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Germination in the marine phanerogam Zostera noltii Hornemann at Golfe Juan, French Mediterranean
Aquatic Botany, 38 (1990) 249-260 Elsevier Science Publishers B.V., Amsterdam
249
Germination in the marine phanerogam Zostera noltii Hornemann at Golfe Juan, French Mediterranean F. Loques, G. Caye and A. Meinesz Laboratoire Environnement Marin Littoral, Universitd de Nice-Sophia Antipolis, Parc Valrose, 06034 Nice Cedex (France) (Accepted for publication 23 May 1990 )
ABSTRACT Loques, F., Caye, G. and Meinesz, A., 1990. Germination in the marine phanerogam Zoslera nollii Hornemann at Golfe Juan, French Mediterranean. Aquat. Bot., 38: 249-260. Seed germination and germling development were investigated in Zostera noltii Hornemann taken from a sublittoral meadow on an open coast. Experimental treatments were imposed involving differing temperature, salinity, stratification and incision of the seed tegument. The speed and rate of success of germination were increased both by low salinity ( 1-10%o) and by incision of the seed tegument. After germination, germlings developed best in the 10%o salinity treatment. They reached the three-leaf stage in 20 days and showed two developmental stages: first, elongation of the cotyledon and the hypocotyl, followed by plumule development. The results show that there is no primary dormancy in Z. noltii and that unfavourable environmental conditions and tegument impermeability are the factors which prevent rapid germination.
INTRODUCTION
While the biology of the marine phanerogam Zostera marina L. is well known, that of Zostera noltii Hornemann is less so. Den Hartog (1970) was the first to elucidate its distribution (Atlantic coasts of Europe and Mauritania, the Mediterranean and the Black Sea), as well as giving some details of its biology. More recently, Jacobs et al. ( 1981 ), in a study of grazing by birds, showed the importance of this phanerogam in littoral ecosystems. Along the Atlantic and North Sea coasts of Europe, Jacobs ( 1982 ) studied the reproduction strategy of Z. noltii. He deduced that its seeds are of little importance in propagation. In the south of England, Tubbs and Tubbs ( 1983 ) estimated that seed production was high, but they were never able to find germlings in situ. Additionally, Hootsmans et al. ( 1987 ) investigated the germination of Z. noltii seeds from the North Sea, comparing it with germination in Z. marina. 0304-3770/90/$03.50
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In the western Mediterranean, Z. noltii is c o m m o n in most of the coastal brackish lagoons. It also occurs on the open coast, particularly on the C6te d'Azur, where it forms dense meadows together with the phanerogam Cymodocea nodosa (Ucria ) Ascherson and the alga Caulerpa prolifera (Forssk~l ) Lamouroux (Meinesz and Simonian, 1983). In the northwestern Mediterranean, Z. noltii flowers every year from mid-May until early September (Loqubs et al., 1988). Seed production is high, but the extent to which the seeds play an active role in reproduction is unknown. To our knowledge, in situ germination of seeds has never been observed in the Mediterranean. To determine the factors necessary to initiate germination in Z. noltii seeds, as well as their development into germlings, we investigated germination under controlled conditions. MATERIAL A N D M E T H O D S
We have found Z. noltii in the Baie de Golfe Juan (C6te d'Azur, France) in open sea. Like other adjacent protected bays of the same area, this phanerogam grows between 0 and - 6 m mixed with Cymodocea nodosa and near Posidonia oceanica (L.) Delile beds. At these sites, the salinity of the sea is constant (38.1-38.2%o) and there are no data about local imprevisible and possible ephemeral fluctuations of these values.
Sampling of seeds Inflorescences of Z. noltii were sampled by SCUBA diving from their first appearance in mid-May until the end of flowering in late August. Approximately 2000 flowering shoots were harvested and stored in an aquarium containing natural seawater. The seeds continued to ripen in their spathes in vitro, the ripe seeds then falling to the floor of the aquarium. The first seeds were sampled in mid-June, a month after the first following shoots were taken, while the last seeds were sampled in early October, ~ 4 months after formation of the first seeds. By these means, a stock of ~ 1300 seeds was built up. A second method for sampling seeds was tested. Sediment was taken from a part of a meadow where flowering is particularly abundant and the sediment was then sieved through meshes of progressively smaller size. Sampling of seeds by this method proved slow and fastidious owing to their small size and their resemblance to sediment grains. Only three seeds were obtained on sieving 0.1 m 3 of sediment.
Investigation of germination For each treatment, seeds were placed in tens in small pill boxes (25 ml) each closed by a mesh lid, and each treatment was repeated in triplicate. The
GERMINATION OF ZOSTERA NOLTII
251
pill boxes were arranged in aquaria containing l 0 1 of seawater of salinity 1, 10, 20, 30 and 38%o. These salinities were tested to refer to a study by Hootsman et al. (1987). Three temperature treatments were investigated: 13, 18 and 25°C (temperature being controlled by means of thermostats) at the same salinity as the Mediterranean sea (38%o). The two extreme temperatures represent the mini m u m and m a x i m u m temperatures of the littoral Mediterranean water of the French coasts (between 0 and - 10 m, where Z. noltii grows in open sea), while the intermediate one represents an average temperature. The experiment with incised seed consisted of making a longitudinal incision in the tegument. These incised seeds were subjected to the same temperature and salinity treatments as those seeds not incised. The effect of the germination hormone, giberellin GAy, was tested at a concentration of 1 mg l- 1 in natural seawater at the ambient temperature, 18 ° C. The stratification treatment consisted of placing the seeds in natural seawater of salinity 38%o and temperature 5 °C for 1 month, after which the temperature was increased to 18 ° C. All experimental work was carried out in natural day/night conditions between October 1987 and January 1988, in aquaria placed under glass. The results are expressed as mean percentage germination and rapidity of germination, as found in the three replicates. Both wet and dry weights were measured. The viability of seeds was tested using 0.25% tetrazolium for 5 h at 30°C, following the m e t h o d of Mayer and Poljakoff-Mayber ( 1975 ). Tegument permeability was assessed by the degree of water uptake by seeds soaked in iodized seawater.
Germling development and growth Development was investigated in germlings which had originated as seeds germinated in media of salinity 1, 10, 20, 30 and 38%0, which were then transferred to natural seawater at 10, 20 and 38%o. The results are expressed as the percentage survival rate of the germlings which developed, and as the growth rate of the cotyledon and hypocotyl. RESULTS
The harvest of seeds The production of seeds by flowering shoots in vitro depended on the time of year at which these shoots were taken. In shoots taken in late May, seed production was low and we were able to harvest the first seeds after 35 days
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F. LOQUESETAL.
in the laboratory. Production did not exceed 0.3 seeds per shoot and, after 90 days in vitro, no further seeds had been produced. To maximize seed production, the best time to take flowering shoots was found to be late June or early July. They then gave a mean of 0.71 + 0.1 seeds per shoot after 60 days in vitro. After late July, since the flowering shoots observed in situ no longer presented spathes of the first and second order, seed production must be negligible. Whatever the time of year when the flowering shoots were taken, the number of seeds finally harvested in vitro was much lower than that estimated from observation of the whole inflorescences originally present. The number of ripening seeds, observed on 30 inflorescences with a mean of 4.1+ 1.2 (s.d.) spathes per shoot, allowed us to estimate a harvest of ~ 3.41 _+ 1.21 seeds per spathe, i.e. 14 seeds per shoot. The proportion of seeds which never mature is thus 95%.
The seed and its germination The embryo of Z. noltii is greenish-blue in overall appearance, due to an expansion of the hypocotyl. Folded inside this formation is the cylindrical cotyledon, and inside the cotyledon sheath occurs the plumule with leaf primordia and stem apex (Fig. 1 ). A longitudinal section of the seed shows the seed coat to be thick, comprising a hard external integument and a more delicate inner integument adhering to the embryo. The tetrazolium test, carried out on a sample of 30 seeds, indicated that 72% were viable. The seed coat of these 30 seeds could be divided into three groups by colour. The first group, consisting of seeds black to dark blue in colour, represented 60% of the total. The second group, representing 22% of the total, consisted of seeds with chestnut brown teguments, and the third group, 18% of the total, had light brown teguments. Group 1 comprised the heaviest seeds with wet weights of 1.2 + 0.3 mg per seed. Group 2 were light, 0.9 + 0.15 mg per seed, and Group 3 contained the lightest seeds, 0.75 + 0.21 mg per seed. Under favourable conditions, the seeds in Group 1 germinated co
s c ~ hp
If
05.mm
Fig. 1. Longitudinal section of seed to show the seed coat (sc), the cotyledon (co) folded back, the hypocotyl (hp), the plumula with the leaf primordia (If) and the stem apex (sa).
GERMINATION OF ZOSTERA NOLTII
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Fig. 2. Germinated seed showing emergence of the cotyledon at the level of the seed micropyle and the hypocotyl.
the most rapidly, while only half of the seeds in Group 2 germinated and in the third group no seeds ever germinated. Only seeds from Group 1 were thus used for the germination experiments. Comparison of the wet and dry weights of 30 seeds indicated that the seeds had a high moisture content, ~ 41%. Seeds soaked in iodized seawater did not discolour, even after 10 days. Seed germination begins by longitudinal rupture of the tegument following a stria. From this split the white cotyledon, folded inside the hypocotyl, then emerges at the same distance along the seed as the micropyle. At this stage, the radicle has not yet emerged (Fig. 2 ). The cotyledon unfolds and elongates to reveal the cotyledon sheath enclosing the plumule. The cotyledon then begins to split along one side and the first leaf appears, projecting through this split. The leaves become green and by the time that three leaves have formed the first roots may have appeared at the base of the first node. These first germination stages, from cotyledon emergence to the development of the first chlorophyll-bearing leaves, take ~ 20 days.
Germination experiments Effects of salinity The experiments carried out showed that the rate of success of germination was related to salinity (Table 1 ). At a salinity of 1%o, 42.1 _+8.8% of seeds had germinated in 63 days, but at 10%o only 12.2 +_2.5% had germinated in the same time. No seeds developed at salinities of 20, 30 or 38%o. During storage of the seeds for 4 months in seawater of salinity 38%o, however, three
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F. LOQUESET AL.
TABLE 1 Germination success rate, expressed as a percentage, in non-incised seeds placed in seawater of different salinities. The results show the mean (with standard deviation) of three replicates of 10 seeds Time (days)
Salinity ( % ) 1
2 7 9 10 13 17 21 31 36 63 68 75
17_+1.1 23.1 _+ 1.6 26.8_+5.8 30.5_+4.8 30.5-+4.8 30.5-+4.8 36.2-+6.1 36.2_+6.1 36.2-+6.1 42.1 -+8.8 42.1 -+8.8 42.1 _+8.8
10
20
30
38
0 0 0 0 0 3.7_+0.5 3.7-+0.5 3.7_+0.5 8.3+_2.2 12.2_+2.5 12.2_+2.5 12.2_+2.5
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
cases of spontaeous germination were observed after storage for 35, 49 and 70 days, respectively.
Effects of incision of the seed tegument In seeds subjected to the incision treatment, the percentage germination was much higher than in the control treatment, reaching 100% in salinities of 1, 10 and 20%o (Table 2). In both incised seeds and in the controls, lowering the salinity gave progressively faster germination with cotyledons emerging in 20% of incised seeds after soaking at 10% for only 9 h. After soaking for 49 h at either 1 or 10%0 salinity, all the incised seeds had germinated, but after the same period in salinities of 30 and 38%0, germination success was only 35.6 _+9.4 and 7.9 + 3.6%, respectively.
Effects of temperature When the culture temperature was changed from 18 ° C for either 13 or 25 ° C, at a salinity of 38%o, no germination occurred whether or not the seeds had been incised. The environmental change treatment, involving 1 month at 5 ° C after which the temperature was increased to 18 °C, was not favourable to germination in seawater of 38%0 salinity. After a total of 3 months at 38%o, germination success was only 1.1%.
Effects of a hormone on germination A trial using the germination hormone GA7 (gibberellin) at 1 mg 1-' did not improve the germination success in seeds grown at 38°o. The percentage
GERMINATION
OF
ZOSTERA NOLTII
255
TABLE 2 G e r m i n a t i o n success rate (as percentage) in seeds subjected to the incision t r e a t m e n t a n d cultured in seawater o f different salinities. T h e results show the m e a n (with s t a n d a r d d e v i a t i o n ) o f three replicates o f 10 seeds Time
Salinity (%0)
1 9h 19h 26 h 36 h 49 h 7 days 13 days 21 days 31 days 36 days 63 days 75 days
0 58.3"+2.3 60.2"+3.5 81.2"+3.8 100 -
10
20
30
38
20.5 _+ 1.8 57.8"+2.3 79.8"+4.1 81.3"+4.9 100
0 0 39.8"+3.2 41.2"+3.5 42 _+3.8 61 -+4.1 78.2 _.%3.1 82.6-+4.8 87.3"+6.4 95.8"+7.2 100 -
0 0 0 0 0 35.6-+9.4 35.6 -+ 9.4 35.6"+9.4 35.6"+9.4 35.6_+9.4 35.6 _+9.4 Necrosis
0 0 0 0 0 4.1 "+2.1 7.87 -+ 3.6 7.87"+3.6 7.87"+3.6 7.87_+ 3.6 7.87 _+3.6 Necrosis
-
TABLE 3 Percentage o f g e r m i n a t e d seeds developing further in seawater o f different salinities. "Salinity o f initial m e d i u m " c o r r e s p o n d s to the salinity o f the m e d i u m in w h i c h the seeds were placed to germinate. "'Salinity o f the final m e d i u m " c o r r e s p o n d s to the salinity o f the m e d i u m in which the resultant germlings were placed to develop further. "'regr." indicates regression o f the germlings Time (days)
Salinity o f final m e d i u m (%0) 10
20
38
Salinity o f initial m e d i u m (%o)
4 8 12 14 22 27 33 54 67
l
10
20
30
1
0 18.2 18.2 27.3 27.3 27.3 regr. -
20 50 50 60 80 80 90 90 90
0 0 0 50 50 50 regr.
0 0 0 60 60 70 75 75 75
0 11 11 11 20 33 regr. -
10
20
30
0 20 30 30 60 60 60 regr. -
0 0 0 40 40 40 40 50 50
0 0 0 33 33 33 40 regr. -
1 0 0 0 0 0 regr. -
10
20
30
38
0 0 0 0 0
0 0 0 0 regr.
0 0 0 0 regr.
0 0 0 regr. -
0
-
-
-
regr.
-
-
-
of germination after 3 months was 6.6 +_0.6. Further, after germination the young germlings showed abnormal development, their cotyledons becoming curved instead of straight, development ceasing after 10 days.
256
v. LOQUES ET AL
0
0
~
0
1
1
1
0
0
0
~
0
~
o
+1+1
"~
~ N.~e~
.~- ~
r;e ~N
+F+I+I+I+I
r
0
~o ~.~ 0
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+l+l+l+l+l+r
i
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GERMINATION OF ZOSTERA NOL171
257
Investigation of germling development Table 3 shows that when seeds germinated in seawater of salinity 1, 10, 20, 30 or 38%o were transferred to a salinity of 38%o, they never developed further. After 33 days at 38%o, all the young germlings had necrosed. The germlings which continued to develop most successfully and most rapidly were those which had germinated and further developed at 10%o. Investigation of the development of young germlings over 67 days (Table 4 ) revealed that growth of the cotyledon and hypocotyl began from the fourth day onward and continued rapidly until the fourteenth day, when the mean growth attained was 15 ± 4.3 mm. Growth then slowed down markedly. At 20%o salinity, up to 60% of the seeds developed, the first signs of development appearing on the eighth day. At this salinity, it was also the seeds that had germinated at 10%o which developed the fastest. Observation of their development until Day 67 showed that the length reached by the cotyledon and the hypocotyl was roughly the same as that reached in plants that had germinated at 10%o salinity (Table 4). DISCUSSION
When flowering shoots of Z. noltii are harvested and stored in an aquarium, the number of ripe seeds obtained per shoot is less than one per shoot, very much lower than would be predicted from the number of ripening seeds originally present. The time at which the flowering shoots are taken and the storage conditions of these shoots can also slightly influence the production of ripe seeds. Hootsmans et al. ( 1987 ) had the same difficulty in creating a stock of seeds, as ripe seed production in Z. noltii from the North Sea appeared to be similarly much less than that predicted. In Z. noltii, the seed is surrounded by a hard and very impermeable seed coat. Germination is initiated by piercing of the teguments by the cotyledon, as in Z. marina (Setchell, 1929; den Hartog, 1970), and not by the radicle. The most important factor directly affecting the rapidity and the rate of success of germination has proved to be salinity. Like Hootsmans et al. ( 1987 ), we have shown that the germination success of Z. noltii seeds is greatly and progressively improved as the salinity is lowered. Similar results have been obtained by other workers for other species (Arasaki, 1950; Phillips, 1972; Lamounette, 1977; Phillips et al., 1983 ). Nevertheless, salinities as low as the ones found to give maximum germination success could never occur on the open coast at Golfe Juan, from where our material originated. Furthermore, in this particular meadow Z. noltii occurs mixed with another marine phanerogam, Posidonia oceanica, known not to tolerate low salinity (Aleem, 1955; Simonetti, 1973). The importance of the seed coat in initiating germination is now demon-
258
F. LOQUES ET AL.
strated. The fact that the greatest germination success was obtained with the incision treatment indicates that the seed coat represents a relatively impermeable barrier, it may prevent ingress of water, or reduced gas exchange between the embryo and its surroundings, or both. In incised seeds, lowering of salinity had the major effect of accelerating germination and improving its success rate. It may do this by causing the seed to swell and burst its teguments, and it may act directly on initiating embryo development by promoting hydration of the tissues and respiration. While several workers (Orth, 1976; McMillan, 1983; Orth and Moore, 1983 ) have shown that temperature is a preponderant factor in the initiation of germination in Z. marina, the present study has shown that in Z. noltii temperature has little or no effect on germination. The water content found in Z. noltii seeds indicates a higher level of hydration than is generally found in terrestrial plants. This suggests that diapause, a state characterized by dehydration, does not exist in the seeds of this species. The results of three experiments further suggest that primary dormancy is absent in Z. noltii seeds from the meadow at Golfe Juan. Firstly, the endogeneous level of the germination hormone gibberellin generally increases in seeds at the time of germination and exogenous application suppresses embryo dormancy (Simpson, 1965). Thus our finding that the application of gibberellin did not increase germination is evidence for a lack of primary dormancy. Secondly, cold treatment is frequently carried out to suppress dormancy in the seeds of aquatic or terrestrial plants (Muenscher, 1936) and indeed Hootsmans et al. ( 1987 ) found that low-temperature treatment influenced the germination rate of seeds of Z. noltii from The Netherlands, reversing the negative effect of high salinities. The observation that low-temperature pre-treatment (the stratification treatment) failed to facilitate germination in the present study is thus further evidence for a lack of dormancy. Lastly, our observation of low-frequency spontaneous germination further supports the contention that primary dormancy is absent in this population. In seeds of Mediterranean origin, there appears to be a dormancy regulated by salinity, an environmental factor. This type of dormancy is considered to be secondary (Crocker, 1916 ). In situ at Golfe Juan, we observed that seeds leave the spathe at maturity and fall immediately to the muddy sand. The seeds, in contact with sand grains, benthic bacteria and fungi, and moved around by waves, may thus have their teguments worn away or abraded. Seed germination could then be initiated by heavy spring and autumn rain, when there would be a lowering of salinities. Nevertheless, these small variations in salinities are unexpected and ephemeral, and thus might be difficult to measure in situ. We have never found a germling of Z. noltii in situ, however, so the time of year when germination occurs naturally remains unknown. Germination, however, could be induced in seeds stored in seawater of salinity 38%o for
GERMINATION OF ZOSTE1L4 NOLTll
259
between a month and a year. This suggests that seeds remain viable in situ for at least 1 year and that germination can be induced at any time by suitable environmental factors. As found by Hootsmans et al. (1987) in Z o s t e r a from The Netherlands, development of Mediterranean Z. noltii germlings was increased by lowering salinity. We obtained the three-leaf stage after 20 days in culture, showing that germling development is rapid. As described for Z. m a r i n a by Orth et al. ( 1981 ), development occurs first by elongation of the cotyledon and the hypocotyl, and then by development of the plumule. At 10%0, the salinity found to be most favourable for germling development, a survival rate of up to 90% was also optimal, unlike in Z. m a r i n a (Keddy and Patriquin, 1978; Churchill, 1983; Hootsmans et al., 1987 ). Given the low production of seeds per flowering shoot, and the combination of conditions, lowered salinity and tegument incision necessary to induce their germination, it is likely that seeds play little part in propagating Z. noltii. Even where this species occurs in areas of low salinity, germlings have been found only exceptionally (den Hartog, 1970; Jacobs, 1982). Our study has shown that low salinity is required for germling development as well as for germination. The multiplication strategy of this phanerogam is thus probably essentially vegetative, particularly as the rhizomes are perennating (Jacobs, 1982; Jacobs et al., 1983 ). Thus, under natural conditions vegetative propagation alone seems capable of increasing this species (Meinesz, 1976; Noten, 1983). ACKNOWLEDGEMENTS We thank Dr. I.R. Jenkinson for his help with the translation. This work was made possible by support provided by the French Ministry of the Environment to the "Groupement d'Int6r6t Scientifique Posidonie" (G.I.S. Posidonie).
REFERENCES Aleem, A., 1955. Structureand evolutionof the seagrasscommunitiesPosidonia and Cymodocea in the SoutheasternMediterranean.Essaysin the Natural Sciencesin Honor of Captain Allan Hancock,Universityof Southern California Edn., Los Angeles,pp. 279-298. Arasaki, S., 1950. Studies on the ecologyof Zostera marina and Zostera nana. Bull. Jpn. Soc. Fish., 16: 70-76. Churchill, A.C., 1983. Field studies on seed germinationand seedlingdevelopmentin Zostera marina L. Aquat. Bot., 16: 21-29. Crocker, W., 1916. Mechanicsof dormancyin seeds. Am. J. Bot., 3: 99-120. Den Hartog, C., 1970. The seagrassesof the world. Verh. K. Ned. Akad. Wet. Afd. Natuurkd. Reeks, 2, 59( 1): 1-275. Hootsmans, M.J.M., Vermaat,J.E. and Van Vierssen, W., 1987. Seed-bankdevelopment,ger-
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mination and early seedling survival of two seagrass species from the Netherlands: Zostera marina and Zostera noltii Hornem. Aquat. Bot., 28: 275-285. Jacobs, R.P.W.M., 1982. Reproductive strategies of two seagrass species (Zostera marina and Zostera noltii) along West European coasts. In: J.J. Symoens, S.S. Hooper and P. Compbre (Editors), Studies on Aquatic Vascular Plants. Royal Botanical Society, Brussels, Belgium, pp. 150-155. Jacobs, R.P.W.M., den Hartog, C., Braster, B.F. and Carri6re, F.C., 1981. Grazing of the seagrass Zostera noltii by birds at Terschelling (Dutch Wadden Sea). Aquat. Bot.: 241-259. Jacobs, R.P.W.M., Noten, T.M.P.A. and Claassen, E., 1983. Population and growth characteristics of the seagrass Zostera noltii Hornem. in the Dutch Waddensea. Proceedings of an International Symposium on Aquatic Macrophytes, at Nijmegen, pp. 95-100. Keddy, J. and Patriquin, D.G., 1978. An annual form of eelgrass in Nova Scotia. Aquat. Bot., 5: 163-170. Lamounette, R.G., 1977. A study of the germination and viability of Zostera marina L. seeds. Masters Thesis, Univ. Microfilms International, Ann Arbor, MI, 40 pp. Loqu6s, F., Caye, G. and Meinesz, A., 1988. Flowering and fruiting of Zostera noltii in Golfe Juan (French Mediterranean). Aquat. Bot., 32:341-352. Mayer, A.M. and Poljakoff-Mayber, A., 1975. The Germination of Seeds. Pergamon Press, Oxford, 192 pp. McMillan, C., 1983. Seed germination for an annual form of Zostera marina from the Sea of Cortez, Mexico. Aquat. Bot., 16:105-110. Meinesz, A., 1976. Notes pr61iminaires concernant quelques exp6riences de repiquage des v6g6taux marins, en particulier de l'algue Caulerpa prolifera (ForsskAl) Lamouroux. Rapp. Comm. Int. Mer. M6dit., 24: 169-170. Meinesz, A. and Simonian, M., 1983. Cartes de la v6g6tation sous-marine des Alpes-Maritimes (C6tes Fran~aises de la M6diterran6e). II - La v6g6tation mixte ~ Cymodocea nodosa-Zostera noltii-Caulerpa prolifera et la limite sup6rieure de l'herbier de Posidonia oceanica entre Juans les Pins et Golfe Juan. Ann. Inst. Oceanogr. (Paris), 59: 21-35. Muenscher, W.C., 1936. Storage and germination of seeds aquatic plants. N.Y. Cornell Agric. Exp. Stn. Bull. 652, 17 pp. Noten, T.M.P.A., 1983. Detached shoots of Zostera noltii Hornem., as a mean of dispersal: a transplantation experiment. Proceedings of an International Symposium on Aquatic Macrophytes, 18-23 September, at Nijmegen, The Netherlands, pp. 161-164. Orth, R., 1976. The demise and recovery of eelgrass Zostera marina in the Chesapeake Bay, Virginia. Aquat. Bot., 2:141-159. Orth, R.J. and Moore, K.A., 1983. Seed germination and seedling of Zostera marina L. (eelgrass) in the Chesapeake Bay. Aquat. Bot., 15:117-131. Orth, R.J., Moore, K.A., Roberts, M.H. and Silberhorn, G.M., 1981. The biology and propagation of eelgrass, Zostera marina, in the Chesapeake Bay, Virginia. Final Report (Contract No. R 805953), U.S. Environ. Prot. Agency, Washington, DC, 227 pp. Phillips, R., 1972. Seed germination in Zostera marina L. Am. J. Bot., 58: 459. Phillips, R.C., Grant, W.S. and McRoy, C., 1983. Reproductive strategies of eelgrass (Zostera marina L. ). Aquat. Bot., 16: 1-20. Setchell, W.A., 1929. Morphological and phenological notes on Zostera marina L. Univ. Calif. Publ. Bot., 14: 389-452. Simonetti, G., 1973. I conzorzi a fanerogame marine nel Golfo di Trieste. Atti Ist. Veneto Sci. Lett. Arti Ital., 131: 459-502. Simpson, G.M., 1965. Dormancy studies in seed of A vena fatua. 4. The role of gibberellin in embryo dormancy. Can. J. Bot., 43: 793-816. Tubbs, C.R. and Tubbs, J.M., 1983. The distribution of Zostera and its exploitation by wildfowl in the Solent, Southern England. Aquat. Bot., 15: 133-144.
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Germination in the marine phanerogam Zostera noltii Hornemann at Golfe Juan, French Mediterranean F. Loques, G. Caye and A. Meinesz Laboratoire Environnement Marin Littoral, Universitd de Nice-Sophia Antipolis, Parc Valrose, 06034 Nice Cedex (France) (Accepted for publication 23 May 1990 )
ABSTRACT Loques, F., Caye, G. and Meinesz, A., 1990. Germination in the marine phanerogam Zoslera nollii Hornemann at Golfe Juan, French Mediterranean. Aquat. Bot., 38: 249-260. Seed germination and germling development were investigated in Zostera noltii Hornemann taken from a sublittoral meadow on an open coast. Experimental treatments were imposed involving differing temperature, salinity, stratification and incision of the seed tegument. The speed and rate of success of germination were increased both by low salinity ( 1-10%o) and by incision of the seed tegument. After germination, germlings developed best in the 10%o salinity treatment. They reached the three-leaf stage in 20 days and showed two developmental stages: first, elongation of the cotyledon and the hypocotyl, followed by plumule development. The results show that there is no primary dormancy in Z. noltii and that unfavourable environmental conditions and tegument impermeability are the factors which prevent rapid germination.
INTRODUCTION
While the biology of the marine phanerogam Zostera marina L. is well known, that of Zostera noltii Hornemann is less so. Den Hartog (1970) was the first to elucidate its distribution (Atlantic coasts of Europe and Mauritania, the Mediterranean and the Black Sea), as well as giving some details of its biology. More recently, Jacobs et al. ( 1981 ), in a study of grazing by birds, showed the importance of this phanerogam in littoral ecosystems. Along the Atlantic and North Sea coasts of Europe, Jacobs ( 1982 ) studied the reproduction strategy of Z. noltii. He deduced that its seeds are of little importance in propagation. In the south of England, Tubbs and Tubbs ( 1983 ) estimated that seed production was high, but they were never able to find germlings in situ. Additionally, Hootsmans et al. ( 1987 ) investigated the germination of Z. noltii seeds from the North Sea, comparing it with germination in Z. marina. 0304-3770/90/$03.50
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In the western Mediterranean, Z. noltii is c o m m o n in most of the coastal brackish lagoons. It also occurs on the open coast, particularly on the C6te d'Azur, where it forms dense meadows together with the phanerogam Cymodocea nodosa (Ucria ) Ascherson and the alga Caulerpa prolifera (Forssk~l ) Lamouroux (Meinesz and Simonian, 1983). In the northwestern Mediterranean, Z. noltii flowers every year from mid-May until early September (Loqubs et al., 1988). Seed production is high, but the extent to which the seeds play an active role in reproduction is unknown. To our knowledge, in situ germination of seeds has never been observed in the Mediterranean. To determine the factors necessary to initiate germination in Z. noltii seeds, as well as their development into germlings, we investigated germination under controlled conditions. MATERIAL A N D M E T H O D S
We have found Z. noltii in the Baie de Golfe Juan (C6te d'Azur, France) in open sea. Like other adjacent protected bays of the same area, this phanerogam grows between 0 and - 6 m mixed with Cymodocea nodosa and near Posidonia oceanica (L.) Delile beds. At these sites, the salinity of the sea is constant (38.1-38.2%o) and there are no data about local imprevisible and possible ephemeral fluctuations of these values.
Sampling of seeds Inflorescences of Z. noltii were sampled by SCUBA diving from their first appearance in mid-May until the end of flowering in late August. Approximately 2000 flowering shoots were harvested and stored in an aquarium containing natural seawater. The seeds continued to ripen in their spathes in vitro, the ripe seeds then falling to the floor of the aquarium. The first seeds were sampled in mid-June, a month after the first following shoots were taken, while the last seeds were sampled in early October, ~ 4 months after formation of the first seeds. By these means, a stock of ~ 1300 seeds was built up. A second method for sampling seeds was tested. Sediment was taken from a part of a meadow where flowering is particularly abundant and the sediment was then sieved through meshes of progressively smaller size. Sampling of seeds by this method proved slow and fastidious owing to their small size and their resemblance to sediment grains. Only three seeds were obtained on sieving 0.1 m 3 of sediment.
Investigation of germination For each treatment, seeds were placed in tens in small pill boxes (25 ml) each closed by a mesh lid, and each treatment was repeated in triplicate. The
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pill boxes were arranged in aquaria containing l 0 1 of seawater of salinity 1, 10, 20, 30 and 38%o. These salinities were tested to refer to a study by Hootsman et al. (1987). Three temperature treatments were investigated: 13, 18 and 25°C (temperature being controlled by means of thermostats) at the same salinity as the Mediterranean sea (38%o). The two extreme temperatures represent the mini m u m and m a x i m u m temperatures of the littoral Mediterranean water of the French coasts (between 0 and - 10 m, where Z. noltii grows in open sea), while the intermediate one represents an average temperature. The experiment with incised seed consisted of making a longitudinal incision in the tegument. These incised seeds were subjected to the same temperature and salinity treatments as those seeds not incised. The effect of the germination hormone, giberellin GAy, was tested at a concentration of 1 mg l- 1 in natural seawater at the ambient temperature, 18 ° C. The stratification treatment consisted of placing the seeds in natural seawater of salinity 38%o and temperature 5 °C for 1 month, after which the temperature was increased to 18 ° C. All experimental work was carried out in natural day/night conditions between October 1987 and January 1988, in aquaria placed under glass. The results are expressed as mean percentage germination and rapidity of germination, as found in the three replicates. Both wet and dry weights were measured. The viability of seeds was tested using 0.25% tetrazolium for 5 h at 30°C, following the m e t h o d of Mayer and Poljakoff-Mayber ( 1975 ). Tegument permeability was assessed by the degree of water uptake by seeds soaked in iodized seawater.
Germling development and growth Development was investigated in germlings which had originated as seeds germinated in media of salinity 1, 10, 20, 30 and 38%0, which were then transferred to natural seawater at 10, 20 and 38%o. The results are expressed as the percentage survival rate of the germlings which developed, and as the growth rate of the cotyledon and hypocotyl. RESULTS
The harvest of seeds The production of seeds by flowering shoots in vitro depended on the time of year at which these shoots were taken. In shoots taken in late May, seed production was low and we were able to harvest the first seeds after 35 days
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in the laboratory. Production did not exceed 0.3 seeds per shoot and, after 90 days in vitro, no further seeds had been produced. To maximize seed production, the best time to take flowering shoots was found to be late June or early July. They then gave a mean of 0.71 + 0.1 seeds per shoot after 60 days in vitro. After late July, since the flowering shoots observed in situ no longer presented spathes of the first and second order, seed production must be negligible. Whatever the time of year when the flowering shoots were taken, the number of seeds finally harvested in vitro was much lower than that estimated from observation of the whole inflorescences originally present. The number of ripening seeds, observed on 30 inflorescences with a mean of 4.1+ 1.2 (s.d.) spathes per shoot, allowed us to estimate a harvest of ~ 3.41 _+ 1.21 seeds per spathe, i.e. 14 seeds per shoot. The proportion of seeds which never mature is thus 95%.
The seed and its germination The embryo of Z. noltii is greenish-blue in overall appearance, due to an expansion of the hypocotyl. Folded inside this formation is the cylindrical cotyledon, and inside the cotyledon sheath occurs the plumule with leaf primordia and stem apex (Fig. 1 ). A longitudinal section of the seed shows the seed coat to be thick, comprising a hard external integument and a more delicate inner integument adhering to the embryo. The tetrazolium test, carried out on a sample of 30 seeds, indicated that 72% were viable. The seed coat of these 30 seeds could be divided into three groups by colour. The first group, consisting of seeds black to dark blue in colour, represented 60% of the total. The second group, representing 22% of the total, consisted of seeds with chestnut brown teguments, and the third group, 18% of the total, had light brown teguments. Group 1 comprised the heaviest seeds with wet weights of 1.2 + 0.3 mg per seed. Group 2 were light, 0.9 + 0.15 mg per seed, and Group 3 contained the lightest seeds, 0.75 + 0.21 mg per seed. Under favourable conditions, the seeds in Group 1 germinated co
s c ~ hp
If
05.mm
Fig. 1. Longitudinal section of seed to show the seed coat (sc), the cotyledon (co) folded back, the hypocotyl (hp), the plumula with the leaf primordia (If) and the stem apex (sa).
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Fig. 2. Germinated seed showing emergence of the cotyledon at the level of the seed micropyle and the hypocotyl.
the most rapidly, while only half of the seeds in Group 2 germinated and in the third group no seeds ever germinated. Only seeds from Group 1 were thus used for the germination experiments. Comparison of the wet and dry weights of 30 seeds indicated that the seeds had a high moisture content, ~ 41%. Seeds soaked in iodized seawater did not discolour, even after 10 days. Seed germination begins by longitudinal rupture of the tegument following a stria. From this split the white cotyledon, folded inside the hypocotyl, then emerges at the same distance along the seed as the micropyle. At this stage, the radicle has not yet emerged (Fig. 2 ). The cotyledon unfolds and elongates to reveal the cotyledon sheath enclosing the plumule. The cotyledon then begins to split along one side and the first leaf appears, projecting through this split. The leaves become green and by the time that three leaves have formed the first roots may have appeared at the base of the first node. These first germination stages, from cotyledon emergence to the development of the first chlorophyll-bearing leaves, take ~ 20 days.
Germination experiments Effects of salinity The experiments carried out showed that the rate of success of germination was related to salinity (Table 1 ). At a salinity of 1%o, 42.1 _+8.8% of seeds had germinated in 63 days, but at 10%o only 12.2 +_2.5% had germinated in the same time. No seeds developed at salinities of 20, 30 or 38%o. During storage of the seeds for 4 months in seawater of salinity 38%o, however, three
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TABLE 1 Germination success rate, expressed as a percentage, in non-incised seeds placed in seawater of different salinities. The results show the mean (with standard deviation) of three replicates of 10 seeds Time (days)
Salinity ( % ) 1
2 7 9 10 13 17 21 31 36 63 68 75
17_+1.1 23.1 _+ 1.6 26.8_+5.8 30.5_+4.8 30.5-+4.8 30.5-+4.8 36.2-+6.1 36.2_+6.1 36.2-+6.1 42.1 -+8.8 42.1 -+8.8 42.1 _+8.8
10
20
30
38
0 0 0 0 0 3.7_+0.5 3.7-+0.5 3.7_+0.5 8.3+_2.2 12.2_+2.5 12.2_+2.5 12.2_+2.5
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
cases of spontaeous germination were observed after storage for 35, 49 and 70 days, respectively.
Effects of incision of the seed tegument In seeds subjected to the incision treatment, the percentage germination was much higher than in the control treatment, reaching 100% in salinities of 1, 10 and 20%o (Table 2). In both incised seeds and in the controls, lowering the salinity gave progressively faster germination with cotyledons emerging in 20% of incised seeds after soaking at 10% for only 9 h. After soaking for 49 h at either 1 or 10%0 salinity, all the incised seeds had germinated, but after the same period in salinities of 30 and 38%0, germination success was only 35.6 _+9.4 and 7.9 + 3.6%, respectively.
Effects of temperature When the culture temperature was changed from 18 ° C for either 13 or 25 ° C, at a salinity of 38%o, no germination occurred whether or not the seeds had been incised. The environmental change treatment, involving 1 month at 5 ° C after which the temperature was increased to 18 °C, was not favourable to germination in seawater of 38%0 salinity. After a total of 3 months at 38%o, germination success was only 1.1%.
Effects of a hormone on germination A trial using the germination hormone GA7 (gibberellin) at 1 mg 1-' did not improve the germination success in seeds grown at 38°o. The percentage
GERMINATION
OF
ZOSTERA NOLTII
255
TABLE 2 G e r m i n a t i o n success rate (as percentage) in seeds subjected to the incision t r e a t m e n t a n d cultured in seawater o f different salinities. T h e results show the m e a n (with s t a n d a r d d e v i a t i o n ) o f three replicates o f 10 seeds Time
Salinity (%0)
1 9h 19h 26 h 36 h 49 h 7 days 13 days 21 days 31 days 36 days 63 days 75 days
0 58.3"+2.3 60.2"+3.5 81.2"+3.8 100 -
10
20
30
38
20.5 _+ 1.8 57.8"+2.3 79.8"+4.1 81.3"+4.9 100
0 0 39.8"+3.2 41.2"+3.5 42 _+3.8 61 -+4.1 78.2 _.%3.1 82.6-+4.8 87.3"+6.4 95.8"+7.2 100 -
0 0 0 0 0 35.6-+9.4 35.6 -+ 9.4 35.6"+9.4 35.6"+9.4 35.6_+9.4 35.6 _+9.4 Necrosis
0 0 0 0 0 4.1 "+2.1 7.87 -+ 3.6 7.87"+3.6 7.87"+3.6 7.87_+ 3.6 7.87 _+3.6 Necrosis
-
TABLE 3 Percentage o f g e r m i n a t e d seeds developing further in seawater o f different salinities. "Salinity o f initial m e d i u m " c o r r e s p o n d s to the salinity o f the m e d i u m in w h i c h the seeds were placed to germinate. "'Salinity o f the final m e d i u m " c o r r e s p o n d s to the salinity o f the m e d i u m in which the resultant germlings were placed to develop further. "'regr." indicates regression o f the germlings Time (days)
Salinity o f final m e d i u m (%0) 10
20
38
Salinity o f initial m e d i u m (%o)
4 8 12 14 22 27 33 54 67
l
10
20
30
1
0 18.2 18.2 27.3 27.3 27.3 regr. -
20 50 50 60 80 80 90 90 90
0 0 0 50 50 50 regr.
0 0 0 60 60 70 75 75 75
0 11 11 11 20 33 regr. -
10
20
30
0 20 30 30 60 60 60 regr. -
0 0 0 40 40 40 40 50 50
0 0 0 33 33 33 40 regr. -
1 0 0 0 0 0 regr. -
10
20
30
38
0 0 0 0 0
0 0 0 0 regr.
0 0 0 0 regr.
0 0 0 regr. -
0
-
-
-
regr.
-
-
-
of germination after 3 months was 6.6 +_0.6. Further, after germination the young germlings showed abnormal development, their cotyledons becoming curved instead of straight, development ceasing after 10 days.
256
v. LOQUES ET AL
0
0
~
0
1
1
1
0
0
0
~
0
~
o
+1+1
"~
~ N.~e~
.~- ~
r;e ~N
+F+I+I+I+I
r
0
~o ~.~ 0
e..
~
e~
;~
e..
+l+l+l+l+l+r
i
i
GERMINATION OF ZOSTERA NOL171
257
Investigation of germling development Table 3 shows that when seeds germinated in seawater of salinity 1, 10, 20, 30 or 38%o were transferred to a salinity of 38%o, they never developed further. After 33 days at 38%o, all the young germlings had necrosed. The germlings which continued to develop most successfully and most rapidly were those which had germinated and further developed at 10%o. Investigation of the development of young germlings over 67 days (Table 4 ) revealed that growth of the cotyledon and hypocotyl began from the fourth day onward and continued rapidly until the fourteenth day, when the mean growth attained was 15 ± 4.3 mm. Growth then slowed down markedly. At 20%o salinity, up to 60% of the seeds developed, the first signs of development appearing on the eighth day. At this salinity, it was also the seeds that had germinated at 10%o which developed the fastest. Observation of their development until Day 67 showed that the length reached by the cotyledon and the hypocotyl was roughly the same as that reached in plants that had germinated at 10%o salinity (Table 4). DISCUSSION
When flowering shoots of Z. noltii are harvested and stored in an aquarium, the number of ripe seeds obtained per shoot is less than one per shoot, very much lower than would be predicted from the number of ripening seeds originally present. The time at which the flowering shoots are taken and the storage conditions of these shoots can also slightly influence the production of ripe seeds. Hootsmans et al. ( 1987 ) had the same difficulty in creating a stock of seeds, as ripe seed production in Z. noltii from the North Sea appeared to be similarly much less than that predicted. In Z. noltii, the seed is surrounded by a hard and very impermeable seed coat. Germination is initiated by piercing of the teguments by the cotyledon, as in Z. marina (Setchell, 1929; den Hartog, 1970), and not by the radicle. The most important factor directly affecting the rapidity and the rate of success of germination has proved to be salinity. Like Hootsmans et al. ( 1987 ), we have shown that the germination success of Z. noltii seeds is greatly and progressively improved as the salinity is lowered. Similar results have been obtained by other workers for other species (Arasaki, 1950; Phillips, 1972; Lamounette, 1977; Phillips et al., 1983 ). Nevertheless, salinities as low as the ones found to give maximum germination success could never occur on the open coast at Golfe Juan, from where our material originated. Furthermore, in this particular meadow Z. noltii occurs mixed with another marine phanerogam, Posidonia oceanica, known not to tolerate low salinity (Aleem, 1955; Simonetti, 1973). The importance of the seed coat in initiating germination is now demon-
258
F. LOQUES ET AL.
strated. The fact that the greatest germination success was obtained with the incision treatment indicates that the seed coat represents a relatively impermeable barrier, it may prevent ingress of water, or reduced gas exchange between the embryo and its surroundings, or both. In incised seeds, lowering of salinity had the major effect of accelerating germination and improving its success rate. It may do this by causing the seed to swell and burst its teguments, and it may act directly on initiating embryo development by promoting hydration of the tissues and respiration. While several workers (Orth, 1976; McMillan, 1983; Orth and Moore, 1983 ) have shown that temperature is a preponderant factor in the initiation of germination in Z. marina, the present study has shown that in Z. noltii temperature has little or no effect on germination. The water content found in Z. noltii seeds indicates a higher level of hydration than is generally found in terrestrial plants. This suggests that diapause, a state characterized by dehydration, does not exist in the seeds of this species. The results of three experiments further suggest that primary dormancy is absent in Z. noltii seeds from the meadow at Golfe Juan. Firstly, the endogeneous level of the germination hormone gibberellin generally increases in seeds at the time of germination and exogenous application suppresses embryo dormancy (Simpson, 1965). Thus our finding that the application of gibberellin did not increase germination is evidence for a lack of primary dormancy. Secondly, cold treatment is frequently carried out to suppress dormancy in the seeds of aquatic or terrestrial plants (Muenscher, 1936) and indeed Hootsmans et al. ( 1987 ) found that low-temperature treatment influenced the germination rate of seeds of Z. noltii from The Netherlands, reversing the negative effect of high salinities. The observation that low-temperature pre-treatment (the stratification treatment) failed to facilitate germination in the present study is thus further evidence for a lack of dormancy. Lastly, our observation of low-frequency spontaneous germination further supports the contention that primary dormancy is absent in this population. In seeds of Mediterranean origin, there appears to be a dormancy regulated by salinity, an environmental factor. This type of dormancy is considered to be secondary (Crocker, 1916 ). In situ at Golfe Juan, we observed that seeds leave the spathe at maturity and fall immediately to the muddy sand. The seeds, in contact with sand grains, benthic bacteria and fungi, and moved around by waves, may thus have their teguments worn away or abraded. Seed germination could then be initiated by heavy spring and autumn rain, when there would be a lowering of salinities. Nevertheless, these small variations in salinities are unexpected and ephemeral, and thus might be difficult to measure in situ. We have never found a germling of Z. noltii in situ, however, so the time of year when germination occurs naturally remains unknown. Germination, however, could be induced in seeds stored in seawater of salinity 38%o for
GERMINATION OF ZOSTE1L4 NOLTll
259
between a month and a year. This suggests that seeds remain viable in situ for at least 1 year and that germination can be induced at any time by suitable environmental factors. As found by Hootsmans et al. (1987) in Z o s t e r a from The Netherlands, development of Mediterranean Z. noltii germlings was increased by lowering salinity. We obtained the three-leaf stage after 20 days in culture, showing that germling development is rapid. As described for Z. m a r i n a by Orth et al. ( 1981 ), development occurs first by elongation of the cotyledon and the hypocotyl, and then by development of the plumule. At 10%0, the salinity found to be most favourable for germling development, a survival rate of up to 90% was also optimal, unlike in Z. m a r i n a (Keddy and Patriquin, 1978; Churchill, 1983; Hootsmans et al., 1987 ). Given the low production of seeds per flowering shoot, and the combination of conditions, lowered salinity and tegument incision necessary to induce their germination, it is likely that seeds play little part in propagating Z. noltii. Even where this species occurs in areas of low salinity, germlings have been found only exceptionally (den Hartog, 1970; Jacobs, 1982). Our study has shown that low salinity is required for germling development as well as for germination. The multiplication strategy of this phanerogam is thus probably essentially vegetative, particularly as the rhizomes are perennating (Jacobs, 1982; Jacobs et al., 1983 ). Thus, under natural conditions vegetative propagation alone seems capable of increasing this species (Meinesz, 1976; Noten, 1983). ACKNOWLEDGEMENTS We thank Dr. I.R. Jenkinson for his help with the translation. This work was made possible by support provided by the French Ministry of the Environment to the "Groupement d'Int6r6t Scientifique Posidonie" (G.I.S. Posidonie).
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mination and early seedling survival of two seagrass species from the Netherlands: Zostera marina and Zostera noltii Hornem. Aquat. Bot., 28: 275-285. Jacobs, R.P.W.M., 1982. Reproductive strategies of two seagrass species (Zostera marina and Zostera noltii) along West European coasts. In: J.J. Symoens, S.S. Hooper and P. Compbre (Editors), Studies on Aquatic Vascular Plants. Royal Botanical Society, Brussels, Belgium, pp. 150-155. Jacobs, R.P.W.M., den Hartog, C., Braster, B.F. and Carri6re, F.C., 1981. Grazing of the seagrass Zostera noltii by birds at Terschelling (Dutch Wadden Sea). Aquat. Bot.: 241-259. Jacobs, R.P.W.M., Noten, T.M.P.A. and Claassen, E., 1983. Population and growth characteristics of the seagrass Zostera noltii Hornem. in the Dutch Waddensea. Proceedings of an International Symposium on Aquatic Macrophytes, at Nijmegen, pp. 95-100. Keddy, J. and Patriquin, D.G., 1978. An annual form of eelgrass in Nova Scotia. Aquat. Bot., 5: 163-170. Lamounette, R.G., 1977. A study of the germination and viability of Zostera marina L. seeds. Masters Thesis, Univ. Microfilms International, Ann Arbor, MI, 40 pp. Loqu6s, F., Caye, G. and Meinesz, A., 1988. Flowering and fruiting of Zostera noltii in Golfe Juan (French Mediterranean). Aquat. Bot., 32:341-352. Mayer, A.M. and Poljakoff-Mayber, A., 1975. The Germination of Seeds. Pergamon Press, Oxford, 192 pp. McMillan, C., 1983. Seed germination for an annual form of Zostera marina from the Sea of Cortez, Mexico. Aquat. Bot., 16:105-110. Meinesz, A., 1976. Notes pr61iminaires concernant quelques exp6riences de repiquage des v6g6taux marins, en particulier de l'algue Caulerpa prolifera (ForsskAl) Lamouroux. Rapp. Comm. Int. Mer. M6dit., 24: 169-170. Meinesz, A. and Simonian, M., 1983. Cartes de la v6g6tation sous-marine des Alpes-Maritimes (C6tes Fran~aises de la M6diterran6e). II - La v6g6tation mixte ~ Cymodocea nodosa-Zostera noltii-Caulerpa prolifera et la limite sup6rieure de l'herbier de Posidonia oceanica entre Juans les Pins et Golfe Juan. Ann. Inst. Oceanogr. (Paris), 59: 21-35. Muenscher, W.C., 1936. Storage and germination of seeds aquatic plants. N.Y. Cornell Agric. Exp. Stn. Bull. 652, 17 pp. Noten, T.M.P.A., 1983. Detached shoots of Zostera noltii Hornem., as a mean of dispersal: a transplantation experiment. Proceedings of an International Symposium on Aquatic Macrophytes, 18-23 September, at Nijmegen, The Netherlands, pp. 161-164. Orth, R., 1976. The demise and recovery of eelgrass Zostera marina in the Chesapeake Bay, Virginia. Aquat. Bot., 2:141-159. Orth, R.J. and Moore, K.A., 1983. Seed germination and seedling of Zostera marina L. (eelgrass) in the Chesapeake Bay. Aquat. Bot., 15:117-131. Orth, R.J., Moore, K.A., Roberts, M.H. and Silberhorn, G.M., 1981. The biology and propagation of eelgrass, Zostera marina, in the Chesapeake Bay, Virginia. Final Report (Contract No. R 805953), U.S. Environ. Prot. Agency, Washington, DC, 227 pp. Phillips, R., 1972. Seed germination in Zostera marina L. Am. J. Bot., 58: 459. Phillips, R.C., Grant, W.S. and McRoy, C., 1983. Reproductive strategies of eelgrass (Zostera marina L. ). Aquat. Bot., 16: 1-20. Setchell, W.A., 1929. Morphological and phenological notes on Zostera marina L. Univ. Calif. Publ. Bot., 14: 389-452. Simonetti, G., 1973. I conzorzi a fanerogame marine nel Golfo di Trieste. Atti Ist. Veneto Sci. Lett. Arti Ital., 131: 459-502. Simpson, G.M., 1965. Dormancy studies in seed of A vena fatua. 4. The role of gibberellin in embryo dormancy. Can. J. Bot., 43: 793-816. Tubbs, C.R. and Tubbs, J.M., 1983. The distribution of Zostera and its exploitation by wildfowl in the Solent, Southern England. Aquat. Bot., 15: 133-144.