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Anthesis and seed production in Zostera marina L. from Great South Bay, New York, U.S.A.
Aquatic Botany, 4 (1978) 83--93 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
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A N T H E S I S A N D S E E D P R O D U C T I O N I N Z O S T E R A M A R I N A L. F R O M G R E A T S O U T H B A Y , N E W Y O R K , U.S.A.
A. COOLIDGE CHURCHILL and MICHAEL I. RINER Adelphi Institute of Marine Science, Adelphi University, Garden City, N.Y. 11530 (U.S.A.)
(Received 8 June 1976)
ABSTRACT Churchill; A.C, and Riner~ M.I., 1978. Anthesis and seed production in Zostera marina L. from Great South Bay, New York, U.S.A. Aquat. Bot., 4: 83--93. Flowering and seed production were studied during the spring and summer of 1975 in a population of Zostera marina L. from Great South Bay, New York. The maturation of stamens and pistils was observed from April, until the completion of anthesis in June. Pollination occurred successively within increasing spathe orders and lasted approximately one month, starting between May 15 and May 21, when the water temperature was 17--20 ° C, and ending by June 17, when the water temperature had reached 21 ° C. Ovary dehiscence and seed release were first observed on June 17, and essentially completed by July 9, together with the deterioration of the flowering shoots. An average of 48 pistils was produced on each flowering shoot, but seed formation occurred in only 72% of these reproductive organs. The remaining 28% aborted, presumably due to the failure of fertilization. The average density of the flowering shoots was 53/m 2 , yielding a potential seed crop of 1802 seeds/m 2 .
INTRODUCTION T h e seagrass Zostera marina L. has b e e n intensively studied d u r i n g r e c e n t years, b u t t h e r e is surprisingly little i n f o r m a t i o n c o n c e r n i n g t h e p r o c e s s o f f l o w e r i n g a n d seed p r o d u c t i o n . T h e classic d e s c r i p t i o n b y Setchell ( 1 9 2 9 ) o f the r e p r o d u c t i v e p e r i o d i c i t y o f Z. marina f r o m E u r o p e and t h e east and w e s t coasts o f N o r t h A m e r i c a still serves as t h e basis f o r m u c h o f o u r u n d e r s t a n d ing o f f l o w e r i n g in this p l a n t . Setchell ( 1 9 2 9 ) p r o p o s e d t h a t w a t e r t e m p e r a t u r e was the p r i m a r y e n v i r o n m e n t a l variable d e t e r m i n i n g the o n s e t and dura t i o n o f sexual r e p r o d u c t i o n . His h y p o t h e s i s t h a t anthesis and seed f o r m a t i o n o c c u r r e d o n l y at w a t e r t e m p e r a t u r e s f r o m 15 t o 2 0 ° C was s u p p o r t e d b y T u t i n ( 1 9 3 8 ) , a n d m o r e r e c e n t l y b y M c R o y ( 1 9 6 6 ) , b u t it d o e s n o t explain t h e p e r i o d i c i t y o f f l o w e r i n g in p l a n t s f r o m P u g e t S o u n d (Phillips, 1969). Setchell ( 1 9 2 9 ) p r e s e n t e d little q u a n t i t a t i v e d a t a o n t h e f l o w e r i n g p r o c e s s a l t h o u g h r e c e n t studies ( M c R o y , 1970; Phillips, 1 9 7 2 ; Felger and M c R o y , 1 9 7 5 ) have d e t e r m i n e d t h e d e n s i t y o f f l o w e r i n g s h o o t s in n a t u r a l p o p u l a tions, a n d in o n e case (Felger and M c R o y , 1 9 7 5 ) h a v e e s t i m a t e d seed p r o d u c -
84 tion. To our knowledge, however, there has been no quantitative analysis of flower production, percent seed set, or seed release. This study describes the flowering of Z. marina from Great South Bay, New York. Weekly collections and observations of plants were made during the flowering period and used to describe floral development, seed production, and the pattern of seed release. MATERIAL AND METHODS Collections of flowering plants were made from an established Zostera meadow on the north side of West Fire Island (Fig. 1). Flowering plants growing 1.0 m below MLW (tidal amplitude 25 cm) were harvested initially by random sampling of terminal shoots and later, when the flowering plants were visible w i t h o u t close inspection, by selective picking from among the vegetative branches. Weekly collections of 30 flowering shoots were made between April 17 and July 9 and were preserved in seawater with formaldehyde (4%). Fifteen shoots were used for evaluation and analysis of flower and fruit development, and the remainder were stored as reference material. On June 3, the entire collection of 30 shoots was used to determine the average number of spathes and the number of stamens and pistils on individual shoots. The density of flowering shoots and their percentage within the population was determined by m o n t h l y harvests of all plants within ten 0.05 m 2 quadrats. Standard deviations of the means are given in the text.
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Fig. 1. Map of Great South Bay, New York showing the study area and West Sayville, site of daily water temperature recordings.
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Water temperature and salinity (by refractometer) were recorded during each visit to the study area. Additional temperature values were kindly provided by the Bluepoints Co. Inc. These were daily recordings made between January 17, 1975 and July 31, 1975 at West Sayville, New York, and were representative of the study area. RESULTS
The flowering shoots differ from the vegetative ones in being erect and w i t h o u t adventitious roots. When mature, they varied from 19 to 48 cm in length with an average of 32 -+ 8 cm and, as described by Setchell (1929), each flowering shoot represented the tip of a main rhizome axis. Only a small percentage (6--9%) of the shoots present flowered, and the average density of flowering shoots on June 3 was 53 -+ 24/m 2 . The reproductive stem produced both leaves and floral branches in a uniform pattern (Fig. 2). Flower-bearing spathes were initiated at the apex of
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Fig. 2. A s c h e m a t i c r e p r e s e n t a t i o n o f t h e f l o w e r i n g s h o o t o f Zostera marina. T h e s p a t h e s (sp) o n each b r a n c h a n d t h e t e r m i n a l i n f l o r e s c e n c e are n u m b e r e d s e q u e n t i a l l y a c c o r d i n g t o t h e i r o r d e r o f m a t u r a t i o n . T h e b r o k e n lines r e p r e s e n t leaf-like s h e a t h s w h i c h successively e n v e l o p smaller s p a t h e clusters d u r i n g t h e i r initial d e v e l o p m e n t o n t h e b r a n c h e s and terminal inflorescence.
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the shoot (terminal inflorescence), and on 1--3 (rarely 4) lateral branches. Frequently, the first branch (branch 1, Fig. 2) was undeveloped and, therefore, evident only after close inspection. Each lateral branch alternated along the stem with ale af, and together with the terminal inflorescence, produced spathes which matured acropetally as indicated by the assigned numbers in Fig. 2. The number of spathes which matured on the branches and terminal inflorescence varied and tended to increase acropetally along the shoot (means: branch 1, 0.3, branch 2, 1.6, branch 3, 2.5, terminal inflorescence, 3.2). The value of 0.3 spathes for branch 1 reflects the frequent undeveloped condition of this branch. The maximum spathe number for a single shoot was 11 and the average 7.6 + 1.8. In most cases, the branches and the terminal inflorescence possessed an additional spathe order which, by the end of the flowering period, was visible only upon microscopic examination. These spathes failed to mature and were, therefore, not included in the present determinations. A spathe ensheathed and protected a single monoecious spadix on which developed the reduced flowers consisting of single stamens (male) and pistils (female). The number of stamens and pistils which developed within each spathe varied. For a specific spathe order, this number increased acropetally from the basal branch to the terminal inflorescence, while on a given branch, it increased successively from the last to the first spathe order (Table I). The number of stamens within a spathe was approximately twice that of the pistils, and they were regularly arranged on a spadix. The maximum number of flowers on a single spadix was 33 (22 stamens, 11 pistils) and on an individual shoot was 247 (160 stamens, 87 pistils). The average number of stamens and pistils produced per flowering shoot was 91 + 24 and 48 -+ 13, respectively, yielding a total of 139 flowers per shoot. Pistils on a single spadix developed simultaneously and, typically, matured before the stamens. This proterogyny was evident by the withering and frequent abscission of the bifurcated stigma prior to anther dehiscence. Flowers TABLE I The m e a n n u m b e r o f s t a m e n s (st) and pistils (p) w i t h i n s p a t h e s o n t h e lateral b r a n c h e s and t h e t e r m i n a l inflorescence Spathe order
Branch n u m b e r
1
1 2 3 4
Terminal inflorescence
2
3
st
p
st
p
st
p
6.5
3.5
9.6 9.0 7.2
4.6 5.0 4.0
13.7 7.0 12.4 6.4 9.5 5.5 8.7 5.0
st
p
14.8 7.6 13.7 7.3 13.1 7.0 8.8 4.7
87 on different branches but within spathes of the same order matured more or less synchronously; the difference, therefore, between branches including the terminal inflorescence lay in the number of flowers and spathes present rather than in the pattern or timing of their development. Flowers of increasing spathe orders on a branch developed sequentially, thereby extending the period of anthesis. This pattern of maturation is demonstrated by size frequency histograms showing changes in the mean length of the largest anther within each spathe on branch 2 (Fig. 3). The anthers within spathe 1 increased in size from April 17 to May 15 and the largest reached a m a x i m u m length of 7.4 mm. Anther dehiscence was first observed on May 21 and was essentially completed by June 3. Development of male flowers within spathe 2 was delayed so that pollen release did not start until the end of May or approximately one week after the flowers on spadix 1, (Fig. 3). The m a x i m u m anther length (4.8 mm) was smaller than on spadix 1, and the time of development as measured by the period between the appearance of class 2 anthers (class mark, 2,95 ram) and the first anther release was reduced from five to three weeks. Maturation of anthers withi~ spathe 3 was further delayed so that pollen release was not observed until June 3. The m a x i m u m anther length was only 4.0 ram, and the time of development was approximately two weeks. The time of development and anther dehiscence in fourth-order spathes was essentially coincident with those on spadix 3. With the exception of a few stamens, pollen release was completed by June 17. Small anthers (less than 1.9 mm) which were present at this time failed to develop further. Development and maturation of the female flowers were examined by observing changes in the length of the pistil (ovary exclusive of the bifurcated stigma). On April 17, the largest ovary (spathe 1) varied in length from 0.1 mm to 0.3 mm. The ovaries increased to a length of 3--4 mm by the start of pollination (May 21) and to 5--7 mm on June 17 when seed release started. Development was similar to that of the stamens in that maturation was delayed within increasing spathe orders and synchronous on a single spadix. Unlike the stamens, however, the mean length of the mature ovary did not decrease within increasing spathe orders. Each mature ovary contained a single elliptical seed with an average length and width of 3.1 + 0.1 mm and 1.5 + 0.1 mm, respectively, and a color that varied from blue-gray to a dark brown. Dehiscence of the ovary and seed release was first observed in the field on June 17, nearly one m o n t h after the start of pollen release. The process occurred initially in the first-order spathes and then successively in the higher spathe orders, and was marked by a lengthwise splitting of the ovary wall and the c o n c o m m i t t a n t deterioration of the enfolding spathe sheath along its edges. The seeds sank immediately after dehiscence from the ovary. Shortly after seed release, or in some cases before, abscission of the spathe occurred just below the spadix, leaving only the narrow, basal stalk of the spathe attached to the shoot.
88
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ANTHER LENGTH (mm) Fig. 3. Size--frequency histograms showing changes in the mean length of the largest anther within each spathe on branch 2. The frequency (ordinate) represents the percentage of total anthers measured for a given spathe order and for dehisced anthers (D) is indicated by solid bars. Each size class (abscissa) has an interval of 2.0 mm and is designated by the class mark (midpoint).
The pattern of seed loss was determined by comparing the n u m b e r of ovaries on shoots prior to and during the period of seed release. The results (Table II) indicate that the greatest loss occurred between June 23 and July 1, and that by July 9, nearly 90% of the seeds had been shed. In addition, it was possible to distinguish by size and appearance between ovaries in which seed formation had occurred and those which had aborted, presumably due to the failure of fertilization. From June 17 to July 1, the percentage of fertile and aborted ovaries on the flowering shoots remained fairly constant; a characteristic which is readily explained by spathe abscission and the resulting simultaneous loss of both fertile and aborted ovaries. By July 9, how-
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TABLE II The mean number and standard deviation of ovaries per flowering shoot during the period of seed release and the percentage of fertile ovaries (containing mature or developing seeds). The percentage of fertile ovaries on June 3 is not given since pollen release was still occurring Date of Collection
Ovaries present
Cumulative ovary loss (%)
Fertile ovaries (%)
3June1975 17 June 1975 23 June 1975 1 July 1975 9 July 1975
48-+ 13 44_+ 12 33-+ 11 14 -+ 7 5 -+ 4
0 8 30 70 89
72± 10 69-+ 15 66 + 19 20 ± 18
ever, t h e p e r c e n t a g e o f fertile ovaries d e c r e a s e d t o 20%. T h e p l a n t s at this t i m e w e r e g r e a t l y r e d u c e d b y the abscission o f all b u t t h e f o u r t h - and occasional t h i r d - o r d e r spathes. T h e t i m e w h e n f l o w e r p r i m o r d i a first a p p e a r e d was n o t d e t e r m i n e d . T e r m i n a l s h o o t s c o l l e c t e d in J a n u a r y 1975 as p a r t o f a n o t h e r s t u d y were exa m i n e d m i c r o s c o p i c a l l y a n d s h o w e d distinct s t a m e n a n d pistil f o r m a t i o n , p a r t i c u l a r l y o n s p a t h e 1 o f the t e r m i n a l inflorescence. Collections in Dec e m b e r a n d N o v e m b e r o f 1 9 7 4 did n o t c o n t a i n f l o w e r i n g shoots, b u t t h e s a m p l e s were t o o small t o d i s c o u n t t h e i r presence. T h e p e r i o d w h e n i m m a t u r e flowers w e r e o b s e r v e d and t h e t i m e o f p o l l i n a t i o n and seed release are illustrated in Fig. 4 t o g e t h e r w i t h t h e r e c o r d e d salinity and t e m p e r a t u r e values. Salinity r e m a i n e d fairly c o n s t a n t d u r i n g the s t u d y p e r i o d , v a r y i n g f r o m 28 t o 3 2 % 0 . W a t e r t e m p e r a t u r e , as e x p e c t e d , s h o w e d m a r k e d seasonal changes. In J a n u a r y a n d F e b r u a r y w h e n small i m m a t u r e flowers were p r e s e n t , t e m p e r a t u r e values ranged f r o m 0.5 t o 3 ° C. F l o w e r m a t u r a t i o n occurred as the w a t e r t e m p e r a t u r e increased to b e t w e e n 17 a n d 2 0 ° C at w h i c h p o i n t anthesis s t a r t e d in t h e f i r s t - o r d e r spathes. Seed release and deteriorat i o n o f the f l o w e r i n g s h o o t s o c c u r r e d as t h e w a t e r t e m p e r a t u r e c o n t i n u e d to increase f r o m 21 to 24°C. DISCUSSION T h e f l o w e r i n g s h o o t s o b s e r v e d in this s t u d y w e r e smaller t h a n t h o s e described b y o t h e r investigators. A l o n g the Pacific c o a s t o f N o r t h A m e r i c a (Setchell, 1 9 2 9 ) and in t h e G u l f o f C a l i f o r n i a (Felger and M c R o y , 1 9 7 5 ) s h o o t s m a y a t t a i n a length o f 3 m , m o r e t h a n six t i m e s the m a x i m u m size (48 c m ) c o l l e c t e d in G r e a t S o u t h Bay. This size v a r i a t i o n p r o b a b l y reflects d i f f e r e n c e s in local c o n d i t i o n s r a t h e r t h a n a d i s t i n c t i o n b e t w e e n A t l a n t i c a n d Pacific varieties (Setchell, 1 9 2 9 ) f o r O s t e n f e l d ( 1 9 0 8 ) c o l l e c t e d flower-
90
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Fig. 4. The relationship of water temperature and salinity in Great South Bay and the time of anthesis (Anth) and seed release (Rel.). The time of flower induction is unknown and is therefore represented by a broken line. It is possible that induction occurs earlier than the time shown on the abscissa. The individual points for salinity and temperature represent single recordings. The solid line for temperature was constructed from weekly averages based on daily measurements.
ing shoots in Danish waters with lengths exceeding 2 m. A large variation occurs also in the density of the flowering shoots. The density observed in this study (53 shoots/m 2 ) falls within the range found in Alaska b y M c R o y (1970), b u t is only a fraction of that (555 shoots/m 2 ) reported by Felger and M c R o y (1975) in the Gulf of California. In both New York and Alaska, the flowering shoots comprise a relatively small percentage (less than 10%) of the total shoot number, whereas in the Gulf of California, 100% of the shoots present during the spring are floral. This is a fascinating difference between populations and one that needs further study. Den Hartog (personal communication, 1976) has suggested that Z. marina may grow as an annual in the Gulf of California and flower precociously under the influence of high water temperatures. Setchell (1929} hypothesized that water temperature determined the periodicity of the reproductive cycle in Z. marina, and that values above 15°C were required for anthesis. The present investigation supports the concept of a minimum 15°C requirement, for anthesis started shortly (4--9 days) after the water temperature had exceeded 15°C. The period immediately prior to anthesis, however, was characterized b y a rapid increase in water
91 temperature (12°C on May 7 to 20°C on May 21) making it difficult, therefore, to define a single, critical temperature. Studies in England (Tutin, 1938), Alaska (McRoy, 1970), and the Gulf of California (Felger and McRoy, 1975) have described anthesis as occurring at water temperatures at or near 15 ° C. In Puget Sound, however, water temperatures do not c o m m o n l y exceed 15°C and flowering occurs when the water is only 8--9°C (Phillips, 1969). This last observation stresses the need to examine the influence of other environmental factors on flowering. Both Phillips (1969) and M c R o y (1970) have emphasized the importance of studying the interaction between light and temperature, while Marmelstein et al. (1968) have demonstrated that photoperiod markedly effects floral development, at least in Thalassia. It is important, however, that future studies carefully distinguish between floral induction, the initiation of primordia, flower development, and anthesis. Each of these stages in the flowering process may be influenced very differently by environmental conditions. At present, there is essentially no information on the nature of floral induction in seagrasses. In Great South Bay, flower primordia were present in January suggesting that induction may occur in the autumn, six to eight months prior to anthesis. Anthesis lasted approximately one month as successive spathe orders matured. Both Setchell (1929) and Tutin (1938) state that flowering ceases when the water temperature increases above 20°C. In Great South Bay, however, the entire period of anthesis occurred while the water temperature fluctuated between 20 ° and 21 ° C. It is unlikely, therefore, that flowering was terminated by unfavorable water temperatures. One may speculate that the duration of anthesis was determined primarily by the ability of the flowering shoot to provide the required nutrients for both seed development and continued flower formation. The existence of such a nutrient stress could explain the progressive decrease in anther size and flower number within increasing spathe orders. It could also explain why the last, microscopic, spathe order present on many of the branches and terminal inflorescences failed to develop. These small spathes remained unchanged and rudimentary throughout the period of anthesis. On June 17, 72% of the ovaries were fertile and contained mature or developing seeds, while the remainder had aborted. The average number of seeds produced by a shoot, therefore, was 34, or 72% of the total ovaries produced (48). This value is small relative to the 500--1000 seeds produced by flowering shoots in California (Setchell, 1929), b u t comparable with the 60 seeds per plant observed by Tutin (1942) in England. Felger and M c R o y (1975) estimated that plants in the Gulf of California produced a minimum of 19,000 seeds/m 2 . This is greater than ten times the value of 1800 seeds/ m 2 determined for Great South Bay. The difference in these two values results primarily from a difference in the flowering shoot densities rather than in the number of seeds produced by individual plants. Felger and M c R o y (1975) observed a density of 555 shoots/m 2 yielding 34 seeds/plant; a value in exact agreement with that observed in the present study.
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The shedding of seeds and c o n c o m m i t t a n t deterioration of the flowering shoots occurred over a period of approximately three weeks. This is a critical period for harvesting seeds. It is our experience that in shallow waters such as Great South Bay, seeds are most reliably and easily collected while still attached to the plant. The question arises, however, as to the best time of collection. The material collected on June 17 had the largest number of seeds per plant (fertile ovaries, Table II), but most of the seeds were immature as judged by embryo size and the soft nature of the seed coat. Plants harvested on July 1 had fewer seeds but nearly all were hard and with embryos that filled the entire volume enclosed by the seed coat. Preliminary experiments on seed viability using the vital dye tetrazolium red [{2, 3, 5-triphenyl-2H-tetrazolium chloride (Baker Chemical)] have yielded results which are relevant to this question. After six months of storage in sea water (28%0) at 4°C, less than 3% of the seeds collected on June 17 and June 23, stained and were thus considered viable (Moore, 1962). A distinct pink staining of the cotyledon and upper h y p o c o t y l region of the embryo (Taylor, 1957), however, was visible in 20--40% of the seeds collected on June 27 and July 1. Further studies on seed viability and germination are currently in progress and will be reported at a later date. We recommend, however, that efforts to collect seed-bearing plants be concentrated during the second week of seed shedding. During the first week those seeds which are collected on the plant are for the most part immature and not viable (at least after six months storage), whereas by the middle of the third week the plants have deteriorated to such a point that they are both difficult to find and contain too few seeds to make collecting profitable. ACKNOWLEDGEMENTS
This reserach was sponsored by the New York Sea Grant Institute under a grant from the Office of Sea Grant, National Oceanic and Atmospheric Administration (NOAA), U.S. Department of Commerce. REFERENCES Felger, R.S. and McRoy, C.P., 1975. Seagrasses as potential food plants. In: G. Fred Somers (Editor), Seed Bearing Halophytes as Food Plants. Proceedings of a Conference at the University of Delaware. NOAA Office of Seagrant, Dept. of Commerce (Grant No. 2-35223). Marmelstein, A.D., Morgan, P.W. and Pequegnat, W.E., 1968. Photoperiodism and related ecol0'gy in Thalassia testudinum. Bot. Gaz. Chicago. 129: 63--67. McRoy, C.P., 1966. The standing stock and ecology of eelgrass in Izembek Lagoon, Alaska. M.S. Thesis, University of Washington, Seattle, 138 pp. McRoy, C.P., 1970. On the biology of eelgrass in Alaska. Ph.D. Dissertation, University of Alaska, 156 pp. Moore, R.P., 1962. Tetrazolium as a universally acceptable quality test of viable seed. Proc. Seed Test. Assoc., 27: 795--805.
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Ostenfeld, C.H., 1908. On the ecology and distribution of the grass wrack (Zostera marina) in Danish waters. Rep. Danish Biol. Station, 16: 3--62. Phillips, R.C., 1969. Temperate grass flats. In: H.T. Odum, B.J. Copeland and E.A. McMahon (Editors), Coastal Ecological Systems of the United States: A source book for estuarine planning. FWPCA Contact Report No. 68-128, Vol. 2, pp. 737--773. Phillips, R.C., 1972. Ecological life history of Zostera marina L. (eelgrass) in the Puget Sound, Washington, Seattle. Ph.D. Dissertation, University of Washington, Seattle, 154 pp. Setchell, W.A., 1929. Morphological and phenological notes on Z o s t e r a marina L. Univ. Calif. Publ. Bot., 14: 389--452. Taylor, A.R.A., 1957. Studies on the development of Z o s t e r a marina L. I. The embryo and seed. Can. J. Bot., 35: 477--499. Tutin, T.G., 1938. The autecology of Z o s t e r a marina L. in relation to its wasting disease. New Phytol., 37: 50--71. Tutin, T.G., 1942. Z o s t e r a L. J. Ecol., 30: 217--226.
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A N T H E S I S A N D S E E D P R O D U C T I O N I N Z O S T E R A M A R I N A L. F R O M G R E A T S O U T H B A Y , N E W Y O R K , U.S.A.
A. COOLIDGE CHURCHILL and MICHAEL I. RINER Adelphi Institute of Marine Science, Adelphi University, Garden City, N.Y. 11530 (U.S.A.)
(Received 8 June 1976)
ABSTRACT Churchill; A.C, and Riner~ M.I., 1978. Anthesis and seed production in Zostera marina L. from Great South Bay, New York, U.S.A. Aquat. Bot., 4: 83--93. Flowering and seed production were studied during the spring and summer of 1975 in a population of Zostera marina L. from Great South Bay, New York. The maturation of stamens and pistils was observed from April, until the completion of anthesis in June. Pollination occurred successively within increasing spathe orders and lasted approximately one month, starting between May 15 and May 21, when the water temperature was 17--20 ° C, and ending by June 17, when the water temperature had reached 21 ° C. Ovary dehiscence and seed release were first observed on June 17, and essentially completed by July 9, together with the deterioration of the flowering shoots. An average of 48 pistils was produced on each flowering shoot, but seed formation occurred in only 72% of these reproductive organs. The remaining 28% aborted, presumably due to the failure of fertilization. The average density of the flowering shoots was 53/m 2 , yielding a potential seed crop of 1802 seeds/m 2 .
INTRODUCTION T h e seagrass Zostera marina L. has b e e n intensively studied d u r i n g r e c e n t years, b u t t h e r e is surprisingly little i n f o r m a t i o n c o n c e r n i n g t h e p r o c e s s o f f l o w e r i n g a n d seed p r o d u c t i o n . T h e classic d e s c r i p t i o n b y Setchell ( 1 9 2 9 ) o f the r e p r o d u c t i v e p e r i o d i c i t y o f Z. marina f r o m E u r o p e and t h e east and w e s t coasts o f N o r t h A m e r i c a still serves as t h e basis f o r m u c h o f o u r u n d e r s t a n d ing o f f l o w e r i n g in this p l a n t . Setchell ( 1 9 2 9 ) p r o p o s e d t h a t w a t e r t e m p e r a t u r e was the p r i m a r y e n v i r o n m e n t a l variable d e t e r m i n i n g the o n s e t and dura t i o n o f sexual r e p r o d u c t i o n . His h y p o t h e s i s t h a t anthesis and seed f o r m a t i o n o c c u r r e d o n l y at w a t e r t e m p e r a t u r e s f r o m 15 t o 2 0 ° C was s u p p o r t e d b y T u t i n ( 1 9 3 8 ) , a n d m o r e r e c e n t l y b y M c R o y ( 1 9 6 6 ) , b u t it d o e s n o t explain t h e p e r i o d i c i t y o f f l o w e r i n g in p l a n t s f r o m P u g e t S o u n d (Phillips, 1969). Setchell ( 1 9 2 9 ) p r e s e n t e d little q u a n t i t a t i v e d a t a o n t h e f l o w e r i n g p r o c e s s a l t h o u g h r e c e n t studies ( M c R o y , 1970; Phillips, 1 9 7 2 ; Felger and M c R o y , 1 9 7 5 ) have d e t e r m i n e d t h e d e n s i t y o f f l o w e r i n g s h o o t s in n a t u r a l p o p u l a tions, a n d in o n e case (Felger and M c R o y , 1 9 7 5 ) h a v e e s t i m a t e d seed p r o d u c -
84 tion. To our knowledge, however, there has been no quantitative analysis of flower production, percent seed set, or seed release. This study describes the flowering of Z. marina from Great South Bay, New York. Weekly collections and observations of plants were made during the flowering period and used to describe floral development, seed production, and the pattern of seed release. MATERIAL AND METHODS Collections of flowering plants were made from an established Zostera meadow on the north side of West Fire Island (Fig. 1). Flowering plants growing 1.0 m below MLW (tidal amplitude 25 cm) were harvested initially by random sampling of terminal shoots and later, when the flowering plants were visible w i t h o u t close inspection, by selective picking from among the vegetative branches. Weekly collections of 30 flowering shoots were made between April 17 and July 9 and were preserved in seawater with formaldehyde (4%). Fifteen shoots were used for evaluation and analysis of flower and fruit development, and the remainder were stored as reference material. On June 3, the entire collection of 30 shoots was used to determine the average number of spathes and the number of stamens and pistils on individual shoots. The density of flowering shoots and their percentage within the population was determined by m o n t h l y harvests of all plants within ten 0.05 m 2 quadrats. Standard deviations of the means are given in the text.
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85
Water temperature and salinity (by refractometer) were recorded during each visit to the study area. Additional temperature values were kindly provided by the Bluepoints Co. Inc. These were daily recordings made between January 17, 1975 and July 31, 1975 at West Sayville, New York, and were representative of the study area. RESULTS
The flowering shoots differ from the vegetative ones in being erect and w i t h o u t adventitious roots. When mature, they varied from 19 to 48 cm in length with an average of 32 -+ 8 cm and, as described by Setchell (1929), each flowering shoot represented the tip of a main rhizome axis. Only a small percentage (6--9%) of the shoots present flowered, and the average density of flowering shoots on June 3 was 53 -+ 24/m 2 . The reproductive stem produced both leaves and floral branches in a uniform pattern (Fig. 2). Flower-bearing spathes were initiated at the apex of
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Fig. 2. A s c h e m a t i c r e p r e s e n t a t i o n o f t h e f l o w e r i n g s h o o t o f Zostera marina. T h e s p a t h e s (sp) o n each b r a n c h a n d t h e t e r m i n a l i n f l o r e s c e n c e are n u m b e r e d s e q u e n t i a l l y a c c o r d i n g t o t h e i r o r d e r o f m a t u r a t i o n . T h e b r o k e n lines r e p r e s e n t leaf-like s h e a t h s w h i c h successively e n v e l o p smaller s p a t h e clusters d u r i n g t h e i r initial d e v e l o p m e n t o n t h e b r a n c h e s and terminal inflorescence.
86
the shoot (terminal inflorescence), and on 1--3 (rarely 4) lateral branches. Frequently, the first branch (branch 1, Fig. 2) was undeveloped and, therefore, evident only after close inspection. Each lateral branch alternated along the stem with ale af, and together with the terminal inflorescence, produced spathes which matured acropetally as indicated by the assigned numbers in Fig. 2. The number of spathes which matured on the branches and terminal inflorescence varied and tended to increase acropetally along the shoot (means: branch 1, 0.3, branch 2, 1.6, branch 3, 2.5, terminal inflorescence, 3.2). The value of 0.3 spathes for branch 1 reflects the frequent undeveloped condition of this branch. The maximum spathe number for a single shoot was 11 and the average 7.6 + 1.8. In most cases, the branches and the terminal inflorescence possessed an additional spathe order which, by the end of the flowering period, was visible only upon microscopic examination. These spathes failed to mature and were, therefore, not included in the present determinations. A spathe ensheathed and protected a single monoecious spadix on which developed the reduced flowers consisting of single stamens (male) and pistils (female). The number of stamens and pistils which developed within each spathe varied. For a specific spathe order, this number increased acropetally from the basal branch to the terminal inflorescence, while on a given branch, it increased successively from the last to the first spathe order (Table I). The number of stamens within a spathe was approximately twice that of the pistils, and they were regularly arranged on a spadix. The maximum number of flowers on a single spadix was 33 (22 stamens, 11 pistils) and on an individual shoot was 247 (160 stamens, 87 pistils). The average number of stamens and pistils produced per flowering shoot was 91 + 24 and 48 -+ 13, respectively, yielding a total of 139 flowers per shoot. Pistils on a single spadix developed simultaneously and, typically, matured before the stamens. This proterogyny was evident by the withering and frequent abscission of the bifurcated stigma prior to anther dehiscence. Flowers TABLE I The m e a n n u m b e r o f s t a m e n s (st) and pistils (p) w i t h i n s p a t h e s o n t h e lateral b r a n c h e s and t h e t e r m i n a l inflorescence Spathe order
Branch n u m b e r
1
1 2 3 4
Terminal inflorescence
2
3
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p
st
p
st
p
6.5
3.5
9.6 9.0 7.2
4.6 5.0 4.0
13.7 7.0 12.4 6.4 9.5 5.5 8.7 5.0
st
p
14.8 7.6 13.7 7.3 13.1 7.0 8.8 4.7
87 on different branches but within spathes of the same order matured more or less synchronously; the difference, therefore, between branches including the terminal inflorescence lay in the number of flowers and spathes present rather than in the pattern or timing of their development. Flowers of increasing spathe orders on a branch developed sequentially, thereby extending the period of anthesis. This pattern of maturation is demonstrated by size frequency histograms showing changes in the mean length of the largest anther within each spathe on branch 2 (Fig. 3). The anthers within spathe 1 increased in size from April 17 to May 15 and the largest reached a m a x i m u m length of 7.4 mm. Anther dehiscence was first observed on May 21 and was essentially completed by June 3. Development of male flowers within spathe 2 was delayed so that pollen release did not start until the end of May or approximately one week after the flowers on spadix 1, (Fig. 3). The m a x i m u m anther length (4.8 mm) was smaller than on spadix 1, and the time of development as measured by the period between the appearance of class 2 anthers (class mark, 2,95 ram) and the first anther release was reduced from five to three weeks. Maturation of anthers withi~ spathe 3 was further delayed so that pollen release was not observed until June 3. The m a x i m u m anther length was only 4.0 ram, and the time of development was approximately two weeks. The time of development and anther dehiscence in fourth-order spathes was essentially coincident with those on spadix 3. With the exception of a few stamens, pollen release was completed by June 17. Small anthers (less than 1.9 mm) which were present at this time failed to develop further. Development and maturation of the female flowers were examined by observing changes in the length of the pistil (ovary exclusive of the bifurcated stigma). On April 17, the largest ovary (spathe 1) varied in length from 0.1 mm to 0.3 mm. The ovaries increased to a length of 3--4 mm by the start of pollination (May 21) and to 5--7 mm on June 17 when seed release started. Development was similar to that of the stamens in that maturation was delayed within increasing spathe orders and synchronous on a single spadix. Unlike the stamens, however, the mean length of the mature ovary did not decrease within increasing spathe orders. Each mature ovary contained a single elliptical seed with an average length and width of 3.1 + 0.1 mm and 1.5 + 0.1 mm, respectively, and a color that varied from blue-gray to a dark brown. Dehiscence of the ovary and seed release was first observed in the field on June 17, nearly one m o n t h after the start of pollen release. The process occurred initially in the first-order spathes and then successively in the higher spathe orders, and was marked by a lengthwise splitting of the ovary wall and the c o n c o m m i t t a n t deterioration of the enfolding spathe sheath along its edges. The seeds sank immediately after dehiscence from the ovary. Shortly after seed release, or in some cases before, abscission of the spathe occurred just below the spadix, leaving only the narrow, basal stalk of the spathe attached to the shoot.
88
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ANTHER LENGTH (mm) Fig. 3. Size--frequency histograms showing changes in the mean length of the largest anther within each spathe on branch 2. The frequency (ordinate) represents the percentage of total anthers measured for a given spathe order and for dehisced anthers (D) is indicated by solid bars. Each size class (abscissa) has an interval of 2.0 mm and is designated by the class mark (midpoint).
The pattern of seed loss was determined by comparing the n u m b e r of ovaries on shoots prior to and during the period of seed release. The results (Table II) indicate that the greatest loss occurred between June 23 and July 1, and that by July 9, nearly 90% of the seeds had been shed. In addition, it was possible to distinguish by size and appearance between ovaries in which seed formation had occurred and those which had aborted, presumably due to the failure of fertilization. From June 17 to July 1, the percentage of fertile and aborted ovaries on the flowering shoots remained fairly constant; a characteristic which is readily explained by spathe abscission and the resulting simultaneous loss of both fertile and aborted ovaries. By July 9, how-
89
TABLE II The mean number and standard deviation of ovaries per flowering shoot during the period of seed release and the percentage of fertile ovaries (containing mature or developing seeds). The percentage of fertile ovaries on June 3 is not given since pollen release was still occurring Date of Collection
Ovaries present
Cumulative ovary loss (%)
Fertile ovaries (%)
3June1975 17 June 1975 23 June 1975 1 July 1975 9 July 1975
48-+ 13 44_+ 12 33-+ 11 14 -+ 7 5 -+ 4
0 8 30 70 89
72± 10 69-+ 15 66 + 19 20 ± 18
ever, t h e p e r c e n t a g e o f fertile ovaries d e c r e a s e d t o 20%. T h e p l a n t s at this t i m e w e r e g r e a t l y r e d u c e d b y the abscission o f all b u t t h e f o u r t h - and occasional t h i r d - o r d e r spathes. T h e t i m e w h e n f l o w e r p r i m o r d i a first a p p e a r e d was n o t d e t e r m i n e d . T e r m i n a l s h o o t s c o l l e c t e d in J a n u a r y 1975 as p a r t o f a n o t h e r s t u d y were exa m i n e d m i c r o s c o p i c a l l y a n d s h o w e d distinct s t a m e n a n d pistil f o r m a t i o n , p a r t i c u l a r l y o n s p a t h e 1 o f the t e r m i n a l inflorescence. Collections in Dec e m b e r a n d N o v e m b e r o f 1 9 7 4 did n o t c o n t a i n f l o w e r i n g shoots, b u t t h e s a m p l e s were t o o small t o d i s c o u n t t h e i r presence. T h e p e r i o d w h e n i m m a t u r e flowers w e r e o b s e r v e d and t h e t i m e o f p o l l i n a t i o n and seed release are illustrated in Fig. 4 t o g e t h e r w i t h t h e r e c o r d e d salinity and t e m p e r a t u r e values. Salinity r e m a i n e d fairly c o n s t a n t d u r i n g the s t u d y p e r i o d , v a r y i n g f r o m 28 t o 3 2 % 0 . W a t e r t e m p e r a t u r e , as e x p e c t e d , s h o w e d m a r k e d seasonal changes. In J a n u a r y a n d F e b r u a r y w h e n small i m m a t u r e flowers were p r e s e n t , t e m p e r a t u r e values ranged f r o m 0.5 t o 3 ° C. F l o w e r m a t u r a t i o n occurred as the w a t e r t e m p e r a t u r e increased to b e t w e e n 17 a n d 2 0 ° C at w h i c h p o i n t anthesis s t a r t e d in t h e f i r s t - o r d e r spathes. Seed release and deteriorat i o n o f the f l o w e r i n g s h o o t s o c c u r r e d as t h e w a t e r t e m p e r a t u r e c o n t i n u e d to increase f r o m 21 to 24°C. DISCUSSION T h e f l o w e r i n g s h o o t s o b s e r v e d in this s t u d y w e r e smaller t h a n t h o s e described b y o t h e r investigators. A l o n g the Pacific c o a s t o f N o r t h A m e r i c a (Setchell, 1 9 2 9 ) and in t h e G u l f o f C a l i f o r n i a (Felger and M c R o y , 1 9 7 5 ) s h o o t s m a y a t t a i n a length o f 3 m , m o r e t h a n six t i m e s the m a x i m u m size (48 c m ) c o l l e c t e d in G r e a t S o u t h Bay. This size v a r i a t i o n p r o b a b l y reflects d i f f e r e n c e s in local c o n d i t i o n s r a t h e r t h a n a d i s t i n c t i o n b e t w e e n A t l a n t i c a n d Pacific varieties (Setchell, 1 9 2 9 ) f o r O s t e n f e l d ( 1 9 0 8 ) c o l l e c t e d flower-
90
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Fig. 4. The relationship of water temperature and salinity in Great South Bay and the time of anthesis (Anth) and seed release (Rel.). The time of flower induction is unknown and is therefore represented by a broken line. It is possible that induction occurs earlier than the time shown on the abscissa. The individual points for salinity and temperature represent single recordings. The solid line for temperature was constructed from weekly averages based on daily measurements.
ing shoots in Danish waters with lengths exceeding 2 m. A large variation occurs also in the density of the flowering shoots. The density observed in this study (53 shoots/m 2 ) falls within the range found in Alaska b y M c R o y (1970), b u t is only a fraction of that (555 shoots/m 2 ) reported by Felger and M c R o y (1975) in the Gulf of California. In both New York and Alaska, the flowering shoots comprise a relatively small percentage (less than 10%) of the total shoot number, whereas in the Gulf of California, 100% of the shoots present during the spring are floral. This is a fascinating difference between populations and one that needs further study. Den Hartog (personal communication, 1976) has suggested that Z. marina may grow as an annual in the Gulf of California and flower precociously under the influence of high water temperatures. Setchell (1929} hypothesized that water temperature determined the periodicity of the reproductive cycle in Z. marina, and that values above 15°C were required for anthesis. The present investigation supports the concept of a minimum 15°C requirement, for anthesis started shortly (4--9 days) after the water temperature had exceeded 15°C. The period immediately prior to anthesis, however, was characterized b y a rapid increase in water
91 temperature (12°C on May 7 to 20°C on May 21) making it difficult, therefore, to define a single, critical temperature. Studies in England (Tutin, 1938), Alaska (McRoy, 1970), and the Gulf of California (Felger and McRoy, 1975) have described anthesis as occurring at water temperatures at or near 15 ° C. In Puget Sound, however, water temperatures do not c o m m o n l y exceed 15°C and flowering occurs when the water is only 8--9°C (Phillips, 1969). This last observation stresses the need to examine the influence of other environmental factors on flowering. Both Phillips (1969) and M c R o y (1970) have emphasized the importance of studying the interaction between light and temperature, while Marmelstein et al. (1968) have demonstrated that photoperiod markedly effects floral development, at least in Thalassia. It is important, however, that future studies carefully distinguish between floral induction, the initiation of primordia, flower development, and anthesis. Each of these stages in the flowering process may be influenced very differently by environmental conditions. At present, there is essentially no information on the nature of floral induction in seagrasses. In Great South Bay, flower primordia were present in January suggesting that induction may occur in the autumn, six to eight months prior to anthesis. Anthesis lasted approximately one month as successive spathe orders matured. Both Setchell (1929) and Tutin (1938) state that flowering ceases when the water temperature increases above 20°C. In Great South Bay, however, the entire period of anthesis occurred while the water temperature fluctuated between 20 ° and 21 ° C. It is unlikely, therefore, that flowering was terminated by unfavorable water temperatures. One may speculate that the duration of anthesis was determined primarily by the ability of the flowering shoot to provide the required nutrients for both seed development and continued flower formation. The existence of such a nutrient stress could explain the progressive decrease in anther size and flower number within increasing spathe orders. It could also explain why the last, microscopic, spathe order present on many of the branches and terminal inflorescences failed to develop. These small spathes remained unchanged and rudimentary throughout the period of anthesis. On June 17, 72% of the ovaries were fertile and contained mature or developing seeds, while the remainder had aborted. The average number of seeds produced by a shoot, therefore, was 34, or 72% of the total ovaries produced (48). This value is small relative to the 500--1000 seeds produced by flowering shoots in California (Setchell, 1929), b u t comparable with the 60 seeds per plant observed by Tutin (1942) in England. Felger and M c R o y (1975) estimated that plants in the Gulf of California produced a minimum of 19,000 seeds/m 2 . This is greater than ten times the value of 1800 seeds/ m 2 determined for Great South Bay. The difference in these two values results primarily from a difference in the flowering shoot densities rather than in the number of seeds produced by individual plants. Felger and M c R o y (1975) observed a density of 555 shoots/m 2 yielding 34 seeds/plant; a value in exact agreement with that observed in the present study.
92
The shedding of seeds and c o n c o m m i t t a n t deterioration of the flowering shoots occurred over a period of approximately three weeks. This is a critical period for harvesting seeds. It is our experience that in shallow waters such as Great South Bay, seeds are most reliably and easily collected while still attached to the plant. The question arises, however, as to the best time of collection. The material collected on June 17 had the largest number of seeds per plant (fertile ovaries, Table II), but most of the seeds were immature as judged by embryo size and the soft nature of the seed coat. Plants harvested on July 1 had fewer seeds but nearly all were hard and with embryos that filled the entire volume enclosed by the seed coat. Preliminary experiments on seed viability using the vital dye tetrazolium red [{2, 3, 5-triphenyl-2H-tetrazolium chloride (Baker Chemical)] have yielded results which are relevant to this question. After six months of storage in sea water (28%0) at 4°C, less than 3% of the seeds collected on June 17 and June 23, stained and were thus considered viable (Moore, 1962). A distinct pink staining of the cotyledon and upper h y p o c o t y l region of the embryo (Taylor, 1957), however, was visible in 20--40% of the seeds collected on June 27 and July 1. Further studies on seed viability and germination are currently in progress and will be reported at a later date. We recommend, however, that efforts to collect seed-bearing plants be concentrated during the second week of seed shedding. During the first week those seeds which are collected on the plant are for the most part immature and not viable (at least after six months storage), whereas by the middle of the third week the plants have deteriorated to such a point that they are both difficult to find and contain too few seeds to make collecting profitable. ACKNOWLEDGEMENTS
This reserach was sponsored by the New York Sea Grant Institute under a grant from the Office of Sea Grant, National Oceanic and Atmospheric Administration (NOAA), U.S. Department of Commerce. REFERENCES Felger, R.S. and McRoy, C.P., 1975. Seagrasses as potential food plants. In: G. Fred Somers (Editor), Seed Bearing Halophytes as Food Plants. Proceedings of a Conference at the University of Delaware. NOAA Office of Seagrant, Dept. of Commerce (Grant No. 2-35223). Marmelstein, A.D., Morgan, P.W. and Pequegnat, W.E., 1968. Photoperiodism and related ecol0'gy in Thalassia testudinum. Bot. Gaz. Chicago. 129: 63--67. McRoy, C.P., 1966. The standing stock and ecology of eelgrass in Izembek Lagoon, Alaska. M.S. Thesis, University of Washington, Seattle, 138 pp. McRoy, C.P., 1970. On the biology of eelgrass in Alaska. Ph.D. Dissertation, University of Alaska, 156 pp. Moore, R.P., 1962. Tetrazolium as a universally acceptable quality test of viable seed. Proc. Seed Test. Assoc., 27: 795--805.
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Ostenfeld, C.H., 1908. On the ecology and distribution of the grass wrack (Zostera marina) in Danish waters. Rep. Danish Biol. Station, 16: 3--62. Phillips, R.C., 1969. Temperate grass flats. In: H.T. Odum, B.J. Copeland and E.A. McMahon (Editors), Coastal Ecological Systems of the United States: A source book for estuarine planning. FWPCA Contact Report No. 68-128, Vol. 2, pp. 737--773. Phillips, R.C., 1972. Ecological life history of Zostera marina L. (eelgrass) in the Puget Sound, Washington, Seattle. Ph.D. Dissertation, University of Washington, Seattle, 154 pp. Setchell, W.A., 1929. Morphological and phenological notes on Z o s t e r a marina L. Univ. Calif. Publ. Bot., 14: 389--452. Taylor, A.R.A., 1957. Studies on the development of Z o s t e r a marina L. I. The embryo and seed. Can. J. Bot., 35: 477--499. Tutin, T.G., 1938. The autecology of Z o s t e r a marina L. in relation to its wasting disease. New Phytol., 37: 50--71. Tutin, T.G., 1942. Z o s t e r a L. J. Ecol., 30: 217--226.