Flowering cycle and seed production of the marine phanerogam Zostera caulescens Miki in Funakoshi Bay, northeast Honshu

Island, Japan.

Dr. Soha SHABAKA Abstract Zostera caulescens, the worlds longest seagrass, is an endangered seagrass species in Japan. This study is the first to elucidate the developmental stages of its flowering cycle and seed production through bimonthly sampling of its flowering shoots and examination of its flowers. The flowering period lasted from April to August. The fruiting stage started by the end of June, and the sexual organs were not recorded in August with only fruiting stage and the flowering season ended in October. In December and January, the flowering shoots were immature. The immature flowering shoots were observed through the year while the mature flowering shoots were observed from June to October. The potential seed production was estimated using flower density and fruit density per unit area from the flowering shoots. The flower and fruit densities reached a maximum of 575 flowers m-2 in June 2008 and 2500 fruits m-2 in August 2007 at 9 m deep at Stn. 2. The unusual height of the flowering shoots of Z. caulescens allow the plant to acquire light under low light conditions and utilize it for seed production at depths reaching to 15 m. Keywords: Zostera caulescens, seagrass, flowering, seed production, Japan

1. Introduction The family Zosteraceae has high species diversity in Japan and Korean coastal waters (Kato et al. 2003). Some of these species were listed in the Red Data Book of threatened marine flora in Japan, e.g. Zostera caulescens Miki and Z. asiatica Miki (Wildlife Division, Agency of the Environment, Japan 2000). Z. caulescens, a dominant species in Funakoshi Bay (Aioi et al. 1996, Sultana and Komatsu 2003), is the worlds longest seagrass with a unique height of its flowering shoots reaching 7 m. This special characteristic has

attracted studies such as biology and morphology (Omori 1989, 1991, 1992, Shabaka and Komatsu 2010) and mapping its distribution (Tatsukawa et al. 1996, Komatsu et al. 2003, Sagawa et al. 2008). However, no studies were conducted to elucidate its developmental stages or to estimate its seed production. Seagrasses have two forms of reproduction: vegetative and sexual reproductions. Sexual reproduction and seed production of seagrasses have an important role in the colonization of new sites (Orth et al. 1994) and recovery after disturbances e.g. seasonal declines and hurricane disturbances (Hammerstrom 2006), destruction caused by red tide algal bloom (Lee et al. 2006) and heavy grazing by dugongs (Peterken and Concher 1997). According to Verhagen and Nienhuis (1983) Z. marina with low rhizome elongation rate can achieve high colonization potential by high reproductive effort, while the combination of slow elongation rate with poor ability to set seeds in some seagrasses, e.g. Posidonia oceanica L., results in extremely slow recovery process (Meinesz and Lefevre 1984). The importance of the forests of Z. caulescens to the temperate coastal waters of Japan together with its threatened existence require studies on its reproductive ecology that contributes directly to its persistence especially for a threatened species. This study elucidates details of flowering cycle of this species and the developmental stages of its flowers from the juvenile stage to the mature stage.

2. Material and Methods. 2. 1. Study area Funakoshi Bay is located on the Sanriku coast of northeast Honshu Island (Figure 1). The bay opens to the Pacific Ocean in a wide mouth with a maximum depth of 30 m (Figure 1). Three seagrass species have been recorded from the southern part of the bay; Z. caulescens, Z. marina and Z. asiatica (Shibatani and Komatsu in preparation). Z. caulescens forms a wide bed extending across the bottom from depths of about 3 to 15 m (Sultana and Komatsu 2003).

2.2. Sampling procedure Bimonthly sampling was carried out from June 2007 to June 2008. Four stations were designed to cover the range of depths across which Z. caulescens is distributed in the bay: Stn. 1 (6 m deep), Stn. 2 (9 m deep), Stn. 3 (12 m deep), and Stn. 4 (15 m deep). At every station, plants were collected randomly by scuba diving from three quadrates (0.04 m2) in each sampling month except in June 2007 (two quadrate samples). In every quadrate, both above-ground parts (vegetative and flowering shoots) and below-ground parts (rhizomes and roots) were collected. Samples were frozen to preserve them for later laboratory measurement. Vertical profiles of water temperature and salinity were taken at Stn. 3 (Figure 1) using a salinity-temperature-depth instrument (STD; Model TCDKU, Alec Co.). Using these data, water temperature and salinity at 1 m above the bottom (at the level of vegetative and small immature flowering shoots) and at mean canopy height of the flowering shoots were depicted in Figures 2 and 3. Solar radiation reaching the sea surface was monitored at 10-min intervals using an automatic weather station (AADI) located on a raft in Otsuchi Bay, which neighbours Funakoshi Bay (Figure 1). Solar radiation data from June 2007 to June 2008 were compiled.

2.3. Flowering cycle and Seed production Each flower of the flowering shoots was examined using a hand lens or a microscope to examine its developmental stage. Flowers in every quadrate sample were separated and counted. Fruits (fertilized ovaries or seeds) in every flower were counted using a hand lens. The potential seed production per unit area was estimated according to the method proposed by Inglis and Waycott (2001). The latter authors proposed two methods to estimate seed production: one is to measure the variation in abundance of many sexual structures containing seed and the other is to estimate seeds density of the seed bank in the sediment. In case of Zostera species, number of fertile fruits per spadix, number of spadices per reproductive shoot, and number of reproductive shoots per unit area must be measured (Inglis and Waycott 2001). The present

study used the first method because the fallen seeds in the sediment may be subjected to dispersal to distant areas by bottom currents.

3. Results 3.1. Environmental parameters Water temperature and salinity at 1 m above the bottom were almost the same as those recorded at mean height of flowering shoots during the sampling period (Figures 2 and 3). At Stn. 3, the highest temperatures in a year were 18.27C and 19.02C in August and October 2007 respectively. The lowest temperature was 5.51C in April 2008. The highest salinities in a year were 34.05 psu and 33.99 psu in December 2007 and January 2008, respectively. The lowest salinity was 33.42 psu in April 2008. Mean daily solar radiation showed a clear seasonal pattern where higher values were observed from April to August 2007 with a peak in June 2007 and lower values in autumn and winter with a trough in December 2007 and January 2008 (Figure 4).

3.2. Frequency distribution of immature and mature flowering shoots. Two kinds of flowering shoots of Z. caulescens, immature and mature flowering shoots, were observed. The immature flowering shoots were characterized by the absence of flowers and/or presence of leaves transforming to flowers without specialized organs near the flowering season (premature shoots). The mature flowering shoots carried flowers with specialized organs. The immature flowering shoots were observed in every month, it had a dominant length between 100 cm and 150 cm (Figure 5); however, it attained a maximum of 400 cm in length in December 2007 and April 2008 (Figures 5 and 6). The percentage of the immature flowering shoots increased from June 2007 to October 2007 (Figure 5). In these months the immature flowering shoots were not observed in the deepest station (Stn. 4). In December 2007, January 2008, and April 2008, all the flowering shoots were immature (Figures 5 and 6). In April 2008, about 73% of the all

immature flowering shoots carried a leaf transforming to a flower (premature flowering shoots). In June 2008, the immature flowering shoots attained its maturity at all stations except in the deepest station (Stn. 4) (Figure 6). In June 2008, 73% of all immature flowering shoots were premature. The mature flowering shoots were observed in June 2007, August 2007, October 2007 and June 2008 (Figure 5 and 6). The length of the mature flowering shoots increased from a maximum of 300 cm in June 2007 to 600 and 500 cm in August and October 2007 respectively (Figure 5). Mature flowering shoots reappeared in June 2008 after its absence in December 2007, January 2008 and April 2008 and it had a maximum length of 500 cm (Figure 6)

3.3. Flowering phases Four flowering phases of different structures in a flower were defined as follows (Figure 7): I. II. III. IV. Immature phase with indistinguishable anthers and pistils Mature phase with mature anthers and pistils Fruiting phase with only fruits Black remains of dead flowers and detached flowering shoots.

In June 2007, flowers of phases I, II and III were observed, with the dominance of phase II (54 %) (Figure 8). In August 2007, phase III was 100%. i.e. all the flowers were in the fruiting phase (Figure 8). In October 2007, all the mature flowering shoots carried black remains of flowers (phase IV) (Figure 8). Detached flowering shoots heavily encrusted with epiphytes were observed in October 2007. In June 2008, phases I (33%) and II (67%) were observed (Figure 8). In April 2008, some immature flowering shoots carried leaves transforming to a flower (without specialized organs).

3.4. Seed production The potential seed production was estimated using flower density, fruit density per unit area (m-2) in every

sampled month. The flower densities ranged between 25 flowers m-2 at Stn. 3 in August 2007 and 575 flowers m-2 at Stn. 2 in June 2008 (Figure 9a). We excluded the grazed flowers from the estimation. The fruit density ranged between 300 to 2500 fruits m-2 in August 2007 at Stn. 1 and Stn. 2 respectively (Figure 9b). In June 2008, the fruiting phase (phase III) was not observed. The flower and fruit density at Stns. 3 and 4 were lower than those at the shallower stations (Stns. 1 and 2) (Figure 9). In June and August 2007, the tanaid crustacean Zeuxo sp was found inside the spathes (Figure 10a). These organisms were observed to be associated only with the fruiting phase (phase III). Formation of bores in seeds demonstrated that Zeuxo sp. ate seeds. Moreover, some of the fruits with expelled inner material were observed which might mean abortion before germination (Figure 10b).

4. Discussion The precise examination of the developmental stages of the flowers and flowering shoots dynamics of Z. caulescens indicated that the flowering season began in April and ended in October. The mature flowering shoots were observed from summer to autumn. While the immature shoots were recorded throughout the year. Water temperatures were higher than 16C from June 2007 to October 2007. Around June 2007, seawater temperature at the flowering shoots canopy level was about 16C while in June 2008 was about 13C. In June 2007, most of the flowering shoots were mature while in June 2008 most of the flowering shoots were immature or premature. Probably water temperature played an important role in the maturity of flowering shoots of Z. caulescens. Omori and Aioi (2000) studied the growth of Z. caulescens flowering shoots in Funakoshi Bay and Otsuchi Bay. They reported that the flowering shoots attaining more than 2 m in length bore inflorescences in mid May and some of these flowers bore fruits in June, then the flowering shoot continued to grow after the fructification and falling of the flowers in autumn. The present study showed that the reproduction cycle started in April and the fruiting phase started at the end of June. There is no relation between the length of

flowering shoots and timing of bearing inflorescence because some flowering shoots of less than 2 m bore flowers, and immature shoots of more than 2 m had no flowers. According to De Cock (1981a, 1981b), under laboratory conditions the flowering mechanism of Zostera marina is controlled by temperature and light synergistically. In Funakoshi Bay, the blooming period of the flowering continued at water temperatures above 16C from June to August. Since solar radiation was at its maximum in Funakoshi Bay in June, flowering shoots accumulate much carbohydrate acquired by leaves around June to produce seeds. In Sagami Bay, central Honshu Island, flowering shoots of Z. caulescens bloomed in April to May when the temperature was between 15 - 20C and terminated blooming when the water temperature exceeded 20C in June and July and the inflorescences began to fall (Omori 1994). Longer flowering season in Funakoshi Bay is due to water temperature lasted suitable for flowering till August colder than that in Sagami Bay where water temperature in summer is too high for Z. caulescens to last fruiting. The potential annual seed production of Z. marina in Ago Bay estimated from the flowering shoots was 6000 2400 no. m-2 at bottom depth of 1 to 4 m deep and that seeds were found in bottom sediments as deep as 8 m, but no deeper (Morita et al. 2007). On the contrary to Z. marina, Z. caulescens can produce seeds at depth of 15 m. According to Yamaki and Ogawa (2009), due to the difference in the habitat between Z. caulescens and Z. marina, the effect of salinity and irradiance on the germination of seedlings of both species was different, where Z. caulescens germinates at lower irradiance and higher salinities than Z. marina since Z. marina grows usually over shallower bottom. The unusual height of the flowering shoots of Z. caulescens allow the plant to acquire light under low light conditions and utilizes it for seed production at deeper stations. In the present study, many fruits were found eaten or aborted before dispersal. The tanaid crustacean was observed in every fructified spadix, this is in addition to the grazing on the flowers as a whole. These factors probably affected the potential seed production of Z. caulescens. Nakaoka (2002) reported that the tanaid crustacean (Zeuxo sp.) fed on the fruits of seagrasses Z. marina and Z. caulescens in Otsuchi Bay and utilized

seagrass spathes for reproduction and as a nursery. Seed predation by the tanaid crustacean has considerable negative impact on seed production (Nakaoka 2002). Other seagrass species were proved to be subjected to considerable seagrass loss due to seed abortion, pre-dispersal seed predation, and physical disturbance of the seeds (Orth et al. 1994, Williams 1995, Fishman and Orth, 1996, Balestri and Cinelli 2002). This is the first study to classify the flowering cycle of Z. caulescens into different developmental stages and measure its seed production. Seed production of this threatened species is very important to maintain its population in Funakoshi Bay and recovery from damages after environmental perturbations. Thus it is important to measure and monitor the reproductive effort of such important and threatened species for its conservation.

Acknowledgements. We are grateful to Mr. Kouichi Morita and the staff of Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo for their help in conducting the field work. We are Thankful to Mr. M. Fukuda the professional diver for sampling the material. Many thanks are also for Prof. Y. A. Michida, Ocean Research Institute for his help with this study.

References Aioi, K., Komatsu, T., and Morita, K. 1996. Giant seagrass, Zostera caulescens Miki, discovered in Funakoshi, Iwate Prefecture. Bull. Japan. Soc. Fish. Oceanogr. 60 (1): 7-10. (In Japanese with an English abstract). Balestri, E. and Cinelli, F. 2003. Sexual reproductive success in Posidonia oceanica. Aquat. Bot. 75: 2132. De Cock A.W.A.M.1981a. Influence of temperature and variation in temperature on flowering in Zostera marina L. under laboratory conditions. Aquat. Bot. 10: 115-123. De Cock A.W.A.M.1981b. Influence of light and dark on flowering in Zostera marina L. under laboratory conditions. Aquat. Bot. 10: 125-131.

Fishman, J. R. and Orth, R. J. 1996. Effects of predation on Zostera marina L. seed abundance. J. Exp. Mar. Bio. Ecol. 198: 11-26. Hammerstorm, K. k., Kenworthy, W. J., Fonseca, M. S., and Whitfield, P. E. 2006. Seed bank, biomass, and productivity of Halophila decipiens, a deep water seagrass on the west Florida continental shelf. Aquat. Bot. 84 (2): 110-120. Inglis, G. J. and Waycott, M. 2001, Methods for assessing seagrass seed ecology and population Genetics. In (F. T. Short, and R. G. Coles, eds.)Global seagrass research methods.. Elsevier Science B. V., Amsterdam. pp. 123-140. Kato, Y., Aioi, K., Omori, Y., Takahata, N., and Satta, Y. (2003). Phylogenetic analyses of Zostera species based on rbcL and matK nucleotide sequences: Implications for the origin and diversification of seagrasses in Japanese waters. Genes Genet. Syst. 78 (5): 329-342. Komatsu T., Igarashi, C., Tatsukawa, K., Sultana, S., Matsuoka, Y., and Harada, S. 2003. Use of multi-beam sonar to map seagrass beds in Otsuchi Bay on the Sanriku Coast of Japan. Aquat. Liv. Res. 16 (3): 223-230. Lee K. S., Park, J. I., Kim, Y. K., Park, S. R. and Kim, J. H. 2006. Recolonization of Zostera marina following destruction caused by a red tide algal bloom: the role of new shoot recruitment from seed bank. Mar. Ecol. Prog. Ser. 342: 105-115. Meinesz, A. and Lefevre, J. R. 1984. Regeneration dun herbier de Posidonia oceanica quarante annees aprs sa destruction par une bombe dans la rade de villefranche (Alpes-Maritimes- France). In (C. F. Boudouresque, A. Jeudy de Grissac and J. Oliver eds) International workshop Posidonia oceanica beds. GIS Posidonie publ. France. 1: 39 44. Morita, T., Okumura, H., Abe, M., Kurashima, A. and Maegawa, M. 2007. Density and distribution of seeds in bottom sediments in Zostera marina beds in Ago Bay, central Japan. Aquat. Bot. 87 (1): 3842. Nakaoka, M. 2002. Predation on seeds of seagrasses Zostera marina and Zostera caulescens by a tanaid crustacean Zeuxo sp. Aquat. Bot. 72 (2): 99-106.

Omori, Y. 1989. Morphology of the flowering-shoot and the leaf of of Zostera caulescens Miki and. Z. asiatica Miki (Zosteraceae). Sci. Rept. Yokosuka City Mus. 37: 55-59. (In Japanese with an English abstract). Omori, Y. 1991. Peculiarity of the flowering-shoot of Zostera caulescens (Zosteraceae). Sci. Rept. Yokosuka City Mus. 39: 45-50. (In Japanese with an English abstract). Omori, Y. 1992. Geographical variation of the size of spadix and spathe and the number of flowers among the four species of the subgenus Zostera (Zosteraceae). Sci. Rept. Yokosuka City Mus. 40: 69-74. (In Japanese with an English abstract). Omori, Y. 1994. Seasonal changes of the reproductive shoot of Zostera caulescens (Zosteraceae) in Segami Bay, central Japan. Sci. Rept. Yokosuka City Mus. 42: 65-69. In Japanese with an English abstract. Omori, Y. and Aioi, K. 2000. Seasonal changes of the erect shoot of Zostera caulescens Miki (Zosteraceae) in Sanriku Kaigan, Northern Honshu. Sci. Rept. Yokosuka City Mus. 47: 67-72. (In Japanese with an English abstract). Orth. R. J., Luckenbach, M. and Moore, K. A. 1994. Seed dispersal in a marine macrophyte: implications for colonization and restoration. Ecology. 75 (7): 1927-1939. Sagawa, T., Mikami, A., Komatsu, T., Kosaka, N., Kosako, A., Miyazaki, S. and Takahashi, M. 2008. Mapping seagrass beds using IKONOS satellite image and side scan sonar measurements: a Japanese case study', Int. J. Remote Sens. 29 (1): 281 291. Shabaka, S. and Komatsu, T., 2010. Phenology and morphology of the marine phanerogam Zostera caulescens Miki in Funakoshi Bay, northeast Honshu Island, Japan. Coast. Mar. Sci. 34 (1). Sultana, S. and Komatsu, T. 2003. Changes in shoot density, biomass and leaf area of seagrass Zostera caulescens Miki from summer to autumn in Funakoshi Bay off Sanriku Coast, Japan. Otsuchi Mar. Sci. 28 : 91-98. Tatsukawa, K., Komatsu, T., Aioi, K. and Morita, K. 1996. Distribution of seagrasses off Kirikiri in Funakoshi

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Bay, Iwate Prefecture, Japan. Otsuchi Mar. Res. Cent. Rep. 21: 38-47. (In Japanese). Verhagen, J. H. G and Nienhuis, P. H. 1983. A simulation model of production, seasonal changes in biomass and distribution of eelgrass (Zostera marina) in Lake Grevelingen. Mar. Ecol. Prog. Ser. 10: 187-195. Wildlife Division, Agency of the Environment, Japan. 2000. Threatened wildlife of Japan : Red Data Book ; vascular plant, Japan Wildlife Research Centre, Tokyo. pp. 662. Williams, S. 1995. Surfgrass (Phyllospadix torreyi) reproduction: reproduction phenology, resource, allocation and male rarity. Ecology. 76 (6): 1953-1970. Yamaki, K. and Ogawa, H. 2009. Characterisation of germination and seedling production of Zostera marina and Z. caulescens. Coast. Mar. Sci. 33: 46-53.

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Figure1. Map showing study area in Funakoshi Bay, the STD station (star) and automatic weather station (closed circle).

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20 18 16 14 12 10 8 6 4 Aug 07 Apr 08 Jun 07 Jan 08 Oct 07 Dec 07 Jun 08

Temperature (C)

At 1 m above the bottom At mean canopy height of flowering shoots

Figure 2.Water temperature at Stn. 3 for each sampling period.

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35 Salinity (psu) At 1 m a bove the bottom At mean canopy height of flowering shoots

34

33

32 Aug 07 Apr 08
14

Jun 07

Jan 08

Oct 07

Figure3. Water salinity at Stn. 3 for each sampling period.

Dec 07

Jun 08

x 10 40 35 Amount of solar radiation (W/ m )

2

2007 2008

30 25 20 15 10 5 0 May Apr Aug Mar Sep Jan Nov Feb Jun Dec Oct Jul

Figure 4. Mean daily solar radiation per month reaching sea surface recorded by the automatic weather station in Otsuchi Bay from 2007 to 2008.

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% 20 40 60 80

% 10 20 30 40 50 0
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30

of flowering shoots.

Figure5. Frequency distributions of Zostera caulescens immature and mature flowering shoots lengths from June 2007 to December 2007. N: total number

0-50 50-100 100-150 150-200

0-50 50-100 100-150 150-200 200-250 Cm December 2007 Station 1 October 2007 Station 1

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October 2007 Station 4

August 2007 Station 4

N= 5

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N= 4
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Immature Mature

Immature Mature

Immature Mature

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500-550 550-600

500-550 550-600

Figure6. Frequency distributions of Zostera caulescens of immature and mature flowering shoots lengths from January 2008 to June 2008. N= total

number of flowering shoots

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April 2008 Station 1

N= 13

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f g

Figure7. Developmental stages of the flower of Zostera caulescens: Phase I: immature reproductive organs enclosed in the spadix (a), Dissected spadix showing the immature reproductive organs (b) an enlarged part of b (c), Phase II: mature reproductive organs (d), Phase III: ripe fruits (e), Phase IV: flowering shoot with dead remains of flowers (f) and detached flowering shoots (g)

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120 100 80 % 60 40 20 0 Aug 07 Apr 08 Jun 07 Jan 08 Dec 07 Oct 07 Jun 08 Phase IV Phase III Phase II Phase I

Figure 8. Frequency distribution of flowering phases of Zostera caulescens. Phase I, II, III, IV are immature phase with indistinguishable anthers and pistils, mature phase with mature anthers and pistils, fruiting phase with only fruits, black remains of dead flowers and detached flowering shoots, respectively. N: total number of flowers.

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3000 Fruit density (no. m )

-2

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Jun-07 Aug-07

2500 2000 Jun-08 1500 1000 500 0 q1 q2 q3 q1 q2 q3 q1 q2 q3 q1 q2 q3 Stn.1 Stn.2 Stn.3 Stn.4

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500 400 300 200 100 0

Jun-07 Aug-07 Jun-08

q1 q2 q3 q1 q2 q3 q1 q2 q3 q1 q2 q3 Stn.1 Stn.2 Stn.3 Stn.4

Figure 9. Fruit density (a) and flower density (b) per unit area of Zostera caulescens in three quadrat samples (q).

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Figure10. Picture of Zeuxo sp. living inside the spadix of Zostera caulescens (a) and aborted fruit with expelled inner material (b).

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