Flowers of the intertidal seagrass Halophila stipulacea ( Forsskål ) Ascherson : A new record from tropical coast of Tanzania , Indo-Pacific

Flowers of the seagrass Halophila stipulacea (Forsskål) Ascherson in Tanzania are currently unreported. The present study was conducted along the coast of Tanzania, Indo-Pacific, Kunduchi intertidal mudflats. Transplanted cuttings from Kunduchi intertidal mudflats were successfully grown in sand-mud substrate in the growth chamber in a 12 h photoperiod (1,250 μmol photons m -2 s -1 ) and an inductive temperature, salinity, and pH range of 24 to 28°C, 34 to 38‰, and 7 to 8, respectively. Plants began to flower after four months of culturing. No flowers were observed in the first three months; 0.229±0.50 staminate and 0.123±0.45 pistillate were recorded between April and June; 0.440±0.65 staminate and 0.221±0.03 pistillate between July and September, and 0.282±0.36 staminate and 0.105±0.78 pistillate between October and December. Although, further research is required to fully assess the pollination success and sexual reproduction including fruiting of the species, our study is the first to report the presence of flowers ex situ in Tanzania.


INTRODUCTION
Flowering has been rarely reported for tropical seagrasses along the East African coast with the exception of Kenya where one flower of Cymodocea serrulata was collected in a beach drift in January 1967, at Diani Beach (Isaac and Isaac, 1968).The recorded collections are over fifty years; August 1965, from plants of Halophila ovalis, Halophila stipulacea (cited as Halophila balfourii Solered.),Thalassodendron ciliatum, and Thalassia hemprichii (den Hartog, 1970); pistillate flowers of Syringodium isoetifolium, Halodule uninervis, and H. stipulacea collected in August of 1968 (Isaac, 1968).These records suggest that observing of H. stipulacea flowers in situ is not common occurrence.
Studies of seagrass reproduction and phenology are therefore important in determining the contribution of reproduction to the population dynamics of different seagrasses (Clores and Agoo, 2013).Knowledge of reproductive biology may also be critical in the reestablishment of declining seagrass populations and in targeting the best species for use in revegetation (Orth et al., 1994).Due to their small sizes and inconspicuous nature, flowers of seagrasses are usually overlooked and not collected (Clores and Agoo, 2013;Sidik et al., 2010).The extent and timing of flowering in seagrasses worldwide is variable, between species, and between locations, making generalizations difficult.In tropical regions, seagrass flowering is a year-round phenomenon but with variations in intensity related to location, while in temperate regions, flowering often occurs in spring, but the timing of the whole reproductive cycle varies with both species and location (Walker et al., 2001).
Culturing of seagrasses in the laboratory had been attempted by many marine biologists.The attempt had been conducted as early as 1922 (Setchell, 1922).Different systems and diverse methods had been deployed on various seagrasses species in order to create a small-scale marine system that simulates the environmental conditions of the natural habitats according to species and locality.Among the successful culture systems are: Setchell (1922Setchell ( , 1924Setchell ( , 1929)), Wood (1959), McMillan (1976McMillan ( , 1978McMillan ( , 1980a, b), b), and Bujang et al. (2008), where representatives of 9 out of 12 genera of seagrasses (Thalassia, Halodule, Halophila, Posidonia, Zostera, Cymodocea, Syringodium, Enhalus, and Thalassodendron) have been successfully cultured in synthetic seawater and under controlled environmental conditions.In these culture systems, studies focused on the biological, ecological, and phenological aspects of seagrasses (McMillan, 1980a, b;McMillan et al., 1981).
Despite the fact that culture systems have been developed, they were mostly established to suit particular seagrasses species in relevant environmental conditions (Hillman et al., 1995;Longstaff et al., 1999;Ralph, 1998;Short, 1985).And thus, these techniques may not apply to all seagrass species.
In the coastal waters of Tanzania, ten seagrasses species have been reported to occur (Lugendo et al., 1999;Oliveira et al., 2005;Richmond, 1997), of which two species belong to the genus Halophila, that is, H. stipulacea and H. ovalis.H. stipulacea is the most common species in the intertidal mudflats, sand-mud substrates and along the shallow intertidal coast and subtidal areas along Dar es Salaam coast.Although, common, information on its biology and phenology is scanty.This study attempts to develop a culture system to assess the reproductive biology (flowering) of H. stipulacea under favorable environmental conditions in order to obtain critical information about the species.

MATERIALS AND METHODS
This experiment was carried out at the Department of Aquatic Science and Fisheries of the University of Dar es Salaam, Kunduchi

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Station, Tanzania, from January to December, 2013.In the growth chamber (air condition controlled room), a small-scale culture system for growing H. stipulacea was comprised of 30 cm × 30 cm × 40 cm glass aquarium, which was provided with 6 cm thick of substrate/sediment and flooded with 20 L of 35‰ ocean water.The culture system was fitted with an external filter system and a submersible pump to provide filtration and circulation of water inside the aquarium.To keep the amount of water constant, water levels were monitored daily, and evaporation was compensated for by adding distilled water.To maintain water clarity, the external filter was cleaned with distilled water twice monthly.Samples of H. stipulacea collected from Kunduchi intertidal mudflats (6°39ʹ-6°41ʹ S and 39°12ʹ -39°13ʹ E) were randomly planted in the 6 cm substrate.Algal growths covering the substrate were removed manually when necessary.Experiments were performed in H. stipulacea native substrate of sand-mud, under Gro-Lux fluorescent lamps (1,250 µmol photons m -2 s -1 ) under a daily 12 h photoperiod; and an inductive temperature, salinity and pH of 24 to 28°C, 34 to 38‰ and 7 to 8, respectively.No artificial nutrients were added, and water was changed every month, with the assumptions that seawater was of uniform physicochemical composition and that other physicochemical parameters do not influence the flowering of H. stipulacea.The culture ran for twelve months monitored at three month intervals: January to March, April to June, July to September and October to December; this coincided with the species flowering pattern (McMillan, 1976).Flowering observations were done twice monthly and monitored as per Short and Coles (2001).The floral density was calculated as per standard method also described by Short and Coles (2001).

RESULTS AND DISCUSSION
Sods of H. stipulacea were successfully grown in the laboratory using native sand-mud substrate under controlled temperature and salinity with minimum aeration.After initial planting in January, plants in culture system began to multiply through vegetative propagation by producing new shoots; a new shoot was produced after every seven to nine days.By the end of the first three months of culture, continuous propagation of H. stipulacea inside the aquarium area had densely populated the plugs.H. stipulacea under the controlled conditions produced flowers.H. stipulacea is a dioecious plant, male and female parts are separated (den Hartog, 1970).Plants began to flower after four months of culturing; that is from April.Flowers (Figure 1) were recorded from April to December.No flowers were observed in the first three months; 0.229±0.50staminate and 0.123±0.45pistillate were recorded between April and June; 0.440±0.65 staminate and 0.221±0.03pistillate between July and September, and 0.282±0.36staminate and 0.105±0.78pistillate between October and December (Figure 2).
No flowering was observed in H. stipulacea during the first three-months of culturing.Although there was insufficient flowering to determine inductive conditions with any accuracy, the recorded floral density was produced under day lengths of 12 hours, temperature of 24 to 28°C, salinity of 34 to 38% and pH range of 7 to 8. It seems likely that these conditions may represent the inductive ones, but a nutrient effect of the water column  and/or sediments may also be involved.
Flowering in other seagrass species of the genus Halophila has primarily been a consequence of temperature.For example, in Halophila engelmannii Aschers., flowers were induced at 22 to 24°C under day lengths ranging from 14 to 24 h while H. stipulacea showed floral induction at 23.5 and 27.5°C (McMillan, 1976).However, in this study, H. stipulacea showed inhibition of flowering under 12 h day lengths at an inductive temperature.Plants that were kept at 28°C discontinued flower production after a brief flowering period and became vegetative, but plants that were at the slightly lower temperature, 25°C, continued to produce new flowering shoots during a nine-month observation period between April and December.
In the studies of H. stipulacea, the non-flowering materials under inductive temperature-salinity-pHphotoperiod conditions suggested that nutrient conditions may also play a role in flowering.Because flowering in laboratory culture has involved natural seawater and marine substrate planting medium, it may be possible to determine the role of nutrient conditions with more exactness.
This study allowed the identification of both male and female H. stipulacea.During field sampling of several seagrass species such as H. ovalis, often due to the short span of flowering in male flowers in particular and tiny flowers of female (Sidik et al., 2010), flowers and fruits are often overlooked.In culture systems, such as the one used in this study, it is easy to identify, separate

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and follow the phenological development of both male and female plants.H. stipulacea produces flowers and propagates by vegetative and reproductive means.

Conclusion
A small-scale marine system had been successfully set up to simulate the natural environmental conditions of the habitat of H. stipulacea.The culture system established permitted observations on the phenological cycle.Future research could probably more critically identify the temperatures or salinities involved in flowering.The use of fixed temperatures, salinities and pH within the suspected inductive range, 24 to 28°C, 34 to 38‰ and 7 to 8, respectively, might permit the identification of possible optimal temperature, salinity and pH ranges for flowering.Because floral development may proceed slowly at the inductive temperature, salinity and pH, the use of a progressively increased temperature, salinity, and pH patterns might aid the determination of inductive differences.Phenophases should be studied for stages that precede the initial appearance of macroscopically visible floral buds.The results of the present investigation suggest that flowering in H. stipulacea is related primarily to temperature, salinity and pH; and that differences in flowering in responses to temperature, salinity or pH account for the nearly synchronous phenological timing in natural H. stipulacea meadows at different locations along tropical coast of East Africa.Our study is the first to document the presence of flowers in the tropical coast of Tanzania, Indo-Pacific through laboratory experiments.
Future investigations should consider the pollination success and sexual reproduction (fruiting) of the species.

Figure 2 .
Figure 2. Floral density of Halophila stipulacea recorded throughout the experimental culture from January to December, 2013.