CALCAREOUS ALGAE of a TROPICAL LAGOON Primary Productivity, Calcification and Carbonate Production

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CALCAREOUS ALGAE OF A TROPICAL LAGOON Primary Productivity, Calcification and Carbonate Production

JUMA WALAKU KANGWE

DOCTORAL DISSERTATION IN PLANT PHYSIOLOGY

DEPARTMENT OF BOTANY STOCKHOLM UNIVERSITY SWEDEN

2006

© 2006 Juma Kangwe ISBN 91-7155-187-5 PrintCenter Stockholm 2005 Front cover: A meadow of Halimeda opuntia exposed to air during lowest spring tides of the day in Chwaka bay. Back cover: Top: A mixed Halimeda meadow and Udotea species can be seen in the middle (Photo by Katrin Österlund). Below: Rhodolith (left) and H. opuntia (right) meadows in Chwaka bay.

2 To my parents; The late father mzee Walaku Kangwe My mummy Kuyeya Mpanjilwa And my wife Mariana Kangwe

3 ABSTRACT

The green algae of the genus Halimeda Lamouroux (Chlorophyta, Bryopsidales) and the encrusting loose- lying red coralline algae (Rhodophyta, Corallinales) known as rhodoliths are abundant and widespread in all oceans. They significantly contribute to primary productivity while alive and production of CaCO3 rich sediment materials on death and decay. Carbonate rich sediments are important components in the formation of Coral Reefs and as sources of inorganic carbon (influx) in tropical and subtropical marine environments. This study was initiated to attempt to assess their ecological significance with regard to the above mentioned roles in a tropical lagoon system, Chwaka bay (Indian Ocean), and to address some specific objectives on the genus Halimeda (Chlorophyta, Bryopsidales) and the loose-lying coralline algae (rhodoliths). Four Halimeda species were taxonomically identified in the area. The species identified are the most common inhabitants of the world’s tropical and subtropical marine environments, and no new species were encountered. Using Satellite remote sensing technique in combination with the percentage cover data obtained from ground-truthing field work conducted in the area using quadrants, the spatial and seasonal changes of Submerged Aquatic Macrophytes (SAV) were evaluated. SAV percentage cover through ground-truthing was; 24.4% seagrass, 16% mixed Halimeda spp., 5.3% other macroalgae species while 54.3% remained unvegetated. No significant changes in SAV cover was observed for the period investigated, except in some smaller regions where both loss and gains occurred. The structural complexity of SAV (shoot density, above-ground biomass and canopy height) for most common seagrass communities from six meadows, dominated by Thalassia hemprichii, Enhalus acoroides and Thalassodendron ciliatum, as well as mixed meadows, were estimated and evaluated. Relative growth of Halimeda species was up to 1 segment tip-1 day-1. The number of segments produced was highest in hot season. Differences between the numbers of segments produced were insignificant between the two sites investigated. The C/N ratios obtained probably shows that Halimeda species experience nitrogen limitation in the area and may be a factor among others responsible for the varying growth of species obtained. However, this can be a normal ratio for calcified algae due to high CaCO3 content in their tissues. Standing biomass of mixed Halimeda species averaged between 500-600 g dw m-2 over the bay, while the mean cover in Halimeda meadows was about 1560 g dw m-2. Carbonate production in Halimeda beds varied between -2 -1 -2 -1 17-57 g CaCO3 m day and for H. macroloba between 12-91 g CaCO3 m day . This indicates a high annual input of carbonate in the area. Decomposition of Halimeda using litter bag experiments at site I and II gave a decomposition rate (k) of 0.0064 and k = 0.0091 day-1 ash-free dry weight (AFDW) respectively. Hence it would take 76-103 days for 50% of the materials to decompose. Adding inhibitors or varying the pH significantly reduced inorganic carbon uptake, and demonstrated that the two photosynthesis and calcification were linked. Addition of TRIS strongly inhibited photosynthesis but not calcification, suggesting the involvement of proton pumps in the localized low pH acid zones and high pH basic zones. The high pH zones were maintained by the proton pumps maintaining high calcification, while TRIS was competing for proton uptake from acid zones causing photosynthesis to drop. Rhodoliths were found to maintain high productivity at a temperature of 34oC, and even at 37oC. It is therefore concluded that, rhodoliths are well adapted to high temperatures and excess light, a behaviour which enables them to thrive even in intertidal areas.

Department of Botany © Juma Walaku Kangwe Stockholm University ISBN 91-7155-187-5 Sweden Doctoral Thesis [email protected] 4 LIST OF PAPERS

This thesis is based on one published paper and three manuscripts. The papers will be referred to by their roman numerals. I Martin Gullström, Bengt Lundén, Maria Bodin, Juma Kangwe, Marcus C. Öhman, Matern S. P. Mtolera and Mats Björk (2005). Assessment of vegetation changes in seagrass communities of tropical Chwaka Bay (Zanzibar) using satellite remote sensing. (In press: Estuarine, Coastal and Shelf Science). II Kangwe, J.W. Mtolera, S.P.M. Kautsky, L and Björk, M. (2005). Growth and standing biomass of Halimeda (Bryopsidales) species and their contribution to sediment production in a tropical bay (In manuscript). III Kangwe, J.W. Mtolera, S.P.M. and Björk, M. (2005). Inorganic carbon uptake into photosynthesis and calcification in two common Halimeda species. (In manuscript). IV Björk, M., Kangwe, J.W. and Mtolera, S.P.M. (2005). Temperature effects on photosynthesis and calcification at varying light levels in rhodoliths from a tropical lagoon (In manuscript).

Paper I is reproduced with the publisher’s permission. My contribution to the papers were: (I) Performing vegetation assessments and ground-thruthing 2000 and 2001, as well as taking part in writing; (II and III) Performing all experiments and surveys, taking major part in planning and writing; (IV) Performing all experiments, taking part in planning and writing.

5 ABBREVIATIONS

ΔF/Fm´ Effective quantum yield ΔF Fm´- Ft AF Absorption factor AZ Acetazolamide – an inhibitor of external carbonic anhydrase CA Carbonic anhydrase

Ci Inorganic carbon DCMU 3-(3,4-dichlorophenyl)-1,1-dimethly-urea. ETR Electron transport rate at photosystem II and onwards to photosystem I Fm Maximal chlorophyll fluorescence in a dark adapted sample, Fm’ As Fm, but in actinic light, Fo Minimal chlorophyll fluorescence in a dark adapted sample Fo’ As Fo, but measured directly after an exposure to irradiance Ft Steady state chlorophyll fluorescence in actinic light

Fv Variable fluorescence (F0-Fm) Fv/Fm Maximal quantum yield IMS Institute of Marine Sciences (in Zanzibar, Tanzania) GPS Global Positioning System PAM Pulse amplitude modulated PAR Photosynthetically active radiation PSI Photosystem I PSII Photosystem II SAV Submerged Aquatic Vegetation TRIS Tris (hydroxymethyl) aminomethane UDSM University of Dar es Salaam (in Tanzania)

6 TABLE OF CONTENTS

ABSTRACT...... 4 LIST OF PAPERS...... 5 ABBREVIATIONS...... 6 TABLE OF CONTENTS...... 7 PREFACE ...... 8 INTRODUCTION...... 9 Study area ………………………………………………………………………………... 10 Algae ...... 11 Calcifying algae...... 12 The genus Halimeda...... 12 Reproduction in Halimeda ...... 15 Rhodoliths………………………………………………………………………………….17 Photosynthesis and sources of inorganic carbon in aquatic environments ...... 18 Algal calcification ...... 20 The link between algal photosynthesis and calcification...... 22 COMMENTS ON MATERIALS AND METHODS...... 24 The use of Satellite Remote Sensing in assessing vegetation cover ...... 24 Contributions from Halimeda species in the bay...... 25 Metabolic inhibitors of inorganic carbon uptake ...... 26 Inhibitor – DCMU...... 27 Inhibitor – AZ...... 28 Inhibitor – TRIS ...... 28 RESULTS AND DISCUSSION ...... 29 CONCLUSIONS AND FUTURE PERSPECTIVES ...... 35 ACKNOWLEDGEMENTS…………………………………………………………………. 36 REFERENCES...... 38

7 PREFACE

Aragonite and calcite depositing calcareous algae are among the most abundant and widely distributed seaweeds in the world’s oceans. They are the main contributors of primary production while alive, and production of carbonate sediments in the marine environments when they die (Bach, 1979; Hillis-Collinvaux, 1980; Drew, 1983; Multer, 1988; Payri, 1988). In places without coral reefs, as in the Mediterranean, calcareous algae still play a major role in the formation of biogenic deposits and build-ups of carbonate materials (Basso, 1998). Little information however exists on species diversity in the Western Indian Ocean (WIO), and most of studies on taxonomy, productivity and calcification (Borowitzka and Larkum, 1976b, c, d; Borowitzka,1977; Borowitzka, 1981; Wefer, 1980; Perry, 2005), growth and sediment generation (Drew, 1983; Drew and Abel, 1985), bioherms (Davies and Marshall, 1985), ecology and distribution (Hillis-Collinvaux, 1980; Drew and Abel, 1988; Basso, 1998) have been reported from other areas outside the WIO region. Chwaka bay is in the east coast of Zanzibar in the WIO region (Fig. 1). Halimeda species are exclusively flourishing over the area growing in substrates ranging from sandy, muddy to rocky substrata. In soft bottom areas the soils are rich in dark decomposing Halimeda flakes forming deep layers of mud which can reach over 3 meters (Muzuka, et al., 2001; Pers obsers). However, their contribution to primary production and carbonate production in the area has not been determined. The rhodoliths are mostly found in the western part of the Chwaka bay towards Mapopwe creek, lying on a flat intertidal area (some in rocky pools) mixed with the green algae Ulva reticulatum and seagrass species, mainly Thalassodendron species, near a fossil rocky shore. For many years, the presence of the large meadows (beds) of Halimeda plants in the bay area have remained an open question. Knowing its ecological importance to the marine environment, and the existing data gap on calcareous algae in the WIO region, this study was initiated with the following objectives: (1) To identify and describe Halimeda species present in the area, study their distribution, standing biomass, growth, rates of calcification in order to get estimates of their contribution to the carbonate deposition of the bay. (2) Search for mechanisms behind and relations between the photosynthesis and calcification processes in Halimeda species. (3) Examine adaptations in the coralline algae (rhodoliths) explaining their ability to withstand low tide exposure to high temperatures and excess light that regularly occur in the area.

8 INTRODUCTION

The worldwide distributed calcareous algae such as the green algae of the genus Halimeda (Chlorophyta, Bryopsidales) and the coralline red algae (Rhodophyta, Corallinales), has long been known as main contributors of sand to mud-size carbonate sediments (Drew, 1983; Payri, 1988; Bosence and Wilson, 2003), primary productivity (Bach, 1979; Multer; 1983) and provide potential shelter and nursery grounds for a number of invertebrates ((Hillis- Collinvaux, 1980; Kamenos, et al., 2004) in tropical and subtropical marine environments. Published information on calcareous algae does indicate that the species are the most diverse occupying different habitats from intertidal areas to deep waters (Adey, 1998; Basso, 1998; Aponte and Ballantine, 2001). A group of coralline algae known as rhodoliths form large dense beds usually referred to as “rhodolith beds” or “maerl grounds” with wide ecological importance (Maudsley, 1990; Chisholm, 2000). Payri, (1988) reported a number of results from the previous studies on calcium carbonate production including those from Moorea reefs, where coralline algae (Porolithon onkoides and Hydrolithon reinboldii) produced -2 -1 between 26-162 g CaCO3 m y . Similarly, Kennedy, et al., (2002) reported 10-53% production of sand-sized carbonate sediments from coralline algae around Lord Howe Island -2 -1 and Balls Pyramid, Southwest Pacific. Potin, et al., (1990) reported 876 g CaCO3 m y production from Lithothamnion corallioides in the bay of Brest, France. Bosence and Wilson, -2 -1 (2003) reported calcium carbonate production between 30-250 g CaCO3 m y in western -2 -1 Island and between 895-1423 g CaCO3 m y from Norway. However, growth of rhodoliths is generally slow and growth rate estimates are rare, even those reported so far, are based on methods which are questionable (Foster, 2001). For example, Bosence and Wilson, (2003) reported growth rate of 0.5-1.5 mm y-1 from northern east Atlantic. Similarly, Foster (2001) gave a list of growth measurements results of rhodoliths from several authors who used 14C dating method, and commented that the results were questionable. Halimeda species are capable of producing extensive biohermal accumulations (Davies and Marshall, 1985) and meadows (Hillis-Collinvaux, et al., 1998), and can be evaluated in the field using different methods for growth (Bach, 1979; Multer, 1983; Ballesteros, 1991) and sediment generation (Wefer, 1980; Drew and Abel, 1985; Payri, 1988). They are known to contribute significantly to the flux of carbon and carbonate sediments in the marine environment (Wefer, 1980; Braga, et al., 1996). For example, in the Great Barrier Reef, a 2,000 km2 (1,250 sq. mile) area covered with coarse gravel from 10-15m (33-50 ft) deep, was found to be primarily Halimeda fragments with vast areas comprising as much as 98% algal

9 deposits (Drew, 1983). The same study on the Great Barrier Reef reported huge meadows of Halimeda produced up to 2 kg calcium carbonate per m2 every year (Drew and Abel, 1985). A number of studies on carbonate sediment generation (Bach, 1979; Wefer, 1980; Multer, 1988; Payri, 1988; Ballesteros, 1991) have reported significant results. Nevertheless, field measurements of growth rates of individual Halimeda species are still limited, possibly due to difficulties in measuring growth in a plant that grows by unpredictable spurts and varies in percentage CaCO3 with age (Hillis-Collinvaux, 1980; Drew and Abel, 1985), and differs in growth rate by species (Hillis-Collinvaux, 1980), and possibly with depth (Böhm, 1973). Such variables combined with disasters such as storm damage to experimental sites (Merten, 1971), mechanical damage through human activities (this study) and sometimes the patch distribution of Halimeda (Drew, 1983) discourage attempts to evaluate production rates quantitatively.

Study Area Chwaka bay (Fig. 1) is a relatively shallow tropical lagoon (mean depth 3.2 m) located in a tropical climate stretching 34 km on the east coast of Unguja Island in Zanzibar, between 39o22’ to 39o30’E and 6o8’ to 6o15’ S (Cederlöf, et al. 1995). The lagoon is mainly characterized by seagrass beds, macroalgae, some remains of hard corals and mangroves. The bay experiences semidiurnal patterns of tides with ebb currents are stronger than the flood currents, and is a potential source of important biological and commercial activities (Wolanski, 1989; Tobison, et al., 1998). The seabed is broadly influenced by a wide network of channels with the water currents predominantly forced in a north-south direction. The vegetative assemblages found in the eastern and south-eastern parts shows distribution with irregular meadows of different seagrass communities dominated by Cymodocea serrulata, C. rotundata, Thalassodendron ciliatum, T. hemprichii, Enhalus species and macroalgae, mainly Halimeda species. Extensive mangrove forests fringes along the Mapopwe creek, in the east, south, southwest and southeast shoreline (Wolanski, 1989; Mohammed, 1998). The middle, east and south-west parts of the bay is characterised by a wide continuous seagrass meadows partly interspersed with a great amount of the macroalgae mainly Halimeda species, and other such as Sargassum, Ulva and Gracilaria species.

10 Tanzania

Tanzania Mainland

Zanzibar Is. 5 58 18 S

Zanzibar Town Chwaka 6 25 26 S

0 Km 20

39 05 26 E 39 32 34 E Fig. 1: Map of Africa (Top right) showing the position of Tanzania in Africa and the position of Zanzibar from Tanzania mainland with enlarged map of Zanzibar (below) showing the position of Chwaka bay in the East coast where this study was conducted.

Algae

Traditionally, the term algae refer to macroscopic, filamentous and multicellular marine red, green and brown halophytes (plants lacking true roots, stems and leaves). Unlike terrestrial plants, algae are photosynthetic organisms with single reproductive structure, lacking vascular systems and their body is referred to as thallus (Jaasund, 1976). They contain a variety of carotenoids depending on the taxonomic group, and all contain chlorophyll a, and some have chlorophyll b or c (Falkowski and Raven, 1997). Most algae are found in aquatic environments (freshwater to marine), but some can be found in other places such as rocks, deserts, soils snow and hot springs. Classification of algae was formerly based on colour, where algal groups were given names such as “red”, “brown” or “green”. However, at present

11 and according to Van den Hoek, et al. (1995), classification is entirely based on specific characteristics such as cellwall composition, photosynthetic pigments, storage products, morphology and ultra structure. Nevertheless, the names of the divisions and classes still reflect the colour of the main pigments; for example Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyta (brown algae) and Bacillariophyceae (diatoms).

Calcifying algae

This is a group of algae with an ability to deposit CaCO3 around or within the algal thalli (Borowitzka, 1982). Among the Rhodophyta, calcification occurs in the Corallinales (Coralline algae), some members of Bangiales, Gigartinaceae and Squamariceae (Borowitzka, 1982; Kangwe, 1999). Members of the Corallinaceae are both the most abundant and best- known calcified red algae (Littler, 1972; Borowitzka, 1982). In marine green algae such as the Halimeda species and the calcifying brown algae Padina, as well as the red algae not belonging to the family Corallinaceae, deposition of CaCO3 form is invariably extracellular aragonite, largely in the shape of needle-like crystals (Borowitzka, et al., 1974; Borowitzka,

1981; Braga, et al., 1996). The aragonite form of CaCO3 isomorph in Halimeda is orthorhombic, whereas in most coralline algae (red algae) the calcite form of CaCO3 is the rhombohedral carbonate mineral (Milliman, et al., 1974). The Ca2+ in calcite can be replaced by cations of smaller radius (Mg, Fe, Zn, Cd), while aragonite accepts cations of larger radius than Ca2+ (such as Pb, Ba, Sr) (Borowitzka, 1977; Kangwe, 1999). High concentrations of

MgCO3 are an indication of calcite, where aragonite is often characterized by relatively large amounts of SrCO3 (Littler, 1972). Recent studies on calcification process in calcareous algae are still rare. Most of the studies focus on coral reef ecosystems which are the most striking example of calcifying ecosystems (Gattuso, et al., 1999).

The genus Halimeda The green algae of the genus Halimeda belongs to the phylum Chlorophyta, order Bryopsidales. The family Byopsidaceae/Halimedaceae where Halimeda belongs is along with their close relatives Udotea and Penicillus, commonly know as Shaving Brush algae (Hillis- Collinvaux, 1980). A typical Halimeda plant is a flexible string of flattened jointed leaf-like structures often referred to as segments (Fig. 2). The plant is sometimes called the “money plants” as it looks somewhat like small coins (Vroom, et al., 2003). Each 'coin' or segment is

12 hard because it is impregnated with calcium carbonate (Drew and Abel, 1985), connected to its neighbours by a thin strand known as genicula, which gives the plant its flexibility (Multer, 1988). Growth is attained by additional of new segments at branch tips which rapidly achieve full size before calcification begins (Batch, 1979; Drew, 1983).

Fig. 2: Halimeda macroloba (large plant) and H. opuntia (smaller plant beneath) in an aquarium tank. The algae were collected from Chwaka bay during field work for use in laboratory experiments. The flattened jointed leaf-like structures is obvious. New segments can appear at the top of each segment.

Halimeda species inhabit a range of habitats from intertidal zone (this study paper I and part of paper II) usually mixed together with seagrasses and other macroalgae, in sandy floors of lagoons and extend to deeper reef slopes (Drew, 1983; Littler, et al., 1985). They are known to be among the deepest living photosynthetic organisms, found at depths up to 130 m (Littler, et al., 1986). These algae are somewhat different in that they are both coenocytic

(lacks cross-walls in its component siphons) and calcareous (composed mainly of CaCO3) (Payri, 1988). The coenocytic thallus, suggest that the genus and other members of Bryopsidales have distinctive branches on the algal evolutionary tree (Hillis, 2001). While a

13 normal plant cell is tiny, enclosed in a cell wall and contains one nucleus with the genetic material, coenocytic plants can be thought of as a single giant cell with multiple nuclei (Payri, 1988). About 14 species of Halimeda have been described for the tropical and subtropical western Atlantic (Hillis-Colinvaux, 1980; Wynne, 1986), and several others have been described for modern reefs based on morphological properties of the thallus (Drew and Abel, 1988; Hillis, et al., 1998). They provide shelter and sometimes food to a number of reef animals, and as a colonizer, facilitates the restoration of damaged or eroded reefs (Bach, 1979; Hillis, et al., 1998). The calcareous nature of Halimeda and the ability to synthesize noxious and potentially toxic secondary metabolites makes them less appetizing meal to grazing fish such as surgeon and parrot fishes than more succulent algae (Paul and Fenical, 1983; Hay, et al., 1988), thus making them protected from herbivory feeders such as parrot fishes. The compounds halimedatrial and halimedatetraacetate are diterpenoid compounds that appear to give Halimeda an extremely noxious taste and could prove toxic in large quantities (Paul and vanAlstyne 1988b). Younger segments have the highest concentration of these compounds, while older segments are protected by heavier calcification that make them rich of CaCO3 in the algal wall which makes the plant less tasty to herbivores (Hay, et al., 1988; Braga, et al., 1996). Death of Halimeda tissues disintegrate into fine, white calcium carbonate particles (Milliman, 1977; Bach, 1979). The white sandy beaches of some coral atolls may be made up mostly of Halimeda and coralline alga (rhodoliths) remains (Grall and Hall-Spencer, 2003).

The genus Halimeda is an important element of tropical reefs (Hillis-Collinvaux, 1980), a contributor of sand and carbonated sediment in tropical reefs since mid-Jurassic to the Holocene period (Hillis, 2001). The greatest pre-Cenozoic species diversity was achieved during the latter part of the cretaceous (Flügel, 1988; Kooistra, et al., 2002). According to Hillis (2001), the long paleohistory is capped by an apparent burst of speciation associated with the Holocene (Fig. 3), and at least three time-periods during the ca. 260 million years ago of Halimeda history are likely to have had major impacts on evolution of the genus; (1) Cretaceous-Tertiary boundary events; (2) closing of the circumtropical Tethyan seaway with associated Messinian crisis; and (3) final closure of the Panama seaway. However, little information can be obtained from the recorded paleohistory of Halimeda, and the first phylogenetic data (evidence) of the genus came from analysis of the 18s DNA sequence, and phylogenetic trees were presented to indicate geographical distribution and separation of the rhipsalian species into Atlantic and Pacific clades (Mankiewicz, 1988; Flügel, 1988).

35 % 30 K T

25 Messinian 20 crisis

15 Number of species of Number 10

e e ne e ous ne ne ce ce ocen oce ioce a leocen E l itocene Permian et ligo Mi P e r Pa O Pl Holocen ly C te Cretaceous ar E La Geological time-scale

Fig.3. Diversity of Halimeda species in geological time-scale determined from the fossil data. The figure suggests peaks of diversity during the time period represented by late cretaceous to Eocene, that is from ca. the last 30 million years ago of the Mesozoic through approximately the first half of the Cenozoic. The comparative species richness is followed by seemingly very low diversity before an apparent burst of speciation in the Holocene (Source: Hillis, 2001).

Reproduction in Halimeda

Halimeda and other closely related members of Bryopsidales have an ability to reproduce both sexually and asexually. Sexual reproduction is rarely seen in Halimeda because it is a short lived phenomenon, and has recently been described for one species from direct observation in the field (Clifton, 1997). The ability of Halimeda to propagate asexually via vegetative fragmentation has been mentioned as one of the reason on why it’s abundant on coral reefs (Walters, et al., 2002). Asexual propagation occurs through vegetative fragmentation when detached live portions of individuals survive and continuous to grow. In the marine environment, fragmentation via fission may be; (1) an endogenous (Yamashiro and Nishihira, 1998) (2) as a result of exogenous processes, such as predation or physical disturbance events (Walters and Smith, 1994). The advantage of fragmentation over sexual reproduction includes extension of the distribution of genets and species, increase in the abundance of the organism and individual biomass, and colonization of areas where sexual

15 propagules are unable to settle or high rate of early post settlement mortality. Nevertheless, recent research shows that fecundity of the organism is reduced especially when the fragments are dispersed in an unfavourable habitat (Smith and Hughes, 1999). Therefore, the costs of fragmentation outweigh the benefits for some marine organisms and this may be related to the organism’s potential to successfully sexually reproduce.

Sexual reproduction by many members of the Bryopsidales, including Halimeda, is holocarpic, with dioecious individuals releasing gametes all at once and then dying within hours (Clifton and Clifton, 1999), and the thallus completely disintegrates after spawning. The spawning process is initiated after sunset (Drew and Abel, 1988). The simple life-history of Halimeda (Fig. 4) is illustrated by a free-living phase that reproduces sexually is seen to have a dynamic, asexual, fragmentation component that allows for long term viability and re- establishment of fragments. Recent observations have shown that sexual reproduction in Halimeda to some extent is synchronised (Hay, 1997), where many individual in a population may become fertile within a period of only few days, or sometimes on the same day.

Fig. 4: Sexual reproduction and the general life cycle of Halimeda species. Halimeda can reproduce very successfully sexually and through vegetative propagation which enables copies of the same plant to be produced. Many species can produce filaments which can grow more than 20 cm long, spread laterally through the substrate, and then push up to form new segments. Eventually the physical connections between the young and parent thallus are lost (Source: http://www.aims.gov.au).

16 Rhodoliths

The term rhodoliths (or maerl) is used to define nodules and detached branched growth with a nodular form composed primarily of coralline algae (Basso, 1998), that occurs not confined to a particular benthic zone. They belong to a group known as coralline red algae

that deposit CaCO3 within their cell walls to form hard structures that closely resemble maerls or pearls, which accumulate to form large beds and have been found throughout the world’s oceans (Wilson, et al., 2004). They are known as rhodoliths when the corallines are made up to more than 50% of the nodule (Basso, 1998) or composed entirely of non-geniculate coralline algae (Foster, 2001). They are critical habitats for many species including fishes, clams and true corals (Basso, 1998; Kangwe, 1999). However, unlike corals, rhodoliths do not attach themselves to the rocky seabed, rather they drift along the seafloor until they grow heavy enough to settle and form brightly colour beds (Basso, 1998). The most difference between rhodoliths and corals is that, the corals filter plankton and other organisms from water for food, whereas rhodoliths produce energy through photosynthesis (Borowitzka, 1981).

Rhodoliths grounds (also known as Maerl grounds) are composed of loose-lying nongeniculate coralline red algae (Foster, 2001), and are mostly found in areas characterised by high water movements (wave action) in the photic zone (Kamenos, et al., 2004). Maerl grounds vary in size and are dense accumulations of unattached coralline algae and occur throughout the world oceans (Woelkerling, 1988). They serve two main functions (1) production of calcareous and carbonate sediments important for reef building and corals (2) with their structural complexity provide relatively stable microhabitats which are important shelters and nursery grounds for the increasing number of refuges from predators (Grall and Hall-Spencer, 2003). Maerl grounds have been found to fulfil the density and refuge prerequisites of a nursery area for a number of invertebrates and vertebrates (Kamenos, et al., 2004). Maerl grounds are important in sustainable fisheries, providing nursery grounds for commercial fish species and shellfishes. Fragments and hard substrate may originate in the

17 bed or be broken from nearby reefs and transported to the site of growth, where new rhodolith beds established in this way. Apart from their ecological importance rhodoliths (maerl) contains resources economic importance which can be extracted and used primarily as soil conditioners instead of ground limestone, and as a water filtration and conditioning agent in Europe (Foster, 2001). For example, in France, maerl beds in Brittany represent the largest resources (Grall and Hall-Spencer, 2003) and accounts for 80% of the total 500,000 t extracted annually. This enables the maerl industry provide hundreds of local jobs to the people (Bosence and Wilson, 2003). However, due to their slow growth rate between 0.5-1.5 mm per year (Bosence and Wilson, 2003), maerl beds are considered as non-renewable resources. To save the maerl from being over-exploited, some efforts are being made by some countries in Europe (example France) by introducing laws through their marine conservation programmes which include maerl grounds for conservation (Grall and Hall-Spencer, 2003).

Photosynthesis and sources of inorganic carbon in aquatic environments

In both terrestrial and marine plants, photosynthesis is usually regarded as the main indicator of performance, adaptation and physiological status. The processes produces energy using certain wavelengths of light, involving two photosystems, PSI and PSII which are mostly active at 680 and 700nm wavelengths. Other wavelengths are also peaks in the action spectrum for photosynthesis. Autotrophs use CO2 and energy from the sunlight to synthesize organic molecules (such as glucose). Plants are autotrophs, which means they are able to synthesize food directly using carbon dioxide gas, water and light to produce sugars and oxygen gas. For instance, the production of glucose can be simply represented in an overall chemical equation;

12H2O + 6CO2 + Light → C6H12O6 + 6O2 + 6H2O

Even though, this equation may appear simple, but it is actually a summary of very complex processes (Falkowski and Raven, 1997). The glucose is variously used to form other organic compounds, such as the building material cellulose, or it may be used as a fuel to drive other physiological processes of the plant. This takes place through respiration found in both animals and plants. The chlorophylls in plants absorb light energy that drives the process of photosynthesis. Contrary to the terrestrial environment where there is a plenty of CO2 available in the atmosphere for the plants to use in photosynthesis, submerged plants experience problems of inorganic carbon acquisition in photosynthesis (Hellblom and Björk,

18 1999). This is due to slow diffusion rate of CO2 in aquatic environment which makes CO2 less available to aquatic macrophytes (Beer, et al., 2002). Thus, depending on the pH of the - 2- aquatic media, four forms of inorganic carbon exist; CO2, HCO3 CO3 and H2CO3. At low pH 6.15-6.5, CO2 is abundant such that it can drive high photosynthetic rates by sole diffusion into the site of carbon fixation from the bulk waters (Borowitzka and Larkum, 1976d). At pH 8.2 sea water, more than 90% of the inorganic carbon available in the aquatic environment is - in the form of HCO3 (Hellblom and Björk, 1999), furthermore the diffusion of CO2 in water is drastically slower than in air (Falkowski and Raven, 1997; Hellblom, 2002).

At the normal pH ranges of sea water (8.1-8.2), poor CO2 availability is thus quite often limiting productivity in marine plants. To solve the problem of CO2 limitation, aquatic plants - have developed mechanisms (modes) for using HCO3 available as the main source of inorganic carbon in photosynthesis. The following mechanisms for inorganic carbon acquisition in aquatic environment have been described and reported (1) direct diffusion of - + CO2 into the cell (2) spontaneous dehydration of HCO3 to CO2 due to locally elevation of H concentration outside the plasma membrane (acid zones)(Hellblom, et al., 2001). This mechanism is based on natural processes, where H+ are pumped from the cytosol resulting into accumulation of protons outside the plasma membrane and consequently lowering the pH - which favours more dehydration of HCO3 to CO2, followed by its diffusion into the cell (Hellblom, et al., 2001; Klenell, et al., 2004). The drawbacks of this natural process (protogenic mechanism) is that, it can be negatively inhibited by the presence of biological inhibitors such as TRIS buffers which competes for uptake of protons (Hellblom, 2002) (3) - Extracellular dehydration of HCO3 catalysed by carbonic anhydrase (CA), an enzyme localized outside or within the plasma membrane (Axelsson, et al., 2000; Hellblom, et al., - - 2001), which speeds the conversion of HCO3 to CO2 under normal pH 8.0-8.2 (4) HCO3 can - be actively transported across the membrane by protogenic HCO3 uptake through a symport + - H /HCO3 or co-transport (Price and Badger, 1985; Hellblom, et al.. 2001). This mechanism is closely similar to the second mechanism, and both can be inhibited if the acid zones are - dissipated by biological buffer (5) CA-catalysed HCO3 dehydration within the acid zones. This mechanism takes advantage of the protons excreted within the acid zones creating a more favourable condition for CO2 conversion. The different forms of inorganic species in sea water are produced when carbon dioxide enters the aquatic environment and reacts water in the following sequence;

19 - 2- CO2 + H2O ↔ H2CO3 ↔ H+ +HCO3 ↔ 2H+ + CO3 (1)

- - OH + CO2 ↔ HCO3 (2)

Equation 1 above starts when CO2 from the atmosphere enters the aquatic environment and reacts with water to form a weak carbonic acid which is less stable and dissociates to - + 2- HCO3 and H , which dissociates further to form CO3 when pH increases. At higher pH - - above 8.2 the OH can combine with available CO2 to form HCO3 (equation 2). In general, sea water at pH 8.2 contains a smaller proportion of CO2 (10 μM ) and more than 95% (about - 2.1 mM) of total Ci is in the form of HCO3 (Falkowski and Raven, 1997).

Algal calcification

The word calcification refers to the precipitation of CaCO3 around or within the algal thalli (Borowitzka, 1982) of calcifying algae such as those in the Rhodophyta, brown algae (Padina) and Chlorophyta (Halimeda). Calcification process in Halimeda is active in light and lower in the dark (Borowitzka and Larkum, 1976b). Respiration reduces calcification, probably due to lowering of pH in the intercellular space as a result of CO2 production (Borowitzka, 1977). Plant metabolism may stimulate calcification by increasing the local 2+ 2- concentration of Ca and/or CO3 ions, or by removing inhibitors of CaCO3 precipitation such as phosphates which are known as crystal poisons (Simkiss, 1964).

To explain calcification in Halimeda, a researcher needs to take into consideration the specialized morphology of the thallus of these algae (Borowitzka and Larkum, 1976c). Calcification takes place when photosynthesis is active and the intercellular space (ICS: which represents a large portion of exchangeable calcium) must be separated from the external medium by loose peripheral utricles (Borowitzka and Larkum, 1976c). This morphology means that the supply of ions to the ICS must be by diffusion through the outer cuticle and the cell walls of the appressed utricles. Multer, (1988) studying the ultra-structure of H. incrassata and H. monile found a unique aspect of development of large intercellular spaces (ICS) in which CaCO3 in form of aragonite was deposited. A typical equation of the process can be represented as follows;

- + - + CO2 + H2O ↔ HCO3 + H ↔ CO3 + 2H (1)

20 2- 2+ CO3 + Ca ↔ CaCO3↓ (2)

- 2+ 2HCO3 + Ca ↔ CaCO3↓ + CO2 + H2O (3)

Equation 1 above shows the hydration of CO2 in ocean waters in equilibrium with air levels of CO2, resulting in different forms of carbon and proportions of the different carbon species of the equilibrium reactions are dependent on pH of the media (Axelsson and Uusitalo, 1988). At lower pH a large proportion of inorganic carbon is present in the form of

CO2 and the reactions to the left are favoured, whereas at higher pH like pH 8-9, the majority of carbon is present as bicarbonate and carbonate (Hellblom, 2002). The third one is often used to emphasize that under normal pH conditions of any natural waters (pH 8.1-8.2), the - 2- HCO3 ions largely dominate over the CO3 ions (Johnston, et al., 1992; Hellblom, 2002).

Photosynthetic CO2 uptake from the intercellular spaces increases intercellular pH where 2- 2+ - CO3 in the presence of Ca combines to form CaCO3 which is deposited as aragonite, thus facilitating calcification process (Borowitzka, 1982). However, this mechanism for calcification in Halimeda is not applicable to all aragonite depositors such as the brown alga Padina, where there are no intercellular spaces and aragonite is precipitated in concentric bands on the outer surface of the thallus (Lobban and Harrison, 1994). Moreover, there are other seaweeds with apparently suitable morphology that do not calcify (example, Enteromorpha) (Borowitzka, 1982).

Despite calcification being one of the important structural processes in the oceans, its mechanism in algae is not fully understood. It is however well known that calcification is directly proportional to photosynthetic rates and is stimulated by light (Borowitzka, 1977), and that, calcification rate is highest in the young tissues (Lobban and Harrison, 1994).

Moreover, taking in consideration the type of CaCO3 deposited, localization and organization of the cell wall matrix, it appears that the calcification process in algae involves more than one mechanism (Borowitzka, 1977), and crystal formation requires two steps; crystal nucleation and crystal growth. Nucleation is the major rate limiting step for the precipitation of CaCO3 and can be used to explain why other algae do not calcify (Borowitzka, 1982).

21 The link between algal photosynthesis and calcification

Knowledge of the source and forms of inorganic carbon for photosynthesis in aquatic environment is important for understanding calcification mechanisms in algae. Previous studies on photosynthesis and calcification processes have shown that the two processes are coupled in a certain way (Borowitzka and Larkum, 1976b, c, d; Borowitzka, 1977; Pentecost, 1978; Gattuso, et al., 1999; Borowitzka, 1982; Marshall and Clode, 2002). Light stimulated 2- algal calcification involves a rise in CO3 as a result of CO2 uptake during photosynthesis or - - due to alkalization of the medium due to OH extrusion from the cell after HCO3 uptake (Borowitzka and Larkum, 1976b). Similarly, it has been suggested that calcification enhances - photosynthesis by providing protons that convert seawater HCO3 to CO2 and H2O, thereby supplying some of CO2 for photosynthesis (Borowitzka and Larkum, 1976c; McConaughey and Whelan, 1997). The rhythm and nature of calcification processes may be estimated in different ways, but the measurement of TA changes in seawater is considered as the most convenient in short time duration experiments (Smith and Key, 1975; Chisholm and Gattuso, 1991).

In the course of development in research on photosynthesis and calcification, Borowitzka (1977) put forward a number theory explaining the link between calcification and photosynthesis mechanisms. The widely accepted theories include; (1) CO2 usage theory: - This suggest that, photosynthetic CO2 uptake spontaneously generated from HCO3 may increase extracellular pH (due to alkalization of the medium caused by OH- extrusion from - 2- the cell after HCO3 conversion to CO2) high enough to elevate the concentration of CO3 2+ which leads to extracellular precipitation of CaCO3 in the presence of Ca ions. However, this process can be inhibited if the acid zones are dissipated by a biological buffer such as TRIS or AZ (Price and Badger, 1985; Hellblom, et al., 2001), or if there is a limited - - spontaneous generation of CO2 from HCO3 (Fig. 5) (2) HCO3 usage theory: Suggest that, by - the aid of the enzyme carbonic anhydrase, the photosynthesizing algae which uses HCO3 as a source of carbon, may extrude OH- to specific zones outside the plasmalemma which will favour the precipitation of CaCO3. (3) Organic matrix: The presence of charged Calcium binding mucilage (polysaccharide complexes) in the cell walls (Borowitzka and Larkum, 2+ 1976b) acts as nucleation sites for the Ca crystals. The form of CaCO3 to be deposited is suggested to be determined by the nature of polysaccharide of the relevant alga (Borowitzka, 1977). Non-calcifying algae may have the same cell wall organisation, but it is not known why they do not calcify (Borowitzka, 1982).

22 Despite such conceptual agreement on the link between photosynthesis and calcification, Yamashiro (1995) showed that bisphosphonate (as a crystal poison) reduced 14C incorporation in into the skeleton (CaCO3 deposition) but not into the tissues (photosynthesis) of zooxanthellate coral, and concluded that calcification is not necessary for photosynthesis. Similarly, Gattuso, et al., (2000) showed that artificial seawater with a low calcium concentration lowered calcification rate but did not reduce the production of photosynthetic oxygen and concluded that “calcification is not a significant source of photosynthetic CO2”.

CELL CO2 Respiration

- Photosynthesis HCO3 - - CO2 + OH OH

- - HCO3 OH CO2

CO2 + H2O CO2 + H2O

- + - + HCO3 + H HCO3 + H

2- + CO3 + H 2- + CO3 + H 2+ Ca Ca2+ CaCO3

SEA WATER INTERCELLULAR SPACE

Fig. 5: Schematic representation of possible mechanisms of Ci uptake and postulated ion fluxes for CaCO3 precipitation in Halimeda species during photosynthesis and calcification processes in seawater. Passage of ions from seawater to the intercellular space is by diffusion through the cell wall of appressed utricles. CO2 for photosynthesis enters the cell by diffusion from both the external medium and from the intercellular spaces - (ICS), and CO2 produced during respiration diffuses out of the cell. HCO3 enters the cell by periplasmic CA- + - - mediated dehydration or mediated by H -ATPase. After dissociation of the HCO3 the OH may leave the cell possibly in much localized region [Modified from Borowitzka and Larkum, 1976c].

23 COMMENTS ON MATERIALS AND METHODS

The use of Satellite Remote Sensing in assessing vegetation cover

Field work Seasonal and spatial distribution of bottom vegetation cover (ground-truthing) was assessed visually using SCUBA facilities. The area was divided into 221 sampling sites, 500 m apart from each positioned using a GPS. Ten 0.25 m2 metal frames were randomly placed at each site, followed by assessment of vegetation cover within the frames. The sites were seasonally assessed in December 2000, March, June and September 2001. The field work for 2002 on SAV coverage (structural complexity, shoot density, above-ground biomass and canopy height) mainly focused on six selected sites representing homogenous and dense meadows within the bay using methods described in paper I. The aim was to use the data to describe the most dominant seagrass communities, and compare well-quantified seagrass habitats with spectral signatures derived from Satellite Remote Sensing. Apart from analysis of seasonal variations between assessment periods, a correlation analysis was made between total submerged aquatic vegetation (SAV) coverage between years using Landsat ETM+ images available. A regression analysis was applied between percentage cover field data (ground truthing) obtained in September 2001 and the digital spectral values from Landsat ETM+ scene taken in the same month. However, due to the effect of cloud cover, only 107 sites out of 221 were used in this analysis. A field work conducted in 2004 aimed at verifying the positions and extensions of the Remote Sensing mapped major habitats. In addition, an interview (discussions) was held between local people and fishermen of Chwaka village to obtain information on the cause of the observed changes in vegetation cover in certain parts of the bay.

Image analysis: The digital spectral values from September 2001 Landsat ETM+ image were compared with vegetation coverage obtained from field work on the same month. The satellite sensors used for this study were (1) Thematic Mapper (TM) on Landsat 5 and (2) Enhanced Thematic Mapper (ETM+) on Landsat 7. The sensors had a resolution of 30 x 30 meters (which was appropriate for this study) for six identical matching spectral bands, including the blue band which is important for water penetration. The TM data is available since 1982; while the ETM+ data is available since 1999. These data creates a possibility of mapping the coastal environment over a longer period. Using a computer program analysis, the digital spectral

24 values from the September 2001 Landsat ETM+ image were compared to the vegetation coverage (ground-truthing) assessed by the field surveys in that month. This was followed by computer-based unsupervised classification (Mather, 2004) image analysis (after creating masks in the two images to exclude features such as land, deep ocean, clouds so that the analysis focuses on the features of interest) to give a clear relationship between the sensitivity of the satellite data spectral response in relation to vegetation coverage on the bottom of the bay. The visible bands of the satellite image from September 2001 were used. Data analysis also involved the use of two geometrically corrected satellite images (Landsat TM from 27 January 1987 and Landsat ETM+ from 30 January 2003), which were used to create a change detection map. Several other procedures were applied during image analysis including the use of the visible wavelengths (blue, green and red) due to their ability to penetrate water, a crude atmospheric correction (which was done by applying “dark pixel subtraction”), selection of training areas for the two classes (SAV and un-vegetated areas) and making a supervised maximum-likelihood classification, where the acquired map was compared to the outcome from the field surveys. Several Landsat images were available from 1986 to 2003, which were used to illustrate the general vegetation changes within that period by performing correlation analysis of digital radiance values from 0-255 for pairs of satellite images.

Contributions from Halimeda species in the bay Species composition, growth, standing biomass and distribution of Halimeda including their contribution to carbonate production (paper II) and associated parameters (tissue nutrient content, decomposition, carbonate production, in situ photosynthesis and calcification) were assessed between the year 2000 and 2003 using methods previously used by other authors (Smith and Kinsey, 1978; Drew and Abel 1988; Payri, 1988; Ballestros, 1991; Ochieng and Erftemeijer; 1999). These methods have proved to yield significant results in the past. Identification of species composition in the area was done primarily using classical morphological descriptions provided by Jaasund (1976) and Hillis-Collinvaux, (1980), followed by final identification and confirmation by a taxonomist. Growth was determined using tagging method (Drew, 1983; Ballesteros, 1991) at two sites differing in ecological characteristics (substrate type, extent of exposure to air during lowest spring tides of the day and inshore vs. offshore). This was important for comparison to see if there is a significant difference in growth rate between the two sites, and between seasons (dry and wet seasons). Currently, most of the biomass studies on Halimeda have been reported from outside the Western Indian Ocean (Bach, 1979; Drew, 1983; Drew and Abel, 1985; Garrigue, 1991;

25 Payri, 1988). Thus, standing biomass was assessed for six months at five sites using method (Bach, 1979; Ballesteros, 1991), where ten, 0.25 m2 quadrants were used. During each visit, the quadrants were thrown in random at each site, followed by quantification of the above ground biomass from each quadrant, dried to constant weight and presented as gdw m-2. Like for the growth assessment, the aim was to investigate if there is a difference in standing biomass between the sites during rain and dry seasons. Carbonate production assessment was conducted using acid leaching method to get estimates on how much carbonate materials are produced by Halimeda species in tissues, and extrapolate (using standing biomass data and in situ calcification values) into carbonate production m-2, and compare with the previous studies from other parts of the world’s oceans (Böhm, 1973; Wefer, 1980; Drew, 1983; Multer, 1988; Payri, 1988). It was also of added advantage to assess tissue nutrient content (C, N and C/N ratio) on Halimeda materials collected from inshore and offshore, so as to study if there is nutrient gradient between inshore and offshore habitats (Hemminga, et al., 1994; Mohammed, 1998), and between seasons (Boto and Bunt, 1981; Ballesteros, 1991). Analysis of dry Halimeda opuntia materials collected monthly was conducted in the Department of Systems Ecology, Stockholm University, Sweden. Duplicates of about 3-3.8 mg of dry and grounded algal materials from each site were analyzed for C, N and C/N ratio using elemental analyzer (LECO CHNS-932) and expressed in % C, N content (dry weight) and the C/N ratio was calculated. Decomposition of Halimeda materials is an important phenomenon regarding sediment generation for development of reefs and tropical lagoons (Milliman, 1977; Drew, 1983). This was conducted at two sites for 56 days using litter bag experiments (Ochieng and Erftemeijer; 1999), with subsequent deployment of 6 litter bags from each site for analysis after every 8 days. The aim was to investigate decomposition rates (time in days) it takes for a given amount of Halimeda materials to decompose into sediments. In situ calcification was determined using total alkalinity (TA) method (Anderson and Robinson, 1946; Smith, 1973; Smith and Kinsey, 1978) as described in paper II. The method was appropriate to this investigation, where dark and light bottles were incubated in situ for 4 hours, followed by pH measurements in the laboratory, and calculations on calcification and carbonate production were made.

Metabolic inhibitors of inorganic carbon uptake The effect of biological inhibitors or varying pH (paper III) on inorganic carbon uptake into photosynthesis and calcification was conducted in the laboratory by total alkalinity method during dark and light incubations using a newly developed device, the Titration

26 manager (TM865, Radiometer analytical, Denmark) equipped with an automatic sample changer (SAC80 Radiometer analytical Denmark). The TIM865 can be programmed and run titration process automatically to completion. Measurements of electron transport rate (ETR) were measured and recorded using a well known device, the Pulse Amplitude Modulation fluorometer (the diving PAM, Walz, Germany). A Clark type electrode was used for measuring dissolved oxygen with a temperature sensor (Oxi 323 electrode connected to a multi 340i meter, WTW Germany) which was immersed into a closed incubation cylinder, through a hole in the lid, sealed with an o-ring. During the experiment the following parameters were measured and recorded; pH, Total alkalinity, ΔF/Fm´ (or Fv/Fm during darkness), dissolved oxygen and temperature. The stock solutions of the inhibitors used (100 µM AZ, 10 mM TRIS, 100 µM DCMU, pH 9.0 and 9.8) were prepared and added to the sample solution to give the required final concentration, and one inhibitor was added at a time. After addition of the inhibitor, at the start of each experiment, the pH of the experimental medium was adjusted to 8.2 using NaOH or HCl. Each exposure lasted for 3 hours in alternating dark and light incubations. Calculations of the results involved the following;

(1) Since for every CaCO3 precipitated, the total alkalinity is lowered by 2, change in

calcification (ΔCcalc) was calculated as change in total alkalinity divide by 2. i.e.

ΔCcalc= ΔmEq/2

(2) Photosynthetic inorganic carbon uptake (ΔCphot) was calculated as the change in total

carbon minus calcification. i.e. ΔCphot=ΔTC-ΔCcalc

(3) The effective quantum yield was calculated as ΔF/Fm’= Fm´-F/Fm´

(4) The maximum quantum yield was calculated as Fv/Fm = Fm-F0/Fm, and the ETR was calculated as ETR = (ΔF/Fm’) x PAR x AF x 0.5 (Beer, et al., 2001).

(5) The area of the Halimeda samples used was determined using a computer programme (Canyon), so as to express photosynthesis in μmol C m-2s-1 and dissolved oxygen was -2 -1 expressed in µmol O2 m s .

Inhibitor – DCMU - The herbicide DCMU (well know as PSII inhibitor) blocks electron flow from QB to PQ, probably by binding at the QB site of D1 protein (Krause and Weis, 1991), so that the electron is unable to leave QA, the first quinone acceptor. Thus, the binding of a herbicide effectively

27 blocks electron flow from PSII to plastocyanin (PC) towards PSI, and therefore inhibiting photosynthesis. This has a further effect of a back-transfer of electrons in a fraction of the centres that are in the state of QAQB (Hodges and Barber, 1986). Therefore, the action of DCMU is consistent, acting primarily as an inhibitor of photosynthetic electron transport (Borowitzka and Larkum, 1976d), causing a drop in photosynthesis and consequently inhibiting calcification process.

Inhibitor – AZ Acetazolamide (AZ) is a sulphonamide anion which binds to zinc ion of the enzyme CA, - making the enzyme inactive, and thus inhibiting the CA-catalysed conversion of HCO3 to - CO2 and OH process. This can lead to inhibition of both photosynthesis and calcification (Stark, et al., 1969; Borowitzka and Larkum, 1976d; Velitchkova and Picorel, 2004).

Inhibitor – TRIS + - TRIS buffers are potent inhibitors of H dependent HCO3 utilization by their capability to take up protons (Hellblom and Björk, 1999; Hellblom, 2002; Uku, 2005). They highly compete for protons uptake and can inhibit other biochemical processes (El Haїkali, et al., 2004). In paper IV, since the rhodoliths are found lying on the intertidal area in Chwaka bay, exposed to high light and temperatures of the midday sun during lowest spring tides of the day, it was of interest to investigate the tolerance limits of rhodoliths by exposing them to excess light and temperature, while measuring photosynthesis and calcification rates. This was determined using a similar experimental set-up as for the above experiments for paper III on the effect of inhibitors and varying pH, except that the Rhodoliths were first exposed to 25°C and then to a set light intensity (0, 150, 500, 850, or 1200 µmol photons m-2s-1), then to an elevated temperature (28, 31, 34, 37, 40, 43 or 46°C), and then the temperature was returned to 25°C again, while all the time keeping the same light. Each exposure lasted for about 3 hours. Temperature-light combination involved 3-4 experiments in alternating dark and light incubations and the calculations were done as in paper III. Difficulties were encountered on calculating the area of the rhodoliths, as they were nearly spherical in shape. Therefore, we estimated the photosynthetic area as the projected area (since light was unidirectional), i.e. the cross-section area.

28 RESULTS AND DISCUSSION I: The use of Satellite Remote Sensing technique has proved to be a useful tool for mapping and monitoring vegetation cover and distribution in a given area over time (Deysher, 1993; Call, et al., 2003). The technique is quickly replacing the use of conversional techniques such as aerial photographs and field mapping (Ferguson, et al., 1993; Robbins, 1997), which allow a very limited spatial coverage and could be very expensive in terms of equipment, time and personnel (Lillesand, et. al., 2004). The results from this study made in Chwaka bay supports the use of this technique for monitoring seasonal changes in distribution patterns and cover of SAV as indicated by the strong relationship between the Satellite- derived sun reflectance and the in situ quantified SAV percent cover. This suggests that frequently repeated satellite image acquisition in combination with regional covering is a great advantage compared to other mapping techniques. Although we found no significant seasonal variation in SAV during 2000/2001 field work (Fig. 6), but the loss and gain results obtained in our study from mapping (Fig. 7) were possibly attributed by environmental parameters such as fluctuations in temperature and salinity, and turbidity as has been reported from other studies (Robbins and Bell, 2000). It was further learnt that, such changes are mainly due to mechanical damage by the ongoing human activities in the area including seaweed farming, intensive fishing activities using modern techniques and shell collection during lowest spring tides of the day (Tobisson, et al., 1998). These activities might indirectly result in loss of seagrass habitat, leading to change into bare sediment in some areas (Fig.7, B and C) while in others into dense SAV (Fig. 7, A). Moreover, severe overgrazing of seagrass communities due to periods of extreme population densities of sea urchins may bring severe SAV loss in some place. Personal observation during field work we found aggregations of sea urchins in several spots within the bay, especially the sea urchin Tripneustes gratilla, known as an effective seagrass grazer (Alcoverro and Mariani, 2002). In addition, the interviews conducted between local residents and fishermen revelled similar observations. Despite the usefulness of Satellite Remote Sensing technique, its use in the aquatic environment is still at early stages of application (McKenzie, et al., 2001), and is limited to shallow and clear waters of tropical and temperate ecosystems (Dahdouh-Guebas, et al., 1999; Lundén and Gullström, 2003). This limitation of its use to shallow depth brings some difficulties in its application, and due to this problem we found difficulties during data analysis on discriminating among seagrass species and to separate seagrasses from macroalgae, which was possibly due to differences in water depth. In addition, the technique is only useful in areas of cloud-free, thus making difficult to be used in cloud-covered areas.

29 Anyhow, this study has proved that it is possible to monitor changes of seagrass and seaweed distribution in tropical environments using repeated mapping with satellite remote sensing. The spectral and spatial resolution acquired by the Landsat TM/ETM+ sensors was appropriate for this purpose. This type of satellite remote sensing data creates a basis for an operational and cost-effective monitoring method for conservation and restoration purposes.

Coverage

100

80 Bare sediment Other macroalgae 60 Halimeda spp. % Seagrass 40

0 Dec 2000 Mar 2001 Jun 2001 Sep 2001

Fig. 6: The mean percentage cover of seagrass, Halimeda spp., other macroalgae and bare sediment in Chwaka Bay during the survey period from December 2000 to September 2001.

Fig 7: Map from satellite image classifications showing the changes in SAV distribution between 1987 and 2003 in Chwaka Bay. The colours represent changed and unchanged areas: yellow = bare sediment to SAV; orange = SAV to bare sediment; dark green = unchanged SAV; brown = unchanged bare sediment. The letters in the map refer to the areas specifically described under results.

30 II: In paper II, four species were identified from the study area; H. opuntia (L.), H. macroloba (Decaisne), H. incrassata (Ellis) and H. tuna (Ellis and Solander). The species are common inhabitants of tropical and subtropical environments (Jaasund, 1976; Hillis- Colinvaux, 1980; Drew, 1983) and no new species were identified. Environmental parameters such as substrate type (sandy, soft boom, muddy or rocky), and high water motion had a major influence on their distribution and segment size. Muddy and soft bottom areas favoured high density of Halimeda with large segment size compared to rocky, high water motion and sandy areas (Muzuka, et al., 2001; pers obser). The relative growth rates obtained in this study was approximately 1 segment tip-1day-1. This is close to the other reported rates outside the Western Indian region (Bach, 1979; Drew, 1983; Garrigue, 1991; Ballestros, 1991), but comparisons are difficult because of the methods used. Water temperature was probably one of the environmental factors that influenced plant growth and development. This is shown by lower growth obtained during cold period assessment (July-August), where the mean growth trend shows a reduction in number of segments produced per tip per day towards the colder period (June-September of every year). Tissue nutrient content (C and N) showed variations between sites without a specified trend with high C/N ratios above the normal Redfield ratio for normal growing macrophytes, which indicate that Halimeda species in the area are nitrogen limited for growth and photosynthesis. Therefore, there is a possibility that nitrogen was among other factors responsible for the observed variations in growth of individuals. However, since there is no information available on C/N ratio of calcified algae, it is possible that this is a normal range for them due to high CaCO3 in their tissues. Salinity fluctuations and light possibly did not pose major influence on growth of Halimeda species due to high growth obtained during rain period at site I which was close to the shore and highly influenced by low salinities during rainfall period due to dilutions by the incoming freshwaters. The standing biomass of 1,563 g m-2 obtained in this study is possibly higher than the previous findings reported elsewhere from other world’s oceans. This is due to the presence of large meadows (beds) covered by Halimeda species over the entire bay (Muzuka, et al., 2001; Gullström, et al., in press) which lead to high calcium carbonate production results per square meter per day. From these findings, it is believed that the bay is annually receiving a high amount of carbonate materials from Halimeda species. However, despite higher CaCO3 production in the area, lack of data on sediment loss during ebbing represents a budget imbalance that needs to be fully addressed. There is a possibility that most of the CaCO3 produced is transported by outgoing tides across the “littoral fancy” to deep waters.

31 Decomposition experiment using litter bags indicates that Halimeda materials decompose faster in the beginning, but slowly in the end. To a larger extent this was due to the reason that, in the end there is only CaCO3 material left in Halimeda segments (tissues) which decompose more slowly (Fig. 8). The differences in number of days (103 and 76 for site I and II respectively) for 50% of materials in the litter bags to decompose into sediments, was possibly due ecological differences between the two sites. Site I remained submerged even during lowest spring tides of the day (~1.5 m deep at lowest tide), while site II became completely exposed to air for long time during lowest spring tides of the day. In addition, the observed large microbial populations comprising polychaetes, amphipods etc, at site II than at site I (pers obser), possibly enhanced the decomposition of Halimeda materials at this site.

1.1

0.9

Site I

Site II 0.8

R2 = 0.9885 0.7

R2 = 0.9147 remaining (g) Proportion AFDW 0.6

0.5 0 8 16 24 32 40 48 56 64

Time (days)

Fig. 8. Proportion of remaining AFDW of H. opuntia in the litter bags deployed from sites I and II as a function of time. Wo and k describes decrease in AFDW (n = 6) at site I and II during 56 days of deployment.

32 In situ calcification experiments at site I, II and III were comparable to the previous reported by Payri, (1988). The differences between sites were possibly caused by the extent of exposure to light between the three sites, which differed in depth and the extent of exposure full sunlight during lowest spring tides of the day. The dark treatment did not give significant results possibly due to two reasons; (1) dark caused the withdraw of the chloroplasts from the exterior of the thallus to the interior (Drew and Abel, 1990), resulting into reduced photosynthesis and calcification (2) there is a possibility that long periods of darkness resulted 2- into a drop in intercellular pH and a low concentration of CO3 , due to CO2 evolution during respiration.

III: In paper III, photosynthesis and calcification processes were found to be linked together as previously suggested by Borowitzka, (1977), and others (Pentecost, 1978; Smith and Roth, 1979; De Beer and Larkum, 2001). The effect of AZ on C-uptake shows the involvement extracellular carbonic anhydrase (CA) in the process of inorganic carbon uptake as previously reported (Borowitzka and Larkum, 1976b; Hellblom, et al., 2001; Uku, 2005). The strong effect of elevated pH in the media suggested that Halimeda calcification is primarily driven by an increase in pH at specific regions in the thallus as suggested by Borowitzka, (1977). The effect of DCMU as a PSII inhibitor agrees with earlier reported studies (For example, Borowitzka and Larkum, 1979d), and it strongly blocked ETR leading to inhibition of photosynthetic carbon uptake, and consequently decreases calcification rate (but not a complete inhibition). However, the effect of TRIS was somewhat un-expected, showing a strong inhibition of photosynthesis but not calcification. This suggests a structure with both high pH alkaline and low pH acid zones, involving proton pumps. The proton pumps from alkaline zones will favour calcification as suggested by Borowitzka (1977), while proton pumps from alkaline zones to acid zones will result into the built-up of protons causing low pH (which favours high CO2 concentration which diffuses into the chloroplast for photosynthesis). The presence of TRIS will compete for proton uptake from the acid zones causing a drop on photosynthesis, whereas the proton pumps from the alkaline zone will be maintained, favouring high calcification rate (Fig. 9)

33 100.00

80.00

60.00

% of control 40.00

20.00

0.00 CalcLgr GrossphotC- ETR Gross O2 prod uptakegraph

Figure 9: Remaining activity (as % of control) in H. macroloba after addition of TRIS buffer at pH 8.2.

IV: In paper IV, red algal rhodoliths were observed to maintain high productivity and calcification, even at combinations of both high temperature and light stress. Rates did not decrease up to 34oC, and even at 37oC rates were high. Inhibition was observed at high temperature above 37oC with increasing light, which suggests that algae are adapted to survive under high temperature and light conditions. At low light level (150 μmol photons m- 2s-1) the algae seemed to be protected from photo damage, and the reduced levels of Fv/Fm were possibly due to photo destruction caused by oxidative stress due to D1 destruction or other photo-destruction (Carr and Björk, unpublished). Thus, temperature tolerance seems to be higher in rhodoliths than corals which calcify at a narrow temperature range (Marschall and Clode, 2004). This conclude that the rhodoliths are better adapted to withstand temperature stress, and able to live in environments with fluctuating light and Temperature like the intertidal area of Chwaka bay where they become exposed to high light and higher temperatures for a long period during lowest spring tides of the day.

34 CONCLUSIONS AND FUTURE PERSPECTIVES The following conclusions were drawn from the data obtained in this study;

(1) The use of Satellite Remote Sensing technique in combination with ground-truthing data lead into mapping Chwaka bay, and generated important estimates on changes and percentage cover of submerged macrophytes including Halimeda species.

(2) The main Halimeda species in the area have been identified and described.

(3) There is a high standing biomass of Halimeda species in the area, high tissue C/N ratios and Halimeda can grow up to 1 segment per tip per day.

(4) A high amount of calcium carbonate production observed indicate that the bay is annually receiving high levels of carbonate sediments from decomposing Halimeda materials, as reflected in their high standing biomass.

(5) Decomposition rate was slow and it could take a longer time for a given amount of Halimeda materials to decompose into sediments.

(6) In situ calcification results were found to be comparable to the previous reports outside the WIO region, with minor differences.

(7) The effect of metabolic inhibitors such as AZ and DCMU or varying pH on inorganic carbon uptake by Halimeda species showed a strong inhibition on both photosynthesis and calcification and the two, photosynthesis and calcification are linked as previously reported. However, TRIS inhibited photosynthesis without affecting calcification, suggesting the involvement of proton pumps, a mechanism not considered by Borowitzka on the link between calcification and photosynthesis processes.

(8) High temperature and excess light treatments on coralline algae species (rhodoliths) suggest that the algae are tolerant to excess light and high temperature than coral reefs, and can maintain productivity up to 37oC. However, more information is needed in the following areas; (1) Since only four Halimeda species were identified and described, there is a possibility that some species are still unidentified from the area which needs further taxonomical work. (2) This study has documented high production of carbonate sediment materials influx in the bay. However, it is possible that most of them are exported to the main ocean waters by the outgoing tides through the littoral fancy during ebbing. Thus,

35 further investigations are needed to document the amount of materials exported to the main ocean with tidal currents. (3) Since the number of microorganisms was not quantified at the study sites during decomposition experiments in this study, there is a possibility that in some areas, the presence of high number of microorganisms facilitates decomposition of Halimeda materials to decompose faster than presented here. This is an important phenomenon for sediment generation in the area, and needs further investigation to address the phenomenon using long term experiments. (4) Although the spectral and spatial resolutions of the Landsat TM/ETM+ used in this study were appropriate, but the use of a High Resolution Multi-spectral Stereo Imager (HRMSI) in combination with other technical advancement could yield high quality data in the future. Nevertheless, it’s expected that, the information contained herein, will form a baseline for future Halimeda studies in the WIO region of East African.

ACKNOWLEDGEMENTS This thesis could have not come into completion without the help from different people: First and foremost, I would like to thank my supervisor, Professor Mats Björk for his tireless guidance, advice, patience and courage even when things looked worrying! You took me from far, since when you became a co-supervisor during my Masters Degrees studies at the University of Dar es Salaam, all the way to Stockholm for my PhD programme. You have been so kind beyond expectations, something I will never forget. Thank you for entrusting me in the project, giving me both great freedom and support when I asked. Secondly, I wish to thank Sida/SAREC for financial support during my studies from Masters Degree to PhD level. Thank you so much the Swedish government and the Swedish people (as the main tax payers) for your assistance to Tanzania as one among the developing countries in Africa. Your contribution to our country is highly appreciated. I am indebted to Professor Birgitta Bergman for her decision to allow me join the Plant Physiology section in Mats group. Your decision is highly appreciated, without which, probably I could have not joined the Botany Department. Thank you my co-supervisor Professor Lena Kautsky of the Department of Botany, for your good comments and advice during writing the manuscript. Dr. Mtolera of IMS, you are acknowledged for your favourable guidance, advice and courage especially during field work of this project. Apart from being a co-supervisor for the Tanzania side

36 (IMS/UDSM), you also acted as a guardian, giving me good advice on how to solve my family problems. I am grateful to Professor Phillip Bwathondi, Director General of Tanzania Fisheries Research Institute (TAFIRI) who is my current employer, for granting me a prolonged study leave from Masters Degree studies to PhD level. I acknowledge with thanks to Dr. Kjell Wannäs for introducing me to Professor Bengt Lundén who made me family with Remote Sensing and GIS techniques. I highly appreciate the efforts made by Professor Bengt to include me in the UN course for Remote Sensing and GIS for the year 2003, and continued to co-operate in research activities with me and my supervisor Professor Mats Björk. The current IMS director, Dr. Dubi and the previous, Dr. Julius Francis, are acknowledged for showing interest on me and continued to retain me at IMS from my Master’s degree studies to PhD. I appreciate your positive recommendations you made in favour of me, in efforts to bring me up. My close friend and fellow PhD student Alex Mamboya, thank you so much for your courage and co-operation during the PhD programme. You always made me feel at home during our long stay in Stockholm, sharing a lot of discussions, jokes and company. A big hug and appreciation to my fellow PhD students from Mats group; Frida Hellblom (The Thing), Herman Carr (Bwana Mkubwa), Jacqueline Uku and a visiting scientist Salamao Bandeira (Ero!) who stayed with us in Mats group at the Department of Botany for one year during 2004/2005 period as a visiting scientist. Thank you all for the good ideas, courage and jokes during boring hours. Thank you Dimitra for your attention and act when asked for help including going to the West Coast of Sweden to collect sea water for my experiments. The current and previous PhD students at the Botany Department, few to mention; Pelle (Hur är läget?), Martin, Johan Klint, Mercedes, Anders (Ecology section), Karolina, Liang, Alphonso, Sara, Mallena, Pernilla, Jenny, Marcus Klenell, Lotta, Mathias Öster, Behnoosh and others. Thank you all for your help of all kinds you offered to me, nice talks, company and help when requested. Other staff members (technicians, librarians, secretaries…… etc) are acknowledged for their positive co-operation. To my fellow students at IMS and University of Dar es Salaam main campus, thank you for your valuable support in different aspects, co-operation and for sharing ideas, sometime jokes and company. I wish you good luck in your studies wherever you are registered. IMS staffs are acknowledged for their co-operation and help of different kinds they offered to me during my studies. I really enjoyed living with you for a long time, and I promise to remember you all wherever I go. Specials regards goes to my “permanent technician” Muhidin Abdallah for your patience and tireless help during my long field days in Chwaka

37 bay. You really helped me a lot, and I appreciate your contribution to my PhD degree programme. Juma Nene and Mweleza thank you so much for your help in field work. Lastly, to my wife Mariana Kangwe; thank you Mama for your extreme tolerance to family problems during my absence and your strong support and courage on my Masters and PhD studies. For many times I was away to Sweden for too long leaving you alone with the kids in Zanzibar, but you managed to keep them healthier and joyful even in my absence. Thank you so much Mama and god bless you.

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Doctoral Thesis Department of Botany Stockholm University