The testa is characteristically sculptured with small peg-like projections (Figs. 1, 2 and 3). The following germination stages were recognised: (1) Splitting of the sculptured testa, which may be shed before the emergence of the cotyledon and radicle. This procedure does not seem to be as c o m m o n as stage 2; a smooth naked seed is left (Fig. 2). (2) Uncoiling of a coiled cotyledon from its pocket at the apex of the seed; the tip of the cotyledon usually carries with it the whole of the dehiscing testa (Fig. 4), unless shed previously as in stage 1. (3) The radicle starts to emerge. (4) Concomitant with stage 3, or even before it, long unicellular superficial hairs develop from the surface of the naked seed (Figs. 5 and 6). (5) Development of root hairs behind the apex of the radicle. (6) Extension of the cotyledon and enlargement of the cotyledonary pocket containing the first true leaf (Figs. 7 and 8). (7) Emergence of the first true leaf from the cotyledonary pocket; this leaf has only one vein, the midrib, and a smooth margin.
Fig. 2. H. spinulosa seeds, one (s) w i t h testa shed (bar scale 0.75 mm).
83
Fig. 3. Peg sculpturing of the testa of H. spinuiosa (bar scale 50 urn).
(8) Development of a lateral root primodium at the base of the first leaf at the node (Fig. 9). (9) Extension of the lateral root and the development of m a n y root hairs from it (Fig. 10). (10) Protrusion of the second leaf (three-veined) in the axil of the first leaf (Fig. 11). (11) Protrusion of the third leaf (three-veined) distichous to the second leaf. Under laboratory conditions it t o o k about 10 weeks for a seedling to reach this stage; all seedlings then died (or previously at stage 10) whatever culture treatments were used. These ranged from continuing in normal seawater or Schreiber's culture solution, to transplanting to sand and shell
84
Fig. 4. Germinating seeds of H. spinulosa in situ, showing the testas carried up on the elongated cotyledons (bar scale 10 mm).
mixtures, or even to loam or clay in seawater. Chloromycin was used in some cultures in an a t t e m p t to control algal growth. DISCUSSION The germinations showed that Halophila spinulosa seeds have a d o r m a n t period of at least one m o n t h before germination starts. This dormancy, coupled with the b u o y a n c y of the capsules m a y enhance propagule dispersal. However, that many capsules can be washed-up above the intertidal zone, can only result in many seeds reaching habitats inappropriate to the ecological range. Both Thalassia testudinum Banks ex KSnig and Zostera marina L. have been reported to be capable of immediate germination (Taylor, 1957 Thorhaug, 1974), though winter dormancy is more normal with Zostera. The tropical Thalassodendron ciliatum (Forsk.) den Hartog is viviparous (Isaac, 1969) as are b o t h the temperate-water species of Amphibolis (den Hartog, 1970). A spectrum of establishment strategies is thus exploited b y the seagrasses. The strategies so far investigated point to rapid, rather than to long delaying mechanisms for the control of germination. The short delay in the germination of Halophila spinulosa can be due to one, or more, of a number of intrinsic or extrinsic factors. Intrinsic factors could include the need for a ripening period of the e m b r y o , and the presence of germination inhibitors in the e m b r y o , endosperm, or testa, which need to be leached o u t before germination can occur. Of these, the ephemeral
85
Fig. 5. Naked seed of H. spinulosa with seed-hairs arising from the epidermis (bar scale
0.25 ram).
testa (see below) makes physical impermeability, and the presence of inhibitots in it, unlikely. This leaves after ripening, or internal inhibition as likely mechanisms for delaying germination. Extrinsic environmental factors as major controls in germination seem unlikely, unless in combinations of wider ranges of the treatments than were used in this exploratory work. It will be recalled that treatments involved combinations of three light levels, t w o temperature regimes, reduced salinity, absence or presence of substrate, scarification, and with or w i t h o u t nutrient solution. L o w oxygen concentration as a major factor can be eliminated because germination eventually occurred after five weeks in b o t h the seedstore beaker, which was always vigorously aerated with an aquarium aerator, and in the treatment vials, which were not artificially aerated. Intrinsic control of d o r m a n c y in H. spinulosa is indicated. The likelihood that extrinsic factors are less important than intrinsic factors in the control of seagrass germination is also suggested b y work on Zostera
86
C
- - S
I
c
Lt
I
R
5
--L
I
MM
!]"
Lt ~ \
C
I
LR
i
Figs. 6--11 (from t o p left to b o t t o m right). Stages in the germination of H. spinulosa from profile projection tracings (6, 8, 9, 10, 11 ) and showing emergence of first leaf from cotyledonary p o c k e t (Fig. 7, bar scale 0.25 mm): (C) = cotyledon; (CF) = cotyledonary pocket; (S) ffi seed; (R) ffi radicle; (L1) = first leaf; (L2) ffi second leaf; (LR) ffi lateral root.
87
marina L. and Z. japonica Aschers. and Graebn. The former germinated equally well in light or dark (Turin, 1938), and germination of neither species was affected by a wide range of chemical and organic substances (Arasaki, quoted in Taylor, 1957b). The complete seedling mortality in culture is unexplained. Lack of water movement is unlikely because the seed-storage beaker was aerated vigorously enough to keep the water moving. Nutritionally, starch was still present in the endosperm when the seedlings started to die, so a mineral or trace element imbalance could be implicated. Damage from microorganisms is also a possibility; seedlings of Thalassia testudinum Banks ex KSnig have been reported to be susceptible to fungal and bacterial attack in laboratory culture though these were not seen in field specimens (Thorhaug, 1974). Finally, the oxidation-reduction potential of the substrate is another possible factor, but seedlings died regardless of the substrate. The seeds of Halophila spinulosa, with their peg-like sculpturings, are smaller than those of H. ovalis (Fig. 12) which have a more or less rectangular sculpturing of the testa (Fig. 13). The coiled cotyledon was recorded first for H. ovalis (R.Br.) Hook. f. (Balfour, 1877), but not apparently until now for H. spinulosa. The uncoiling and expansion of the cotyledon during germination is, in H. spinulosa, a means of discarding the testa, so facilitating an early
Fig. 12. Comparison of seedlings of (A) H. ovalis and (B) H. spinulosa.
88
Fig. 13. H. ovalis seed; rectangular sculpturing of the testa (bar scale 0.25 mm). development of the hairs from the seed surface. In culturing on a sandy substrate these seed-hairs can be seen to be efficient anchors of the seed before the radicle emerges. The m e t h o d of discarding the testa, and the anchoring seed-hairs are t w o remarkable morphological attributes which are unique n o t only to H. spinulosa b u t probably to the genus Halophila as a whole. This can be predicted because coiled cotyledons are unlikely to have different functions in different species. It should be n o t e d that specimen A (Fig. 12) was observed growing among H. spinulosa seedlings; it was recognised by its greater c o t y l e d o n and seed size. Comparisons of locally harvested authentic H. ovalis seeds left no d o u b t that specimen A was H. ovalis even though the scultured testa was n o t attached; it was however, loose nearby in the vial. The nearest morphologically comparable plants would appear to be aquatic freshwater monocotyledons;Limnocharis flava (L.) Buch. has been shown to produce anchoring root hairs from the base of the h y p o c o t y l while Lophotocarpus, Alisma, and Echinodorus spp. rely on adventitious roots for early anchorage rather than the radicle (Kaul, 1978); in this respect they resemble Zostera marina, which does n o t develop its radicle properly (Taylor, 1957a). Zannichellia polycarpa Nolte. and Elatine hexandra D.C. also
89 produce hairs from the hypocotyl or 'collett' (Arber, 1920). By contrast, Thalassia testudinum KSnig (which has a loose seed-coat which slips off easily) apparently has a flattened basally-positioned cotyledon which does not uncoil, and from whose lower surface there develops a profuse growth of anchoring hairs (Orpurt and Bored, 1964). All produce roothair-like hairs, but none from the seed. Zostera is remarkable in its vascularless laterally developed tuber-like hairy hypocotyl (Taylor, 1957b); the lack of vasculation in this organ is suggestive of the vascularless, often hairy, epiblast in some Gramineae (Birch, 1963). The wall sculpturings may be of some functional significance in providing surface friction against the substrate whereby the testa is loosened around the seed before emergence of the coiled cotyledon. It may be significant here that at seed shedding there is, functionally, only a single integument which is only one cell thick in Halophila spinulosa. In H. ovalis the double integuments, each two-celled thick, which have been demonstrated in its early post-fertilisation stages later become compressed and only the single outer layer of cells of the outer integument remains intact; the other cells disintegrate to form a discontinuous papery layer (Lakshmanan, 1963), and so, as with H. spinulosa, finally provide a delicate loosely-fitting testa which can easily be ruptured by frictional forces. A similar disintegration of the inner integument and the freeing of it from the outer integument was reported in Zostera marina L; at maturity, however, the outer integument became lignified in this species (Taylor, 1957a). The morphological identity of the cotyledon in Halophila is of some interest. Under low light intensities the 'hypocotylar' region can elongate greatly (Fig. 12) carrying with it the node from which the plumule and leaves develop laterally. This agrees with Lakshmanan's (1962) figures for H. ovata Gaud. in which he clearly shows a recognisable lateral cotyledon. However the relative positions of cotyledon and epicotyl at the earlier proembryo stages were more terminally placed. Zostera marina also has this capability for hypocotylar extension; it helps overcome too deep burial of the seeds in silt (Taylor, 1957b). The same function is likely in Halophila. Finally, the seasonality of fruiting in Halophila spinulosa should be mentioned. Observations in 1979, the year after the present study, confirmed a fruiting period in October, but it was not nearly so marked as the year before. October has been noted as a fruiting period elsewhere (den Hartog, 1970), but some fertile specimens have also been collected in March, and October--December. The evidence, so far, suggests that H. spinulosa has a main fruiting period in spring, about three months after the tropical winter which occurs in June--July in southern latitudes. ACKNOWLEDGEMENTS
The author is grateful to J. Darley for his help with the electron microscopy and to his wife for her help with the collection of material.
90 REFERENCES Arber, A., 1920. Water Plants. A Study of Aquatic Angiosperms. Cambridge University Press, Cambridge, 436pp. Balfour, I.B., 1877--78. On the genus Halophila. Bot Soc. Edinburgh, 13: 290--343. Birch, W.R., 1963. Epiblast in Gramineae. Nature (London), 198 (4877): 304. Den Hartog, C., 1970. The Seagrasses of the World. North-Holland, Amsterdam, 275 pp. Isaac, F.M., 1969. Floral structure and germination in Cymodocea cil~ta. Phytomorphology, 19: 44--51. Kaul, R.B., 1978. Morphology of germination and establishment of aquatic seedlings in Alismataceae and Hydrocharitaceae. Aquat. Bot., 5: 139--147. Lakshmanan, K.K., 1962. The origin of epicotylary meristems and cotyledons in H. ovata Gaud. Ann. Bot., 26: 243--249. Lakshmanan, K.K., 1963. Embryological studies in the Hydrocharitaceae. II. H. ovata Gaudich. Indian Bot. Soc. J., 42: 16--18. Orpurt, P~A. and Baral, L.L., 1964. The flowers, fruit and seeds of Thalassia testudinum K~nig. Bull. Mar. Sci. Gulf Caribb., 14: 296--302. Taylor, A.R.A., 1957a. Studies on the development of Zostera marina L. I. Embryo and seed. Can. J. Bot., 35: 477--499. Taylor, A.R.A., 1957b. Studies on the development of Zostera marina L. II. Germination and seedling development. Can. J. Bot., 35: 681--695. Thorhaug, A., 1974. Transplantation of the seagrass Thalassia testudinum KSnig. Aquaculture, 4: 177--183. Tutin, T.G., 1938. The autecology of Zostera marina in relation to its wasting disease. New Phytol., 37 : 50--71.