LW (a)
LL
NS PL
(b) Water filter
25 cm water level Air pump
Tray 1 Tray 2 Tray 3 Tray 4
SEAGRASSES RESOURCE STATUS AND TRENDS IN INDONESIA, JAPAN, MALAYSIA, THAILAND AND VIETNAM
OGAWA H., JAPAR SIDIK B., MUTA HARAH Z.
Japan Society for The Promotion of Science (JSPS)
Atmosphere and Ocean Research Institute (AORI) The University of Tokyo Published by Seizando‐Shoten Publishing Co., Ltd. Seizando Building, 4‐51 Minamimotomachi Shinjyuku‐ku, Tokyo 160‐0012, Japan
Copyright © 2011 Chatcharee Kaewsuralikhit, Edna T. Ganzon‐ Fortes, Hisao Ogawa, Japar Sidik Bujang, Katsunori Yamaki, Ken‐ ichi Hayashizaki, Muta Harah Zakaria, Nguyen Huu Dai, Tatsudou Senshu, Tatsuyuki Sagawa, Teruhisa Komatsu, Wawan Kiswara
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the permission of the publisher and copyright holder.
International Standard Book Number (ISBN): 978‐4‐425‐90211‐8
Book Citation: Ogawa, H., Japar Sidik, B. and Muta Harah, Z. 2011. Seagrasses: Resource status and trends in Indonesia, Japan, Malaysia, Thailand and Vietnam. Japan Society for The Promotion of Science (JSPS) and Atmosphere and Ocean Research Institute (AORI), The University of Tokyo. Seizando‐Shoten Publishing Co., Ltd., Tokyo, Japan.
INITIATIVES TOWARDS SEAGRASS REHABILITATION AND RESTORATION
SEAGRASS CULTURE Japar Sidik Bujang and Muta Harah Zakaria
INTRODUCTION
The attempt of growing seagrass had been caused by freshwater runoff and wet monsoon conducted as early as 1959 (Wood 1959). season (Benjamin et al. 1999). There are no Various methods had been employed on various available best methods for culturing the species of seagrasses in order to create a small‐ seagrasses (Fuss and Kelly 1969) especially for scale marine system that simulates the tropical species. environmental conditions of the natural habitats according to species and locality. In Malaysia, there are 15 species of Among the successful initiatives was done in seagrasses of which 6 species belong to the Austin, Texas where representatives of 9 out of Halophila genus. H. ovalis is the most common 12 genera of seagrasses: Thalassia, Halodule, species which frequently occur along the Halophila, Posidonia, Zostera, Cymodocea, shallow intertidal coasts. Although common, its Syringodium, Enhalus and Thalassodendron biology and phenology have rarely been have been successfully cultured in artificial examined (Japar Sidik et al. 2008). Seagrass seawater under controlled environmental beds of Malaysia have declined to a greater conditions (McMillan et al. 1981). Such facilities extent due to coastal development activities enabled studies involving the biological, such as land reclamation, sand mining, ecological and phenological aspects of harvesting of food resources, pollution, public seagrasses (McMillan et al. 1981, Ralph and recreational activities, and shipping (Japar Sidik Burchett 1995, 1998a, 1998b, Ralph 1998, and Muta Harah 2003). The above mentioned Benjamin et al. 1999, Longstaff et al. 1999, activities continue to cause degradation and Prange and Dennison 2000). decline of seagrasses. To conserve these resources it is important to know their biology Various culture techniques applied by and ecology, i.e., to know how they tolerate seagrass researchers indicate that seagrasses disturbance and adapt to such conditions. can be studied under certain laboratory conditions. Many methods have been used for One can perform culture studies and at the seagrass culturing (Table 11.1). However, these same time assess the uncertainty of vegetative methods appeared to be suitable only for the morphology derived from the plasticity within growth of particular species collected in specific the species, e.g., Halophila ovalis‐minor habitats. The origin, biology, distribution and complex, or study other aspects of their biology, habitat of seagrasses used in the studies were life cycle and phenology on sustainable basis. By not similar. For example, H. ovalis (R. Br.) Hook manipulating environmental factors in the that live in estuaries can tolerate lower salinities culture conditions, their tolerance range to than deep water H. ovalis because they have certain environmental factor can also be been exposed to low salinities in their habitat determined.
108
ovalis
stress
tub
loam (1998)
) 3
90 Sprigs ‐ Culture Terrestrial sandy (11.5×5.5×17 cm Indoor Thermal Ralph Halophila
al.
ovalis each gravel
et
aquaria in
sand
plastic
5 1982
liter
liter
Sprigs 42 April 1 30 Indoor overlying Salinity, temperature, pots, Sterile Hillman (1995) Halophila
(1994) tanks
capricorni al.
1992 diameter deep
intensity
et
cm cm
plugs 60 10 March Light Outdoor fiberglass Mud 40 Abal Zostera
and (1984) tubes
culture
glass
solid
‐ ml
‐ Axenic Seeds ‐ rooting substrate Semi Indoor Moffler culture Durako 75 Thalassia testudinum
metal
and
(1977)
marina
Substrates 28 ‐ Manipulated Kenworthy Fonseca Indoor Rectangular boxes (35.5×12.7×12.7 cm) Zostera Sprigs seagrasses.
S.
,
H.
, area
box,
2 ‐ and H.
culturing (1967)
concrete
, m
tolerance
and
4 for
June liters sediment ‐
testudinum
Wooden Ovoid
Salinity 55 b. Bay McMillan Mid Moseley Indoor outdoor 648 a. tanks, T. filiforme engelmannii wrightii Plugs
techniques
culture
form cultured studied
Various type
of
transplanting 1. study container (days)
‐
of of of
11
Type Duration studies Time Substrate Author(s) Type Outdoor/Indoor Seagrasses Seagrasses Parameter Table 109
1 ‐ s
2 ‐ m
(1998)
µmol
psu hours
35 Aerated Manipulated ‐ 16 120 Filtered seawater Ralph
‐
‐ ‐
60
al. 8
ASP12
Phillips vapour
water
et
white” Metalarc
W and ‐
‐ ‐ W ‐ 40 hours cm Phillips
cm
12 “warm fluorescent tubes 12 Manipulated Modified Aerated 8 Distilled ‐ ‐ Manipulated mercury incandescent bulbs, 400 lamps W Manipulated Hillman Artificial seawater (1995) 14
(1994)
al.
filtered et ‐
psu
23°C
‐ ‐ ‐ ‐ 35 ‐ ‐ ≈ Manipulated Abal Sand seawater
1 ‐ s
2
and ‐ (1984)
Vitalite
groove synthetic
m
in
‐ E 15
‐ hours
48
14 power 4 fluorescent lamps ‐ ‐ Monthly ‐ 27°C ‐ 690 Moffler Durako NH seawater
1 ‐ bulbs s and
2 ‐
(1977)
m
watt µE
‐ hours
40
8 fluorescent 12 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 23.4 Kenworthy Fonseca
bulbs
tubes,
added
and
‐ (1967) langleys
‐ ‐ ‐
(indoor) (outdoor)
cm
656 controlled water ‐
hours cm cm
7.5 ‐
Fluorescent incandescent 12 Tap ‐ ‐ Manipulated 30 Not 292 45 McMillan Moseley Seawater 5
11.1.
‐
above
studied Table
depth
added
dioxide
exchange depth type
intensity
Illuminance Photoperiod Water Nutrient Salinity Carbon Aeration Temperature Light substrate Author(s) Water Water Substrate Parameter Continue….
110
Phillips Natural Daylight lighting tubes
Artificial seawater Submersible pump
External filter Substrate Plug of H. ovalis Figure 11.1. Culture system‐1 for growing of Halophila ovalis in the laboratory. The substrate covered the bottom of the aquarium. Source: Japar Sidik et al. 2008.
A B
C D E Plate 11.1. The developmental stages of H. ovalis; A. Plugs forming plant population are able to adapt and exhibit continuous growth development in native substrate, at ~20% of the ambient light; B. A shoot with a female flower (arrow); C. A shoot with an open male flower (arrow); D. Successful pollination of female flowers producing fruits (arrow); E. With increase in shoot density, plants shade their leaves, thus, showing symptom of decreasing in shoot density. 111
The following chapter deals with the CULTURE SYSTEM –1 development of a laboratory culture system for Halophila ovalis, to be able to study its growth, Culture of Halophila using explants and population, phenology and seedling reproductive biology development from seed to mature plant. This culture study could be the basis for providing To study the growth and development of H. information on the transplantation and ovalis population, as well as, its reproductive restoration of seagrasses. biology as affected by substrate and light, the following experiments were conducted. GENERAL CULTURE METHODOLOGY Two aquarium/experimental set‐ups were The materials used were: for container: 0.42 m prepared separately and maintained (as x 0.42 m x 1.22 m glass aquarium; for media: 45 described above) (Figure 11.1), each has liters of artificial seawater (prepared from different substrate: (1) native substrate of commercial sea salt (Instant Ocean or calcareous sandy mud collected from Marinemix, i.e., a formulation containing all Merambong shoal, and (2) commercial artificial essential major, minor and trace elements) at sand, and under separate light regimes of (1) 30 psu; for substrates (3‐4 cm thick): native ~90 µmol m‐²s‐1 (9% of the ambient light in the substrate, commercial sand, local river sand, field), and (2) ~200 µmol m‐²s‐1 (20% of the beach sand; for light source: Phillips Natural ambient light in the field) supplied by Phillips Daylight lamps or shaded natural light: Natural Daylight lighting tubes with 12 hours of photoperiod is 12L:12D. The culture system is photoperiod. Then, plugs of H. ovalis explants fitted with an external filter and a submersible collected from Merambong shoal (Lat. 1° 19’ pump to provide filtration and circulation of 50.9” N, Long. 103° 36’ 45.0” E), Johore were water inside the aquarium. The Instant Ocean planted randomly in the aquarium covering artificial seawater provides the nutrients to the approximately 7% of the substrate’s surface cultures to sustain their growth and area. Observations on the growth and development (while in culture). development of H. ovalis population were recorded. Water level in each of the aquarium was maintained by adding distilled water. To RESULTS AND DISCUSSION maintain the water clarity, the external filter was cleaned with distilled water bimonthly. Plugs of explants were successfully grown in Algal growths covering the substrate were native substrate and under both light regimes of removed manually when necessary. After six ~90 μmol m‐2s‐1 and ~200 μmol m‐2s‐1 (Plate months, 25% by volume of the water was 11.1A). The sequence of events on the growth replaced to replenish the nutrients and to and development, and reproductive biology of improve the water quality of the aquarium. H. ovalis plug to the formation of the population Light, temperature, pH and salinity were is described below. recorded daily.
112
A B C D
G F E
H
Plate 11.2. The different stages of development, germinating seeds, seedlings to juvenile plants. A‐germinating seed with a cotyledon, radicle and hypocotyl with hairs. B‐seedling with a pioneer shoot having a single leaf. C‐seedling with a pioneer shoot having two leaves. D‐seedling with a pioneer shoot having three leaves. E‐seedling with a pioneer shoot possessing five leaves. F‐seedling with a pioneer shoot that continued to propagate producing a new rhizome with a new developing shoot. G‐seedling with two shoots although the hypocotyl still intact but pioneer shoot leaves have dropped off. H‐seedling still maintain shrunken hypocotyls borne a total of 3 shoots. The seedling at this stage is referred to as young or juvenile plant. Other than the seedling stage having more than a pair of leaves, a mature plant has a pair of leaves (23.5 + 0.18 mm in length, 13.8 + 0.11 in width) at each rhizome node. C‐cotyledon, H‐hypocotyl, R‐radicle, 1‐L ‐first leaf of pioneer shoot, 2‐L‐second leaf of pioneer shoot, LR‐lateral root, 1‐LR‐first lateral root, 2‐LR‐second lateral root, PS‐pioneer shoot, NS‐new shoot. Source: Japar Sidik et al. (2008).
113
Better growth performance of plugs was Seedlings up to the protrusion of the third observed when grown in native substrate (the leaf essentially showed the same development substrate where the plant occurred), and under as described for H. spinulosa by Birch (1981), H. the light regime of ~200 μmol m‐2s‐1. By the first engelmannii by McMillan (1987), H. decipiens by week after initial planting, plants in the plugs McMillan (1988), H. ovalis by Kuo and Kirkman began to propagate by producing new shoots. A (1992), H. tricostata by Kuo et al. (1993), and H. new shoot was produced every two days. By the beccarii by Muta Harah et al. (2002). second month, some plants propagated inside the plug, such that the plugs became dense. As In this experiment, the number of leaves the plugs were grown randomly, vegetative (=five) in the pioneer shoot of H. ovalis propagation occurred all around the side of the seedlings is less than those reported for other plugs colonizing the bare adjacent substrate Halophila species, i.e., six in H. decipiens areas. (McMillan 1987, 1988), and eight in H. beccarii (Muta Harah et al. 2002). Moreover, in the Female flowers (Plate 11.1B) were produced pioneer shoot of the seedlings, a second lateral after two months in culture, and male flowers root developed next to the first lateral root; the (Plate 11.1C) for another three months. Fruits cotyledon persisted up to this stage. (Plate 11.1D) were detected 2 months after flowering, and the event continued for 2 more CULTURE SYSTEM –2 months. After this time, many plants shade their leaves (Plate 11.1E), thus, showing symptoms of Culture of Halophila using rhizomes decreasing shoot density. Thereafter, living plants were absent. It was observed that eight Except for some experimental variables (i.e., months from initial planting, the plants covered substrate types), the procedures for the set‐up 75‐80% of the substrate’s surface area. and maintenance of this culture system basically follow the general procedure described above Results showed that the plugs were able to (also refer to Plate 11.3A). The aquarium used adapt and continued their growth and was slightly larger, measuring 0.45 m x 0.45 m x development under laboratory condition. Also, 1.22 m. No substrate was placed at the bottom when exposed to 20% of ambient light, new of the aquarium. Instead, the non‐native plants sprouted with the germination of seeds substrates of different textures, i.e., fine beach forming seedlings. The presence of viable seeds sand and coarse river sand, were placed in small and seedlings demonstrated the successful experimental plastic trays (33 cm x 26 cm x 9 pollination and sexual reproduction of H. ovalis cm, Plate 11.3A), that were suspended at in culture. This occurs as both male and female varying depths (10, 20, 30 and 40 cm from the plants are in the same aquarium. water surface), then immersed in separate aquaria containing water of different salinities— The morphology of progressive development 25, 30 and 40 psu. Rhizome axes with or of H. ovalis seedlings to juvenile or young plants without leaves of H. ovalis (Plate 11.3B‐C), that in culture is categorized into 8 stages, as were collected from Pantai Bangat (Lat. 04o illustrated in Plate 11.2 A‐H. 54.473’ N, Long. 115o 22.299’ E), Lawas,
114
Artificial seawater
A
B C
Plate 11.3. A. Culture system‐2. The planting materials; B. rhizome axes with leaves; C. rhizome axes devoid of leaves, may be planted in different substrates in plastic containers, immersed at different depths, or different salinities in separate glass aquariums. Source: Japar Sidik et al. 2010.
Table 11.2. Halophila ovalis propagated from rhizomes devoid of leaves, and grown in coarse river sand and fine beach sand, under shaded out‐door sunlight, and water salinity of 30 psu. Morphological variations of leaf size and cross veins number after eight weeks of culture. Means with the different alphabet in column are significantly different at p<0.05 (t‐test). Source: Japar Sidik et al. 2010.
Leaf width Leaf length Leaf petiole length Cross veins (mm) (mm) (mm) (paired no.) Substrate Mean ± s.d Mean ± s.d Mean ± s.d Mean ± s.d (Range), N (Range), N (Range), N (Range), N
Coarse 11.00±1.67a 26.06±5.52a 29.92±7.30a 15.58±2.09a river sand (7.23‐14.55), 72 (16.79‐47.53), 72 (16.10‐45.92), 72 (11‐20), 72
Fine beach 6.88±2.59b 15.15±5.22b 17.16±7.28b 11.56±1.88b sand (3‐11.86), 72 (6.72‐25.98), 72 (8.12‐38.69), 72 (5‐17), 72
115
Sarawak, were planted in the trays with the Based on leaf dimensions, plants showed better substrate. growth and development in coarse river sand (Table 11.2). Halophila ovalis from the native The culture system was kept under a shaded environment grow on different substrates, i.e. out‐door natural light regimes that fluctuated muddy, sandy, and sandy‐mud substrates from 160 μmol m‐2s‐1 (10% of the ambient light respectively. Plants collected from the wild intensity outside) to ~240 μmol m‐2s‐1 (15% of growing on sandy‐mud were similar in the ambient light intensity outside) recorded morphological measurements with those grown between 12.00 noon to 2.00 pm. Water in coarse river sand (compare Table 11.3 with temperature and pH were recorded as 24‐29oC Table 11.2). Den Hartog (1970) reported that H. and 8.0‐8.5 respectively. Water level height of ovalis was found to occur on all kinds of 40 cm (the water salinity maintained) in each of substrate ranging from coarse rubble to soft the aquarium was maintained by adding mud. Halophila ovalis exhibited an array of leaf distilled water. morphology with respect to sizes and shapes and has been attributed to substrate (Young The indicators for the growth performance and Kirkman 1975) and also environmental and development monitored were: (1) the factors such as salinity (Benjamin et al. 1999) morphological changes of the vegetative parts and shade (Japar Sidik et al. 2001). formed, e.g., leaves, rhizomes in adapting to the created conditions as compared to the native The feasibility of using different forms of environment, (2) sustained growth, and (3) the planting materials development of the population, (4) reproductive biology (flowering, anthesis and Rhizomes with leaves or devoid of leaves fruiting), and (5) the pattern of seedling (Plate 11.3B‐C) were successfully grown in the development from seeds to mature plants. tested substrates maintained in artificial seawater of various salinity and water depth, The data were analyzed to test the with minimum aeration exposed in shaded out‐ significant variation in leaf width, length, petiole door natural condition (Tables 11.2, 11.4). length, number of cross‐veins for H. ovalis for Rhizomes devoid of leaves were never used in each treatment using ANOVA and if significant any of seagrass culture studies. This offers an differences were detected, where appropriate advantage where rhizomes devoid of leaves can this was followed by either independent t‐test be sampled without having to collect masses of or Duncan’s Multiple Range test (SPSS for plants together with substrates employed in Windows Release 10.0). other studies where the planting materials were sampled as cores (Prange and Dennison 2000), RESULTS AND DISCUSSION sprigs (Kenworthy and Fonseca 1977, Hillman et al. 1995, Benjamin et al. 1999), plugs (McMillan The growth and development of the and Moseley 1967, Abal et al. 1994, Longstaff et plants under non‐native substrates al. 1999). However, Moffler and Durako (1984) used seeds in the culture study of Thalassia Plants grew and developed equally well in testudinum. the fine beach sand and coarse river sand. 116
Table 11.3. Halophila ovalis from the wild or native habitat and water salinity 29 psu. Morphological variations of leaf size and cross veins number. Means with different alphabet in column are significantly different at p<0.05 (DMRT). Source: Japar Sidik et al. 2010.
Leaf width Leaf length Leaf petiole length Cross veins (mm) (mm) (mm) (paired no.) Substrate Mean ± s.d Mean ± s.d Mean ± s.d Mean ± s.d (Range), N (Range), N (Range), N (Range), N
Sandy‐mud 10.44±1.18a 20.57±4.07a 29.81±6.67a 14.45±2.35a (8.38‐128‐.86), 31 (10.88‐7.91), 31 (13.02‐4.81), 72 (10‐19), 20 Sandy 8.72±0.73b 16.19±2.1b 22.27±5.11b 11.6±1.81b (7.55‐10.13), 31 (10.34‐9.03), 31 (10.27‐7.74), 144 (8‐15), 20 Muddy 6.72±0.76c 12.31±1.15c 16.00±6.03c 10.65±1.87b (5.63‐8.89), 31 (10.38‐4.30), 31 (3.95‐34.4), 149 (7‐15), 20
Table 11.4. Halophila ovalis propagated from rhizomes devoid of leaves, grown in coarse river sand at three salinity levels, 4 depths (salinity kept at 25 psu), kept in shaded out‐door natural conditions. Means with different alphabet in column are significantly different at p<0.05 (DMRT). Source: Japar Sidik et al. 2010.
Leaf width Leaf length Leaf petiole length Cross veins (mm) (mm) (mm) (paired no.) Substrate Mean ± s.d Mean ± s.d Mean ± s.d Mean ± s.d (Range), N (Range), N (Range), N (Range), N
11.27±1.55a 26.69±6.52a 30.59±7.83a 15.04±2.63a 25 (8.76‐14.45), 48 (16.67‐47.53), 48 (17.56‐45.73), 48 (11‐20), 48 Coarse river sand 7.81±3.42b 17.25±6.70b 20.74±3.79b 12.60±2.66b 30 (salinity, (3.00‐14.55), 48 (6.72‐27.35), 48 (8.29‐45.92), 48 (5‐18), 48 psu 7.76±2.25b 17.88±5.83b 19.30±7.60b 13.08±2.63b 40 (4.36‐12.2), 48 (10.09‐29.21), 48 (8.12‐36.45), 48 (9‐18), 48
8.75±2.62b 19.77±6.16b 24.84±9.80ab 13.03±1.99b 10 (4.36‐12.94), 36 (10.09‐32.08), 36 10.34‐42.59, 36 (10‐17), 36
9.52±3.24a 21.65±6.82a 25.48±11.33a 13.78±2.69a 20 Coarse (3.21‐14.45), 36 (9.59‐36.12), 36 (9.52‐45.92), 36 (9‐20), 36 river sand (water 8.89±3.38b 21.50±10.07a 20.76±7.95c 13.52±2.87ab 30 depth, cm) (3.19‐14.55), 36 (8.59‐47.53), 36 (8.12‐31.73), 36 (9‐19), 36
8.61±2.73b 19.51±7.06b 23.09±9.05b 13.97±3.54a 40 (3.00‐11.69), 36 (6.72‐30.35), 36 (9.50‐45.73), 36 (5‐20), 36
117
The tolerance range of plants under the assessment for the growth and development of different salinity and depth the plants under the varying conditions, e.g., substrates, salinities and depth, the feasibility of Halophila plants have no problem in adapting using planting materials, e.g., rhizomes with and to higher salinities compared to the ambient devoid of leaves, plugs, sprigs, detecting the salinity of 29 psu in the native habitat (Table morphological changes of the vegetative parts, 11.3). Better growth performance was observed e.g., leaf, petiole dimensions in the created at salinity 25 psu (Table 11.4). Halophila ovalis is conditions were compared to those from the rather euryhaline and penetrates water with an native environment and the sustain growth and average salinity and even less saline waters into development of the population. In addition, the estuaries and sea‐inlets and is not uncommon reproductive biology (flowering and fruiting) in the brackish coastal lagoons. On the island of can be observed and studied, e.g., Halophila Oahu (Hawaii), the species has been found ovalis is a dioecious seagrass, i.e., male and under hyperhaline conditions according to den female plants are separated. Male flowers lasted Hartog (1970). Distribution and abundance of for brief period of 2 days, and female flowers seagrasses in an environment is controlled by lasted about 1 month and flowering is seasonal. range of environmental conditions including This maybe the reason plants collected from the light availability, one of the most important wild are usually devoid of flowers. In the wild it environmental parameters, controlling the is difficult to distinguished between male and depth to which seagrasses can grow (Longstaff female plants as they can only be identified and Dennison 1999). The culture system was when they are in flowering season. Under both kept under a shaded out‐door natural light culture systems, we can identify, separate and regimes of 160 μmol m‐2s‐1 to ~240 μmol m‐2s‐1 follow the progressive phenology development and plants at water depth of 20 cm from surface of male and female Halophila ovalis plants. The water level showed better growth performance pattern of seedling development from seeds to compared to other depths (Table 11.4). It is mature plants can also be examined. recognized in this experiment that H. ovalis is tolerant to low light, however, the actual In Culture System‐1, the assessments were quantity required by the plant is not known. achieved by planting plants in the tested The average requirement of seagrasses as a substrates in the aquarium maintained in group of plant has been calculated to be 11% of artificial seawater of various salinity (in separate surface light by Duarte (1991). Based on this aquariums), with minimum aeration and present experimental setup, the water level and exposed in shaded out‐door natural condition hence the volume and quantity of artificial or different light treatment. The aqueous seawater salt can be reduced to halved. In medium prepared from artificial seawater addition the shallower culture tank could be permitted a standardization of the medium as used for growing of H. ovalis. opposed to natural sea water which may be variable, e.g., in salinity and composition, and COMPARISON BETWEEN THE CULTURE SYSTEMS altered by human through various activities (Japar Sidik et al. 2008). Both culture systems can be used in seagrass studies and permitted observations and The Culture System‐2 set‐up is more flexible 118
and enable one to manipulate the placement and estuarine plants of Halophila ovalis (R. of substrates in the aquarium (i.e., the planting Br.) Hook. f. to long‐term hyposalinity. Aquat. of seagrass materials into individual containers Bot. 64: 1‐17. then immersed into the aquarium) facilitates Birch, W. R. 1981. Morphology of germinating observation, recording of data and moving seeds of the seagrass Halophila spinulosa (R. plants from one test condition to another. It Br.) Aschers. (Hydrocharitaceae). Aquat. Bot. also aids in the task of cleaning the aquarium 11: 79‐90. without disturbing the sediment. However, this den Hartog, C. 1970. The Sea‐grasses of the culture system is only appropriate for small‐ World. North‐Holland Publishing Company, sized seagrass, e.g., Halophila, Halodule and Amsterdam. Ruppia. Big‐sized seagrass such as Enhalus, Duarte, C. M. 1991. Seagrass depth limits. Thalassia and Cymodocea are more suited to be Aquat. Bot. 40: 363‐377. culture in Culture System‐1, where the Fuss, C. M. and Kelly, J. A. 1969. Survival and substrate at the bottom of the aquarium is growth of sea grasses transplanted under more stable when compared to the one placed artificial conditions. Bull. Mar. Sci. 19: 351‐ in small containers as in Culture System‐2. 365. Hillman, K., McComb, A. J. and Walker, D. I. The culture system is being used under the 1995. The distribution, biomass and primary low light or shaded environment. This is to production of the seagrass Halophila ovalis in minimize the overgrowth of algae, which are in the Swan/Canning Estuary, Western competition with seagrasses if grown under the Australia. Aquat. Bot. 51: 1‐54. normal out‐side light. Japar Sidik, B. and Muta Harah, Z. 2003. The Seagrasses of Malaysia, Chapter 14. In World ACKNOWLEDGEMENTS Atlas of Seagrasses. Green, E. P. and Short, F. T. (eds.), pp. 152‐160. University of California This research is funded by the Ministry of Press, Berkeley, Los Angeles, London. Science, Technology and Environment Malaysia, Japar Sidik, B., Muta Harah, Z., Arshad, A. and under the ScienceFund Priority Areas’ Mohd. Pauzi, A. 2001. Responses of Halophila programme entitled “Indoor and outdoor ovalis and Cymodocea serrulata under the culture of tropical spoongrass, Halophila ovalis shade of Enhalus acoroides. In Aquatic R. Brown: Project number: 04‐01‐04‐SF0867. resource and environmental studies of the Straits of Malacca: Current research and REFERENCES reviews. Japar Sidik, B., Arshad, A., Tan, S. G., Daud, S. K., Jambari, H. A. and Sugiyama, S. Abal, E. G., Loneragan, N., Bowen, P., Perry, C. (eds.), pp. 111‐115. Malacca Straits Research J., Udy, J. W. and Dennison, W. C. 1994. and Development Centre (MASDEC), Physiological and morphological responses of Universiti Putra Malaysia, Serdang, Malaysia. the seagrass Zostera capricorni Aschers. to Japar Sidik, B., Lim Lai, H., Muta Harah, Z., light intensity. J. Exp. Mar. Biol. Ecol. 178: Arshad, A. and Ogawa, H. 2008. Laboratory 113‐129. culture of the seagrass, Halophila ovalis (R. Benjamin, K. J., Walker, D. I., McComb, A. J. and Br.) Hooker f. Mar. Res. Indonesia 33(1): 1‐6. Kuo, J. 1999. Structural response of marine Japar Sidik, B., Muta Harah, Z., Mohd. 119
Fakhrulddin, I., Khairul Anuar, M. S. and Bot. 29: 247‐260. Arshad, A. 2010. Growth performance of Moffler, M. D. and Durako, M. J. 1984. Axenic Malaysian’s spoongrass, Halophila ovalis (R. culture of Thalassia testudinum Banks ex Br.) Hooker f. under different substrate, Konig (Hydrocharitaceae). Amer. J. Bot. 71: salinity and light regime. Coastal Marine 1455‐1460. Science. 34(1): 103‐107. Muta Harah, Z., Japar Sidik, B. and Arshad, A. Kenworthy, W. J. and Fonseca, M. 1977. 2002. Flowering and seedling of annual Reciprocal transplant of the seagrass Zostera Halophila beccarii Aschers. in Peninsular marina L. effect of substrate on growth. Malaysia. Bulletin of Marine Science 71(3): Aquaculture 12: 197‐213. 1199‐1205. Kuo, J. and Kirkman, H. 1992. Fruits, seeds and Prange, J. A. and Dennison, W. C. 2000. germination in the seagrass Halophila ovalis Physiological responses of five seagrass (Hydrocharitaceae). Botanica Marina 35: 197‐ species to trace metals. Marine Pollution 204. Bulletin 41: 327‐336. Kuo, J., Lee Long, W. J. and Coles, R. G. 1993. Ralph, P. J. 1998. Photosynthetic response of Occurrence and fruit and seed biology of laboratory‐cultured Halophila ovalis to Halophila tricostata Greenway. Aust. J. Mar. thermal stress. Mar. Ecol. Prog. Ser. 171: 123‐ and Freshwat. Res. 44: 43‐58. 130. Longstaff, B. J., Loneragan, N. R., O’Donohue, M. Ralph, P. J. and Burchett, M. D. 1995. J. and Dennison, W. C. 1999. Effects of light Photosynthetic responses of the seagrass deprivation on the survival and recovery of Halophila ovalis (R. Br.) Hook. f. to high the seagrass Halophila ovalis (R. Br.) Hook. f. irradiance stress, using chlorophyll a J. Exp. Mar. Biol. Ecol. 234: 1‐27. fluorescence. Aquat. Bot. 51: 55‐66. McMillan, C. 1987. Seed germination and Ralph, P. J. and Burchett, M. D. 1998a. seedling morphology of the seagrass, Photosynthetic response of Halophila ovalis Halophila engelmannii (Hydrocharitaceae). to heavy metal stress. Environmental Aquat. Bot. 28: 179‐188. Pollution 103: 91‐101. McMillan, C. and Moseley, F. N. 1967. Salinity Ralph, P. J. and Burchett, M. D. 1998b. Impact of tolerances of five marine spermatophytes of petrochemicals on the photosynthesis of Redfish Bay, Texas. Ecol. 48: 503‐506. Halophila ovalis using chlorophyll McMillan, C. 1988. Seed germination and fluorescence. Marine Pollution Bulletin 36: seedling development of Halophila decipiens 429‐436. Ostenfeld (Hydrocharitaceae) from Panama. Wood, E. J. F. 1959. Some East Australian Aquat. Bot. 31: 169‐176. seagrasses communities. Proc. Linn. Soc., McMillan, C., Williams, S. C., Escobar, L. and N.S.W. 84: 218‐226. Zapata, O. 1981. Isozymes of secondary Young, P. C. and Kirkman, H. 1975. The seagrass compounds and experimental cultures of communities of Moreton Bay, Queensland. Australian seagrasses in Halophila, Halodule, Aquat. Bot. 1: 191‐202. Zostera, Amphibolis and Posidonia. Aust. J.
120 Coastal Marine Science 34(1): 103–107, 2010
Special Section “Oceanography” Growth performance of Malaysian’s spoongrass, Halophila ovalis (R.Br.) Hooker f. under different substrate, salinity and light regime
1 1 1 1 B. Japar SIDIK *, Z. Muta HARAH , I. Mohd FAKHRULDDIN , M. S. Khairul ANUAR and 2 A. ARSHAD
1 Department of Animal Science and Fishery, Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia Bintulu Sarawak Campus, 97008 Bintulu, Sarawak, Malaysia 2 Faculty of Agriculture, Universiti Putra Malaysia 43400 Serdang, Selangor Darul Ehsan, Malaysia * E-mail: [email protected]
Received 20 December 2009; Accepted 18 February 2010
Abstract — Halophila ovalis plants collected from the native environment (Lat. 04°54.473 N, Long. 115°22.299’E-Pantai Bangat, Lawas, Sarawak, Malaysia) were grown in aquarium culture system to assess: (i) the feasibility of using planting materials e.g. rhizomes with and devoid of leaves, (ii) the growth and development of the plants under non-native substrates, fine beach sand and coarse river sand, (iii) the sustain growth and development of the population, and (iv) the tolerance range of plants under the tested salinity and depth. These assessment were achieved by planting rhizomes with or devoid of leaves in the tested sub- strates in containers, maintained in artificial seawater of various salinity, with minimum aeration and exposed in shaded out- door natural condition. The artificial seawater permitted a standardization of the medium. By manipulating the placement of substrates in the container (and planting of rhizomes into individual container) then submerged at different depths into the aquarium facilitated observation, recording of data and transferring of plants from one test condition to another. The morpho- logical changes of the vegetative parts e.g. leaf, petiole dimensions and paired cross-veins numbers of H. ovalis in the created conditions were compared to those of H. ovalis from native environment. Key words: Halophila ovalis, laboratory culture, substrate, tolerance, salinity, depth
studies were not similar. For example, H. ovalis (R.Br.) Hook Introduction f. that live in estuaries can tolerate lower salinities than that of deep sea H. ovalis because they have to adapt to low salin- The attempt of growing seagrass had been conducted as ities caused by freshwater runoff and wet monsoon season early as 1959 (Wood 1959). Various methods had been em- (Benjamin et al. 1999). There are no available best methods ployed on various species of seagrasses in order to create a for culturing the seagrasses (Fuss and Kelly 1969) especially small-scale marine system that simulates the environmental for tropical species. In Malaysia, there are 15 species of sea- conditions of the natural habitats accordingly to species and grasses of which 6 species belong to Halophila and H. locality. Among the successful one is for example at Austin, ovalis, being the most common and frequently occurred Texas where representatives of 9 out of 12 genera of sea- along the shallow inter-tidal coast. Although common its bi- grasses; Thalassia, Halodule, Halophila, Posidonia, Zostera, ology and phenology have rarely been examined (Japar Sidik Cymodocea, Syringodium, Enhalus and Thalassodendron et al. 2008). The aim of this present study is to assess the have been successfully cultured in synthetic seawater and growth and development of Malaysian H. ovalis using a cul- under controlled environmental conditions (McMillan et al. ture system and techniques that could provide conditions fa- 1981). Using the culture system, seagrasses were maintained vorable to the plants. that facilitated studies involving the biological, ecological and phenological aspects of seagrasses (McMillan et al. 1981). Although such culture has been developed they were Materials and Methods only suitable for a particular type of seagrasses and their con- ditions of growth under studied. In addition, the origin, biol- A culture system for growing H. ovalis comprises 0.45 ogy, distribution and the habitat of seagrasses used in the m 0.45 m 1.22 m glass aquarium, flooded with artificial
103 Coastal Marine Science 34 seawater prepared from commercial sea salt (Marinemix, from the water surface) facilitates observation, recording of Marine Enterprises International Inc., Baltimore, USA). The morphological data, tolerance and adaptation of plants from Marinemix is a formulation containing all essential major, one test condition to another. The culture system was kept minor and trace elements for sustaining the growth and de- under a shaded out-door natural light regimes that fluctuated velopment of H. ovalis. The culture system is fitted with an from 160 mmol/m2/sec (10% of the ambient light intensity external filter system and a submersible pump to provide fil- outside) to 240 mmol/m2/sec (15% of the ambient light in- tration and circulation of water inside the aquarium (Fig. 1). tensity outside) recorded between 12.00 noon to 2.00 pm. Rhizome axes with leaves of H. ovalis collected from Pantai Water temperature and pH were recorded as 24–29oC and Bangat (Lat. 04°54.473 N, Long. 115°22.299 E), Lawas, 8.0–8.5, respectively. Water level height of 40 cm (the water Sarawak were planted in a substrate placed in a 33 cm 26 salinity maintained) in each of the aquarium was maintained cm 9 cm plastic tray. Using these techniques and in the cul- by adding distilled water. To maintain the water clarity, the ture system setup (Fig. 1), by manipulating the planting ma- external filter was cleaned with distilled water bimonthly. terials (e.g. using rhizome axes devoid of leaves), placement After six months, 25% by volume of the water was replaced of non-native substrates of different textures and sources to replenish the nutrients and to improve the water quality of (fine beach sand taken from Tanjung Batu beach, Bintulu; the aquarium. The indicators for the growth performance and coarse river sand from a small stream at Universiti Putra development were the morphological changes of the vegeta- Malaysia Bintulu Sarawak Campus) in the plastic tray (i.e. tive parts formed e.g. leaves, rhizomes in adapting to the cre- the planting of rhizomes into individual container) then sub- ated conditions as compared to the native environment, sus- merged in the separate aquaria containing water of different tain growth and the development of the population, reproduc- salinity (25, 30 and 40 psu) and depth (10, 20, 30 and 40 cm tive biology (flowering, anthesis and fruiting) and the pattern of seedling development from seeds to mature plants. The data were analyzed to test the significant variation in leaf width, length, petiole length, number of cross-veins for H. ovalis for each treatment using ANOVA and if significant dif- ferences were detected, this was followed by Duncan’s Multi- ple Range test (SPSS for Windows Release 10.0).
Results and Discussion
The feasibility of using different forms of planting materials Rhizomes with leaves or devoid of leaves (Fig. 1) were successfully grown in the tested substrates maintained in arti- ficial seawater of various salinity and water depth, with mini- mum aeration exposed in shaded out-door natural condition. Fig. 1. The planting materials, rhizome axes with leaves (A) or Rhizomes devoid of leaves were never used in any of sea- rhizome axes devoid of leaves (B) are planted in a substrate in plastic containers, immersed at different depths in a culture sys- grass culture studies. This offers an advantage where rhi- tem (C) containing artificial seawater. zomes devoid of leaves can be sampled without having to
Fig. 2. Halophila ovalis propagated from rhizome axes devoid of leaves in (a) fine beach sand (b) coarse river sand.
104 Sidik B. J. et al.: Growth perfomance of Malaysian’s spoongrass, Halophila ovalis (R.Br.) Hooker f. collect masses of plants together with substrates employed in tively (Fig. 3). Den Hartog (1970) reported that H. ovalis was other studies where the planting materials were sampled as found to occur on all kinds of substrate ranging from coarse cores (Prange and Dennison 2000), sprigs (Kenworthy and rubble to soft mud. Halophila ovalis exhibited an array of Fonseca 1977, Hillman et al. 1995, Benjamin et al. 1999), or leaf morphology with respect to sizes and shapes and has plugs (McMillan and Moseley 1967, Abal et al. 1994, been attributed to substrate (Young and Kirkman 1975) and Longstaff et al. 1999). However, Moffler and Durako (1984) also environmental factors such as salinity (Benjamin et al. used seeds in the culture study of Thalassia testudinum. 1999) and shade (Japar Sidik et al. 2001).
The growth and development of the plants under non- The tolerance range of plants under the different native substrates salinity and depth Plants grew and developed equally well in the fine beach Halophila plants have no problem in adapting to higher sand (Fig. 2a) and coarse river sand (Fig. 2b). Comparatively salinities compared to the ambient salinity of 29 psu in the based on leaf sizes, plants showed better growth and develop- native habitat. Better growth performance was observed at ment in coarse river sand (Table 1 compare with Table 2, Fig. salinity 25 psu (Table 1). Halophila ovalis is rather euryha- 3 for plants from the wild). Halophila ovalis from the native line and penetrates water with an average salinity and even environment can be categorized into three morphological less saline waters into estuaries and sea-inlets and is not un- forms (small-, intermediate- and big-leaved). They were common in the brackish coastal lagoons. On the island of found at similar depth of 1.5 m but growing on different sub- Oahu (Hawaii), the species has been found under hyperhaline strates, i.e. muddy, sandy, and sandy-mud substrates respec- conditions according to den Hartog (1970). Distribution and
Table 1. Halophila ovalis propagated from rhizomes devoid of leaves, exposed to different treatments in shaded out-door natural condi- tions. Morphological variations as demonstrated by the leaf size and cross veins number after eight weeks of culture. Means with the same letter in rows are not significant different at p 0.05 by Duncan Multiple Range Test.
Leaf width (mm) Leaf length (mm) Leaf petiole (mm) Cross veins (no.)
Parameters Mean s.d Mean s.d Mean s.d Mean s.d (Range) N (Range) N (Range) N (Range) N
Coarse 11.00 1.67a 26.06 5.52a 29.92 7.30a 15.58 2.09a river (7.23–14.55) (16.79–47.53) (16.10–45.92) (11–20) Substrate Water sand 72 72 72 72 salinity 30 psu Fine 6.88 2.59b 15.15 5.22b 17.16 7.28b 11.56 1.88b beach (3–11.86) (6.72–25.98) (8.12–38.69) (5–17) sand 72 72 72 72
11.27 1.55a 26.69 6.52a 30.59 7.83a 15.04 2.63a 25 (8.76–14.45) (16.67–47.53) (17.56–45.73) (11–20) 48 48 48 48 Halophila ovalis Coarse river 7.81 3.42b 17.25 6.70b 20.74 3.79b 12.60 2.66b in culture sand Water (3.00–14.55) (6.72–27.35) (8.29–45.92) (5–18) salinity (psu) 48 48 48 48 7.76 2.25b 17.88 5.83b 19.30 7.60b 13.08 2.63b 40 (4.36–12.2) (10.09–29.21) (8.12–36.45) (9–18) 48 48 48 48
8.75 2.62b 19.77 6.16b 24.84 9.80ab 13.03 1.99b 10 (4.36–12.94) (10.09–32.08) (10.34–42.59) (10–17) 36 36 36 36 9.52 3.24a 21.65 6.82a 25.48 11.33a 13.78 2.69a 20 (3.21–14.45) (9.59–36.12) (9.52–45.92) (9–20) Coarse river sand 36 36 36 36 Water depth 8.89 3.38b 21.50 10.07a 20.76 7.95c 13.52 2.87ab (cm) 30 (3.19–14.55) (8.59–47.53) (8.12–31.73) (9–19) 36 36 36 36 8.61 2.73b 19.51 7.06b 23.09 9.05b 13.97 3.54a 40 (3.00–11.69) (6.72–30.35) (9.50–45.73) (5–20) 36 36 36 36
105 Coastal Marine Science 34
Table 2. Halophila ovalis from the wild or native habitat. Morphological variations as demonstrated by the leaf size and cross veins number. Means with the same letter in rows are not significant different at p 0.05 by Duncan Multiple Range Test.
Leaf width (mm) Leaf length (mm) Leaf petiole (mm) Cross veins (mm)
Parameters Mean s.d Mean s.d Mean s.d Mean s.d (Range) N (Range) N (Range) N (Range) N
Sandy mud 10.44 1.18a 20.57 4.07a 29.81 6.67a 14.45 2.35a Halophila ovalis (8.38–12.86) (7.91–10.88) (4.81–13.02) (10–19) from the wild or 31 31 72 20 native habitat Substrate Sandy 8.72 0.73b 16.19 2.1b 22.27 5.11b 11.6 1.81b Water salinity (7.55–10.13) (9.03–10.34) (7.74–10.27) (8–15) 29 psu 31 31 144 20 Muddy 6.72 0.76c 12.31 1.15c 16.00 6.03c 10.65 1.87b (5.63–8.89) (4.30–10.38) (3.95–34.4) (7–15) 31 31 149 20
Fig. 3. Variation in leaf morphology of Halophila ovalis from the native habitat categorized as (a) small-leaved from muddy substrate, (b) intermediate-leaved from sandy substrate and (c) big-leaved from sandy mud substrate. abundance of seagrasses in an environment is controlled by maybe the reason plants collected from the wild usually de- range of environmental condition including light availability void of flowers. In the field, inconspicuous flowers and fruits which is considered as one of the most important environ- have probably been overlooked because of the short lasting mental parameters, controlling the depth to which seagrasses of particularly the male flowers and the tiny flowers of the fe- can grow and excluding seagrasses from areas with low light male. In the wild it is also difficult to distinguish between conditions (Longstaff and Dennison 1999). The culture sys- male and female plants as they can only be identified when tem was kept under a shaded out-door natural light regimes they are in flowering season. Under the culture system, we of 160 mmol/m2/sec to 240 mmol/m2/sec and plants at water can identify, separate and follow the progressive phenology depth of 20 cm from surface water level showed better development of male and female H. ovalis plants. Few sea- growth performance compared to other depths (Table 1). It is grasses produce flowers under culture conditions except for recognized in this experiment that H. ovalis possesses toler- studies by McMillan (1980, 1987). Viable seeds were pro- ance to low light intensity, however, the actual quantity re- duced in the culture as demonstrated by the progressive de- quired by the plant has not studied. The average requirement velopment of seeds to seedlings, juvenile plants and to ma- of seagrasses as a group of plant has been calculated to be ture and flowering plants sustained in the system. Halophila 11% of surface light by Duarte (1991). Based on this present ovalis produces flowers and fruits, and propagates by vegeta- experimental setup, the water level and hence the volume and tive and reproductive means. quantity of articicial seawater salt can to be reduced to halve. In addition the shallower culture tank could be used for Acknowledgements growing of H. ovalis. We would like to thank Vice-Chancellor, Universiti Putra Malaysia, for encouragement and facilities. This research is funded Production of reproductive materials by the Ministry of Science, Technology and Environment Malaysia, Plants under the varying conditions, e.g. substrate, salin- under the ScienceFund Priority Areas’ programme entitled “Indoor ity and depth were producing flowers and fruits. Halophila and outdoor culture of tropical spoongrass, Halophila ovalis R. Brown: Project number: 04-01-04-SF0867 and some financial and ovalis is dioecious, i.e. male and female plants are separated. travel supports from Japan Society for the Promotion of Science Male flowers lasted for brief period of 2 days, and female (JSPS) are acknowledged. flowers lasted about 1 month and flowering is seasonal. This
106 Sidik B. J. et al.: Growth perfomance of Malaysian’s spoongrass, Halophila ovalis (R.Br.) Hooker f.
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