Seagrasses and Eutrophication

Seagrasses and eutrophication

Interactions between seagrass photosynthesis, epiphytes, macroalgae and mussels

Esther Francis Mvungi ©Esther F. Mvungi, Stockholm 2011

Front cover: Photo showing eutrophic seagrass bed in the Western Indian Ocean. Photo: Mats Björk

ISBN 978-91-7447-250-9

Printed in Sweden by Universitetservice, US-AB, Stockholm 2011 Distributor: Department of Botany, Stockholm University

To my husband, Denis and my daughter, Doreen

Abstract

Seagrass meadows are highly productive, ecologically and economically valuable ecosystems. However, increased human activities along the coastal areas leading to processes such as eutrophication have resulted in the rapid loss and deterioration of seagrass ecosystems worldwide. This thesis focuses on the responses of seagrasses to increases in nutrients, subsequent increases in ephemeral algae, and changes in the physical- chemical properties of seawater induced by interaction with other marine biota. Both in situ and laboratory experiments conducted on the tropical seagrasses Cymodocea serrulata and Thalassia hemprichii revealed that increased concentrations of water column nutrients negatively affected seagrass photosynthesis by stimulating the growth of the epiphytic biomass on the seagrass leaves. Interaction between seagrasses and other marine organisms induced different responses in seagrass photosynthesis. Ulva intestinalis negatively affected the photosynthetic performance of the temperate seagrass Zostera marina both by reducing the light and by increasing the pH of the surrounding water. On the other hand, the coexistence of mussels Pinna muricata and seagrass Thalassia hemprichii enhanced the photosynthetic activity of the seagrass, but no effect on the mussels’ calcification was recorded. This study demonstrates that seagrass productivity is affected by a multitude of indirect effects induced by nutrient over-enrichment, which act singly or in concert with each other. Understanding the responsive mechanisms involved is imperative to safeguard the ecosystem by providing knowledge and proposing measures to halt nutrient loading and to predict the future performance of seagrasses in response to increasing natural and human perturbations.

Keywords: CO2, epiphytes, eutrophication, mussels, pH, photosynthetic activities, seagrasses, Ulva

List of Papers

This thesis is based on the following four papers, which will be referred to in the text by their Roman numerals.

I. Mvungi EF, Lyimo TJ and Björk M (2011). When Zostera marina is intermixed with Ulva, its photosynthesis is reduced by increased pH and lower light, but not by changes in light quality (Submitted).

II. Hamisi MI, Mvungi EF, Lyimo TJ, Mamboya FA, Österlund K, Björk M, Bergman B and Diez B (2011). Nutrient enrichment affects the seagrass Cymodocea serrulata and induces changes to its epiphytic cyanobacterial community (Manuscript).

III. Mvungi EF and Mamboya FA (2011). Photosynthetic performance, epiphyte biomass, and nutrient content of two seagrass species in areas with different levels of nutrient along the Dar es Salaam coast (Submitted).

IV. Semesi IS, Mvungi EF, Beer S, Jidawi N and Björk M (2011). Interactions between seagrasses and mussels: CO2, pH and calcification (Submitted).

My contributions were: for papers I and III planning, performing all experiments, executing fieldwork, analysing data, and writing the manuscripts; for paper II planning, performing experiments (except the morphological and molecular characterization of epiphytic cyanobacteria), conducting part of the data analysis, and writing part of the manuscript in cooperation with the first co-author; and for paper IV planning, executing fieldwork, analysing data, and writing the manuscript in cooperation with the first co-author.

Table of Contents

Abstract ...... iv List of Papers ...... v Table of Contents ...... vi Abbreviations ...... vii 1. Introduction ...... 8 1.1 General overview of seagrasses ...... 8 1.2 Nutrient fluxes in marine environments ...... 9 1.3 Photosynthesis in seagrass meadows ...... 10 1.4 Chlorophyll a fluorescence ...... 11 1.5 Inorganic carbon flux in marine environments ...... 12 1.6 Seagrasses interact with other marine organisms ...... 14 2. Aim ...... 16 3. Comments on the methods used ...... 17 3.1 Species used ...... 17 3.2 Experimental setups ...... 19 3.3 Chlorophyll fluorescence measurements ...... 20 3.4 Nutrient content (CNP) analysis ...... 21 3.5 Physical-chemical characteristics of seawater ...... 21 4. Results and Discussion ...... 22 4.1 Effects of water column nutrient gradients ...... 22 4.1.1 Effects on seagrass photosynthesis ...... 23 4.1.2 Effects on epiphyte biomass and composition ...... 26 4.1.3 Effects on nutrient contents of plant tissues ...... 26 4.2 Ulva overgrowth affects seagrass photosynthesis ...... 28 4.3 Mussels enhance seagrass photosynthesis ...... 30 5. Conclusion ...... 32 Acknowledgements ...... 34 References ...... 36

Abbreviations

AF Absorption factor C:N:P ratio Carbon:nitrogen:phosphorus ratio Ci Inorganic carbon ETR Electron transport rate Fv/Fm Maximum quantum yield Ik Minimum saturation irradiance PAM Pulse amplitude modulation/modulated PAR Photosynthetically active radiation PSI Photosystem one PSII Photosystem two rETR Relative electron transport rate RLC Rapid light curve TA Total alkalinity

1. Introduction

1.1 General overview of seagrasses

Seagrasses are defined as flowering plants (angiosperms) living their full lifecycle submersed in marine environments. They are found in all coastal areas of the world except along the Antarctic shores (Green and Short 2003, Duarte and Gattuso 2010, Fig. 1). They are descendants of terrestrial plants that re-entered the ocean about 100 million years ago (Duarte and Gattuso 2010). Thus, unlike other marine vegetation, they maintained both their vascular system (Kuo and den Hartog 2006) and their ability to produce flowers, fruits, and seeds (Ackerman 2006). They comprise less than 0.02 % of the angiosperm species, and have relatively fewer species than other marine organisms (Short et al. 2007).

Despite their low diversity, seagrass beds are ecologically and economically highly important. They are among the world’s most productive coastal ecosystems (Duarte and Chiscano 1999) and support a high diversity of autotrophic and heterotrophic organisms. They serve as habitats for a broad diversity of invertebrate and fish species, many of which are of commercial and recreational importance, as food source for marine herbivores (Heck and Valentine 2006), they stimulate nutrient recycling and buffer wave action, hence minimizing coastal erosion (Heiss et al. 2000). They also serve as substrata for a diverse array of epiphytic organisms that contribute to the productivity of the seagrass beds in different ways (Borowitzka et al. 2006). Seagrasses are not only supportive to marine biota, but also to human populations, especially in the tropical regions (in particular the Western Indian Ocean region) where people along the coasts depend largely on the seagrass ecosystem for their daily subsistence in terms of food and per capita income (de la Torre-Castro and Rönnbäck 2004).

Regardless of their biological and economical importance, reports on seagrass decline have increased in numbers in recent years and the major cause attributed is the increase in human-related activities along the coastal zone (Short and Wyllie-Echeverria 1996, Orth et al. 2006, Waycott et al. 2009). Nutrient over-enrichment (eutrophication) from various sources, such as domestic waste, industrial discharge, agriculture, and urbanization of coastal zones (Moore and Wetzel 2000, Burkholder et al. 2007, Duarte et al. 2008), concomitant with global climate change (Short and Neckles 1999), pose serious threats to seagrass ecosystems worldwide. 8

Fig. 1. Global seagrass distribution (shown in blue) and their geographic bioregions: 1. Temperate North Atlantic, 2. Tropical Atlantic, 3. Mediterranean, 4. Temperate North Pacific, 5. Tropical Indo-Pacific, 6. Temperate Southern Oceans. The figure is reproduced from Short et al. (2007), with the kind permission from the publisher, Elsevier. Copyright © (2007), Elsevier.

1.2 Nutrient fluxes in marine environments

Seagrasses as any other plants require nutrients for their growth, development, and metabolism, but too much of some nutrients may limit rather than promote growth. The primary and most common nutrients that limit seagrass growth in natural systems are nitrogen and phosphorus (Duarte 1990, Romero et al. 2006). These essential nutrients may be derived from the decomposition of organic matter in the water column and sediments (Holmer and Olsen 2002, Kilminster et al. 2006), in which seagrass litters are the main source (Holmer and Olsen 2002, Romero et al. 2006). However, in tropical areas, bacterial nitrogen fixation may also provide substantial amount of nitrogen in the seagrass meadows (Moriarty and O’Donohue 1993, Hamisi et al. 2009). Unlike terrestrial plants, seagrass can absorb nutrients through both leaves and roots (Touchette and Burkholder 2000a, Gras et al. 2003, Nielsen et al. 2006).

Seagrasses can be adversely affected by eutrophication of the water in different ways. Increased nutrient levels in the water column promote proliferation of phytoplanktonic, epiphytic, and bloom-forming macroalgal species (Short et al. 1995, Borum and Sand-Jensen 1996, Moore and Watzel

2000, McGlathery 2001). Blooms formed lead to reduction in the light available to seagrasses (Brun et al. 2003, Liu et al. 2005, Lamonte and Dunton 2006), and alteration of the surrounding water chemistry (Björk et al. 2004, Beer et al. 2006). Thus, nutrient over-enrichment caused by anthropogenic activities has been considered to be a major cause of seagrass decline worldwide (Green and Short 2003, Orth et al. 2006). Many studies have been carried out to investigate the effects of increased nutrient levels on seagrass performance (e.g. Short et al. 1990, Moore and Wetzel 2000, Touchette and Burkholder 2000a; 2000b, Romero et al. 2006, Burkholder et al. 2007, Lee et al. 2007) but still not conclusive.

When the nutrient is absorbed into the cell, it can either be assimilated immediately into organic matter or stored in inorganic form in the plant tissues for future use. Increased nutrient availability has been shown to correlate with increased nutrient acquisition and assimilation in seagrasses of both N and P, which is reflected by the increased levels of N and P content in the plant tissues (Duarte 1990, Bulthuis et al. 1992, Peréz-Lloréns and Niell 1995, Borum and Sand-Jensen 1996, Pedersen et al. 1997, Alcoverro et al. 1997, Udy and Dennison 1997, Johnson et al. 2006). Therefore, analysis of tissue nutrient content is thought to be a more reliable way of determining fluxes in nutrient availability in the ecosystem than water nutrient analysis, as the latter is influenced by short-lived temporal fluctuations which are difficult to trace (Sjöö and Mönk 2009, Lourenço et al. 2006).

1.3 Photosynthesis in seagrass meadows

Oxygenic photosynthesis is responsible for all biochemical production of organic matter in marine and terrestrial ecosystems, and about 45% of the photosynthesis on the earth occurs in aquatic environments (Field et al. 1998). The photosynthetic reaction in plants starts with absorption of light by chlorophylls, which are composed of large protein complexes with light harvesting antennae where light energy is converted into chemical energy and charge separations of electrons take place. The electron is then transported through and along the thylakoid membrane via series of electron carriers to the photosystem I (PSI), where trans-membrane proton gradient and reduction of NADP+ to NADPH occurs and ATP is formed via proton gradient. The electrons are resupplied from the water-splitting complex in photosystem II (PSII), in which water molecule is cleaved, yielding 4 electrons and 4 protons for each molecule of O2 generated. The NADPH and ATP formed through these processes are then used in a Calvin cycle where CO2 is reduced into sugars.

In the seagrass beds, however, light required for photosynthesis must penetrate the water column. With increasing water depth, light becomes more and more attenuated and scattered due to dissolved substances, phytoplankton, algal blooms, and physical properties of water itself. Consequently, light is also one of the limiting factors for seagrass productivity (Hemminga and Duarte 2000).

1.4 Chlorophyll a fluorescence

When light energy is absorbed by PSII, it can be used through one of three directly competing pathways: in photochemistry, as heat dissipation or can be re-emitted back as fluorescence (Krause and Weiss 1991). When absorbing a photon, the chlorophyll molecule becomes excited and moves to a higher energy level. If the energy of the excited electron is not utilized in photochemistry (or heat dissipation), the electron falls back to the ground state and the energy will be emitted as fluorescence (Krause and Weiss 1991).

Chlorophyll fluorescence techniques are a convenient and non-destructive way to assess processes of photosynthesis, both in situ and in the laboratory, and have been used extensively in recent years (Krause and Weiss 1991, Maxwell and Johnson 2000, Schreiber 2004). When a leaf is exposed to a saturating irradiance the fluorescence yield initially increases rapidly to a peak and then slowly declines. This fluctuation in the fluorescence signal is known as the “Kautsky” curve (Kaustsky et al 1960, Fig. 2), from which dynamic changes in photosynthesis can be estimated.

When a plant sample is dark-adapted for sufficient time (ca.10–15 minutes), all its PSII reaction centres open, providing the maximum quantum yield of PSII (Maxwell and Johnson 2000), estimated as: Fv/Fm = (Fm−Fo)/Fm where Fv is the variable fluorescence, Fm is the maximum fluorescence following a saturating light pulse, and Fo is the minimum fluorescence (Fig. 2). The Fv/Fm ratio has been used as a simple and rapid measure of stress status of the plants (Krause and Weiss 1991, Baker 2008).

When a sample is light-adapted, its reaction centres are closed, and it provides the proportion of absorbed energy that being used for photochemistry (Genty et al. 1989, Baker 2008), and is calculated as ФPSII (Y) = (Fm′ – F′) / Fm′, where F′ and Fm′ are the corresponding light- acclimated minimum and maximum fluorescence (Fig. 2). The electron transport rate (ETR) can be obtained from the ФPSII value as follows: ETR = Y * PAR * 0.5 * AF; where Y is the effective quantum yield, PAR is the irradiance at the leaf surface, 0.5 is a factor that accounts for partitioning of 11 absorbed photons between PSII and PSI, and AF is the proportion of incident light that is absorbed by photosynthetic pigments (Beer et al. 2001).

Photosynthesis has long been measured using gas exchange techniques, which are destructive because they require the removal of the plant from its natural environment. However, the development of underwater pulse amplitude modulated (PAM) fluorometer made it possible to conduct in situ photosynthetic measurements in the ambient environments (Schwarz et al. 2000). Because PAM has the advantages of being non-intrusive, fast, reliable, and able to provide real-time data, it has become very popular.

Fig. 2. A typical fluorescence curve for a seagrass leaf measured with pulse amplitude modulated (PAM) fluorometer (Walz, Germany) showing light and dark- adapted measurement of quantum yield. Fv is variable fluorescence, F0 is the minimum fluorescence, Fm is maximum fluorescence in dark adapted state, Fm’ is maximum fluorescence in the light and Ft is the steady state value of fluorescence. ML is measuring light, SP is saturating light purses and AL is the actinic light which drives the photosynthesis. The figure is modified from Larkum et al. (2006).

1.5 Inorganic carbon flux in marine environments

Unlike in terrestrial photosynthesis, where plants utilise gaseous CO2 from the atmosphere, in the marine environment, upon entering the aqueous environment, atmospheric CO2 reacts with water leading to the formation of

12 ionic forms of carbon (carbonic acid, bicarbonate, and carbonate) in equilibrium with CO2, as described by Falkowski and Raven (2007):

+ - + 2- H2O + CO2 ↔ H2CO3 ↔ H + HCO3 ↔ 2H + CO3

However, depending on pH changes in the seawater, the equilibrium reaction can shift, with high concentrations of CO2 at lower pH and relatively more bicarbonate and carbonate at higher pH. At pH 8.2, salinity 35, and water temperature 25oC, more than 95% of inorganic carbon present is in the form of bicarbonate (Falkowski and Raven 2007).

Unlike in the terrestrial ecosystem, in marine photosynthesis CO2 can often be a limiting factor due to its low solubility and slow diffusion rates (Björk et al. 1997, Beer et al. 2002). Some species have developed mechanisms that enable them to use the readily available bicarbonate (Beer and Rehnberg 1997, Invers et al. 2001). One example is the use of extracellular carbonic - - anhydrase (CA), which catalyses the conversion of HCO3 to CO2 and OH . This can be further accelerated by the use of proton pump-mediated acid - zones and by active uptake of HCO3 (possibly by an AE protein, Drechsler et al. 1993). Although most marine macrophytes have been reported to be − able to use both CO2 and HCO3 for photosynthetic carbon fixation (Beer and Rehnberg 1997, Invers et al. 2001), the use of bicarbonate in some seagrass species is still under some dispute and is thought to be less efficient as compared to the macroalgae (Beer and Rehnberg 1997, Björk et al. 1997). Seagrass photosynthesis has been shown to be carbon-limited for both temperate and tropical species (Björk et al. 1997, Invers et al. 1997).

Effect of increasing global CO2 on marine biota

The concentration of atmospheric CO2 has increased in the past 200 years due to anthropogenic activities, and almost one third of it goes into the ocean (Sabine et al. 2004). There it causes ocean acidification, in which the pH and concentration of carbonate ions decrease while concentrations of hydrogen and bicarbonates increase. These chemical changes brought about by the dissolution of CO2 are expected to affect many biological processes in marine organisms (Fabry et al. 2008). Despite the fact that these changes are predicted to continue rapidly as the oceans take in more CO2 from the atmosphere, little yet is known about how marine organisms will cope with such changes at individual or ecosystem levels.

Several studies have been carried out focusing on the processes likely to be affected by ocean acidification. For instance, marine calcifying organisms have been shown to be negatively affected by the increase in CO2 concentration through the marked decrease in rates of calcification (e.g. Fabry et al. 2008, Ries et al. 2009). Unlike calcifying organisms, marine 13 photosynthetic organisms have been shown to have enhanced photosynthetic activities in response to higher levels of CO2 (Palacios and Zimmerman 2007, Riebesell et al. 2000, Schneider and Erez 2006). However, this observation is based on only a few studies on a few species, hence more investigations at individual and ecosystem levels are called for.

1.6 Seagrasses interact with other marine organisms

Seagrass–mussel interactions

Seagrass ecosystem forms a highly complex habitat that harbours more faunal assemblages than unvegetated habitats (Duarte and Gattuso 2010). Mussels, for example, are a conspicuous feature of intertidal habitats on both soft and hard substrata in many coastal areas. Through their filter-feeding activity, mussels may be capable of lessening both light and nutrient limitation in the seagrass meadows (Newell 2004). The presence of filter- feeding mussels can significantly reduce suspended algal biomass and inorganic particles, thereby increasing light availability and penetration through the water column, and subsequently increasing the growth rates of seagrasses (Wall et al. 2008). They also link water column productivity to the benthos by removing pelagic organisms, increase deposition of solid and faecal materials, and therefore promote a flux of organic matter and nutrient recycling from the water column into the sediments, thus stimulating the uptake and growth of seagrasses (Kotta and Møhlenberg 2002, Lauringson et al. 2007, Peterson and Heck 1999, 2001a & 2001b).

Co-habitation of mussels and seagrasses is commonly seen in the coastal areas of Tanzania; however knowledge about their interactions is generally scarce. Thus, paper IV was designed to investigate the potential influence that the dissolved CO2 released during the respiration of mussels (Pinna muricata) might have on seagrass photosynthesis.

Seagrass–epiphytes and –macroalgae interactions

Seagrasses grow in a variety of sediment types in shallow water and usually provide the solid substrata for the attachment of a diversity of epiphytic organisms. Most seagrass epiphytes are primary producers and hence an important component of seagrass ecosystems. They account for up to 30% of the ecosystem’s productivity and the epiphytic algae contribute more than 30% of the total above-ground biomass in many seagrass ecosystems (Borowitzka et al. 2006). Epiphytes constitute a valuable food source for herbivores as they can be consumed selectively, accidentally, or through detritus food webs (Borowitzka et al. 2006). Higher levels of N2-fixation in 14 the seagrass ecosystems have been linked to bacteria and cyanobacteria, both in the rhizosphere and the phyllosphere (Welsh et al. 2000, Hamisi et al. 2009), of which epiphytic cyanobacteria have been estimated to supply up to 38% of the nitrogen needed for primary production in some seagrass beds (Welsh et al. 1996).

Calcareous algae on the other hand, are also common epiphytes of seagrasses and contribute to the production of calcareous sediments (Chisholm 2000). It has been shown that seagrass photosynthesis influences calcification rates in coralline algae (Semesi et al. 2009), thus it was interesting to explore whether the same effect occurred on mussels calcification when grow intermixed with seagrasses (paper IV).

Despite their important beneficial roles in seagrass ecosystems, epiphytes can also have detrimental effects on seagrasses. Epiphyte loads reduce the productivity of the seagrass by shading, competing for nutrient availability, and creating physical barriers around the leaf surface, which may impede light absorption (Romero et al. 2006). Similarly, in eutrophic environments, green macroalgae frequently occur in masses, forming dense canopies or intermixed with seagrasses, reducing the amount of light reaching the seagrasses beneath (Brun et al. 2003, McGlathery 2001). They also tend to alter the light spectrum as they absorb mainly in the blue and red regions, meaning that the light transmitted through the algal mats is mainly in the green spectrum (Vergara et al. 1997), which is regarded to be less effective for photosynthesis (e.g. Mazzella and Alberte 1986). Based on this, paper I investigated how photosynthetic activity in the temperate seagrass Zostera marina is affected by changes both in water chemistry and in light quality due to shading attributable to the green macroalgae Ulva intestinalis. Paper III addresses the photosynthetic activities of two seagrasses in relation to epiphytic biomass in two areas with different levels of nutrients.

2. Aim

The major aim of this thesis was to achieve a better understanding of the response of seagrass species to elevated levels of nutrients in the water column and the possible influences of interactions between seagrasses and other organisms in the seagrass beds. To achieve this, in situ and laboratory experiments were designed and conducted with the following specific objectives:

i. To evaluate the effect of increased nutrient concentrations in the water column and the associated effects of increased epiphytic loads on seagrass performance (papers I, II, and III).

ii. To investigate effects on seagrass photosynthesis by interactions between seagrasses and other flora and fauna (papers I and IV).

3. Comments on the methods used

This thesis is based on experiments conducted both in situ and in the laboratory. This section summarises the methodology used in the different experiments (refer to papers I–IV for detailed descriptions).

3.1 Species used

Zostera marina (Eelgrass) is the dominant seagrass species in the temperate coastal waters of both the Pacific and the Atlantic oceans (Fig. 1). Eelgrass is very abundant in the Baltic sea and can colonize depths up to 30 m in clear waters, but is typically restricted to shallows or the intertidal depths of estuaries (Borum and Greve, 2004). It grows on a wide range of soft and mixed sediments. (Photo: Fort Wetherill)

Cymodocea serrulata belongs to the family Potamogetonaceae, commonly found along the coasts of the tropical Indo-West Pacific region (Fig. 1). It is one of the twelve species in the Western Indian Ocean. C. serrulata can be distinguished from other seagrass species by their shoots with distinctive open leaf scars, triangular, flat leaf sheath, fibrous roots on the shoot, and serrated leaf tips (Oliviera et al. 2005, Photo: Katrin Osterlund).

Thalassia hemprichii is a member of the family Hydrocharitaceae, widely distributed in Indo-Pacific regions (Fig. 1). It is a common species in many coastal areas of the Western Indian Ocean, growing within soft substrates in the intertidal and subtidal areas. The distinctive features of T. hemprichii is the presence of short black bars of tannin cells on the leaf blade running parallel to the long axis of the leaf and the thick rhizome with conspicuous scars between successive erect shoots (Oliviera et al. 2005, photo: Katrin Osterlund).

Ulva intestinalis is a cosmopolitan green macroalga species frequently found in many habitats along the coastal zones of most oceans. In favourable conditions U. intestinalis can quickly occupy any empty space in the uppermost littoral zone (Martins and Marques 2002, photo: Satogawa-cho).

Pinna muricata: is a bivalve species in the family Pinnidae, which occurs intermixed in the seagrass beds and in unvegetated areas in the Indo-Pacific region. P. muricata in the Western Indian Ocean region live buried in sandy

18 muddy substrates in subtidal and shallow areas covered by different seagrass species including Thalassia hemprichii (Richmond 2002, photo: E. Mvungi).

3.2 Experimental setups

Experiments to investigate the combined effects of Ulva-induced changes in light quality and changes in water chemistry on the photosynthetic response of Zostera marina were conducted in the laboratory, at the Botany Department of Stockholm University. Shoots of Zostera marina were collected from west coast of Sweden. At the laboratory, the photosynthetic characteristics of the seagrass and the changes in water chemistry were assessed under normal light and under Ulva-filtered light (paper I).

To investigate the effects of increased nutrient levels on performance of the seagrass Cymodocea serrulata and the associated diazotrophic community, an outdoor laboratory experiment was set at the Institute of Marine Sciences (IMS), Zanzibar, Tanzania (paper II). In this setting, seagrass samples were collected from the Chapwani seagrass bed in the Indian Ocean, about 2.5 km from IMS and transported to the laboratory, where they were transplanted to buckets containing sediments in a flow-through water system. Seagrass growth, photosynthetic characteristics, and epiphytic characterization were evaluated during the experiments.

Paper III was designed to assess the response of seagrasses in their natural environments to varying nutrient levels in relation to epiphytic loads. A field experiment was conducted during low tides in the intertidal seagrass beds at Mjimwema and Ocean Road along the Dar es Salaam coast. Ocean Road is located at 6o49' S, 39o19' E. The area is considered to be a nutrient-rich site as it receives untreated domestic sewage through the pipes that drain the city centre. Mjimwema is located on the same coast about 4 km south of Ocean Road at 6o50' S, 39o21' E. This area is considered to be nutrient poor and less impacted by anthropogenic disturbances from the coastal population. These sites were selected based on their contrasting nutrient status (paper III). 19

Seagrass interaction with other marine animals is common in many parts of the world; however the nature of these interactions has not been fully explored, in particular in the Western Indian Ocean region. Thus, to investigate the potential influence of the mussels Pinna muricata on the photosynthesis of the seagrass Thalassia hemprichii, and consequently on the water chemistry, a field experiment was set at the Fumba Bay seagrass beds (paper IV). Fumba Bay is located on the south-west of Unguja Island, Zanzibar, Tanzania (06o 19´S, 39o 17´ E). The bay is characterized by shallow waters, and during low tides it has large exposed intertidal flats, dominated by large monospecific stands of Thalassia hemprichii and mixed patches of other seagrass species.

3.3 Chlorophyll fluorescence measurements

In all experiments (papers I–IV) chlorophyll a fluorescence was measured with a diving pulse amplitude modulated (PAM) fluorometer (Walz, Germany). Photosynthetic activities of seagrass were measured in terms of rapid light curve (RLC), maximum quantum yield of PSII (Fv/Fm), and effective quantum yield at different parts of the leaves depending on the study question (see papers I–IV). Prior to any chlorophyll measurements, the PAM flourometer’s light sensor was calibrated against a light meter (IL1400A, International Light Technologies).

Rapid light curves were generated automatically by the diving PAM in a pre- programmed incremental sequence of actinic illumination periods, with light intensities increasing in 8 steps of photon m-2 s-1 according to the methods described by Ralph and Gademann (2005). The maximum rate of electron transport (ETRmax) and initial slope (α) were calculated by fitting the RLC data to an exponential function described by Jassby and Platt (1976). The onset of light saturation (Ik) was calculated by dividing the ETRmax by initial slope (papers I, II, and III).

The Fv/Fm was determined by dark-adapting the seagrass leaves for 10 min using the dark leaf clip (Walz, Germany) and saturating pulses of light from the diving PAM (papers II and III). The effective quantum yield (Y) was determined under ambient light conditions by the saturating-light method (Beer and Björk 2000). The value of Y was then used to estimate the electron transport rate (ETR) through photosystems II and I (paper IV). The use of PAM reflects photosynthetic rates in the light reaction only, but it correlates well with oxygen evolution technique (Beer and Björk 2000, Silva and Santos 2004). Thus, it permits estimation of primary productivity in the field, on intact plants, which is very important. 20

3.4 Nutrient content (CNP) analysis

Samples used for elemental content analysis were dried at 60oC and ground to fine powder (papers II and III). Carbon and nitrogen (CN) contents of seagrass tissues (and epiphytes in paper I) were analysed using a CHN analyser (CHNS 932) and phosphorus (P) content was determined by dry- oxidation acid hydrolysis extraction followed by colourimetric analysis (Fourqurean et al. 1992). The carbon, nitrogen, and phosphorus contents are given in percentages of dry weight, and ratios are mol:mol.

3.5 Physical-chemical characteristics of seawater

In both in situ and laboratory experiments (papers I and IV), pH measurements were performed with a Multi 340i pH meter (WTW, Germany). Concentrations of total inorganic carbon and total alkalinity (TA) in the seawater were determined following the procedures of Anderson and Robinson (1946): whereby 4 ml of water sample was taken, the pH was measured before addition HCL, then 1 ml of 0.01 M HCL was added and the pH was recorded again. The pH values obtained before and after addition of HCL were used to calculate TA and total carbon using the formula and constants of Riley and Skirrow (1965) and Smith and Kinsey (1978).

The calcification rates of mussels were estimated using alkalinity anomaly technique (Smith and Key 1975) by dividing changes in total alkalinity (TA) by 2. Rapid titration method is not as precise as an end point titration, but: it is easy, possible to perform in the field and good enough for our purposes.

For analyses of nutrient concentrations in the water, samples were collected from the experimental buckets (paper II) and from the field (paper III). The concentrations of nitrate, nitrite, and phosphates were determined following the procedures described by Parsons et al. (1989). Samples not immediately analysed were stored at −20oC.

4. Results and Discussion

Tropical marine environments are characterized by low nutrient levels (Hemminga and Duarte 2000), however, with the increasing rate of human- related activities in coastal areas, the rate of nutrient loading in marine environments is increasing, in many parts of the world. Nutrient over- enrichment stimulates the overgrowth of macroalgae and epiphytic loads on seagrass leaves, both of which have been found here and in previous studies (e.g. Short et al. 1995, McGlathery 2001) to affect the performance of seagrasses.

4.1 Effects of water column nutrient gradients

The results obtained in the studies reported here showed that responses of seagrasses to nutrient gradients vary, both in the laboratory and in the field. The nutrient enrichment experiments revealed no significant difference in growth characteristics between of seagrasses growing in different nutrient levels. However, the biomass of the seagrass decreased when exposed to the higher nutrient level for an extended time (paper II). Furthermore, in the natural environment the studied seagrasses showed different growth characteristics in the two sites with different levels of nutrient (Table 1). While Cymodocea serrulata showed significantly higher shoot densities and canopy height at the nutrient-poor site than at the nutrient-rich site, shoot density for Thalassia hemprichii did not vary significantly between the two sites, but its canopy height was significantly higher at the nutrient-rich site than at the nutrient-poor site (Table 1).

The two species varied in their biomass partitioning in the two sites. Species at the nutrient-poor site showed significantly higher allocation of biomass in the below ground than above ground tissues, while at the nutrient-rich site, the partitioning was more or less the same for above and below ground tissues. Thus, the above- to below-ground biomass ratio for both species was lower at the nutrient-poor site than at the nutrient-rich site, reflecting a strategy to cope in low nutrient availability by expansion of the surface area for nutrients uptake (paper III).

Table 1. Characteristics of the seagrasses Thalassia hemprichii and Cymodocea serrulata in areas with different levels of anthropogenic nutrient inputs. Data are means ± Standard deviation).

Shoot density Canopy Above ground Below ground Above to below- Dry weight of Site Species (no. shoots m-2) height (cm) biomass (gDWm-2) biomass (gDWm-2) ground (AG/BG) epiphytes (gg-1) biomass ratios

Mjimwema T. hemprichii 1038.1 ± 288.4 14.8 ± 2.1 267.0 ± 43.8 1177.4 ± 265.2 0.23 ± 0.03 0.08 ± 0.01 C. serrulata 1622.7 ± 304.5 16.7 ± 1.8 352.2 ± 144.7 737.2 ± 260.8 0.51 ± 0.21 0.07 ± 0.04

Ocean Road T. hemprichii 792.0 ± 84.1 17.6 ± 2.7 307.0 ± 74.9 412.1 ± 93.3 0.77 ± 0.15 0.25 ± 0.04 C. serrulata 819.8 ± 203.8 14.1 ± 2.0 202.7 ± 69.6 267.7 ± 147.9 0.81 ± 0.13 0.34 ± 0.29

4.1.1 Effects on seagrass photosynthesis

Photosynthetic performance of the seagrass Cymodocea serrulata was negatively affected by prolonged exposure to increased level of nutrients in the water column, as revealed by the reduction of maximum quantum yield of PSII during long-term enrichment (Fig. 3, paper II). This could probably be associated with the development of nitrate toxicity (Burkholder et al. 1992), which led to a decrease in photosynthetic capacity. The levels of nutrient additions used in this study resulted in water concentrations that were within the range of those previously reported for islands about 3 to 5 km offshore in the area where seagrass were collected (Björk et al. 1995, Mohammed and Mgaya 2001) and likely to be found in places influenced by untreated sewage.

1.0 1.0 A B

0.8 0.8

0.6 0.6

Fv/Fm 0.4 0.4 0:0 µM 0:0 µM 1:0.1 µM 1:0.1 µM 0.2 5:0.5 µM 0.2 10:1 µM 25:2.5 µM 100:10 µM 125:12.5 µM 1000:100 µM 0.0 0.0 Day 0 Day 4 Day 7 Day 11 Day 15 Day 21 Day 0 Day 23 Day 52 Day 95 Measurement days Measurement days

Fig. 3. Maximum quantum yield (Fv/Fm) of Cymodocea serrulata in respect to nutrient enrichment (combined N and P). A and B = short term (28 days) and long term (95 days) experiments. Error bars are means ± SE, n = 4.

The impact of epiphytic biomass on the photosynthetic characteristics of the seagrasses Thalassia hemprichii and Cymodocea serrulata was studied in two intertidal areas with different nutrient levels (paper III). Generally, the photosynthetic characteristics of these species were higher in leaves devoid of epiphytes (younger leaves) than in leaves under the epiphytes (Fig. 4). This may indicate the suppressive effect of epiphytic mass on seagrass leaves, which is also supported by the lower maximum quantum yield (Fv/Fm) observed in leaves under the epiphytes. On the other hand, it could also be that these leaves are shade-adapted and thus seagrasses are protected from strong sunlight.

Of the two species, Thalassia hemprichii showed higher photosynthetic rates than C. serrulata at both sites (Fig. 4). This could probably be explained by their different morphological aspects (e.g. leaf thickness, chlorophyll content, or inherent internal differences). The observed higher absorption factor in T. hemprichii than in C. serrulata could also explain the differences in photosynthetic capacity between the two species. Another possible reason for T. hemprichii’s higher rates of photosynthesis could be that during low tides T. hemprichii are mostly exposed to the air and hence receive much more light for longer periods than does C. serrulata, which grows mostly in pools covered with water and is rarely fully exposed.

40 A

)

-1 30

-2

ETR (umol electron m Un-epiphytised-ORD Under the epiphytes-ORD Un-epiphytised-MJI Under the epiphytes-MJI

0 0 200 400 600 800 1000 1200 1400 1600

40 Uni-epiphytised-ORD B Under the epiphytes-ORD Un-epiphytised-MJI

) Under the epiphytes-MJI

-1 30

-2

ETR (umol electron m m electron (umol ETR

0 0 200 400 600 800 1000 1200 1400 1600 PAR (umol photons m-2 s-1)

Fig. 4. Thalassia hemprichii (A) and Cymodocea serrulata (B): Light curves for leaves under the epiphytes and for un-epiphytised leaves in the areas with contrasting levels of nutrient loading along the Dar es Salaam coast. Vertical bars denote mean ± SE, n = 18. ORD = Ocean Road, MJI = Mjimwema.

4.1.2 Effects on epiphyte biomass and composition

The occurrence and abundance of epiphytes on seagrass leaves has been shown to vary depending on environmental conditions (Hays 2005, Ruíz et al. 2009). Epiphytic composition and biomass varied in relation to different levels of nutrient in both laboratory and field experiments (papers II and III). In the nutrient enrichment experiments, epiphyte biomass did not vary significantly during short term exposure to increased levels of nutrients. However, in the long term experiments, epiphyte biomass varied significantly in relation to nutrient added (paper II). Moreover, in the in situ experiment, higher epiphyte biomass was observed for both species at the nutrient rich-site than at the nutrient-poor site, indicating that epiphytes respond more quickly to nutrient availability than their host seagrasses. A similar trend has been reported in many studies (e.g. Hauxwell et al. 2003, Hays 2005, McGlathery 2001).

The composition of epiphytes varied between species and between sites. Cymodocea serrulata had more epiphytic species at the nutrient-poor site, while T. hemprichii had more species at the nutrient-rich site (paper III), probably indicating the specific preferences of the various epiphytes for different structural and physical-chemical factors (Frankovich and Fourqurean 1997). In general, there were more cyanobacteria species at the nutrient-poor site than at the nutrient-rich site, indicating their contribution to sustained nitrogen demand in this area, supporting the previous observation by Hamisi et al. (2004).

4.1.3 Effects on nutrient contents of plant tissues

The common response of the plants to nutrient fluctuation is storage of respective nutrient in plant tissues (Romero et al. 2006), however the response seems to vary between species. There was no clear pattern in nutrient content in relation to the levels of nutrient added in the flow-through experiments (paper II), even though an increase in %N and %P content of the leaves was observed following nutrient addition in all treatments. Changes in nutrient contents were also observed in the control plants, reflecting the fact that the nutrient concentration in the incoming water was higher than concentrations in the habitat from which they were collected.

The in situ study showed variation in %C, %N, and %P content both within and between species and between sites (paper III). Percentages of N and P, as well as C:N:P ratios, correlated well with the nutrient concentration in the water column, indicating more availability of dissolved nutrient levels at Ocean Road than at Mjimwema, and hence increased acquisition and retention in the plant parts (Duarte 1990, Peréz-Lloréns and Niell 1995, 26

Lourenço et al. 2006 ). The C:P and C:N ratios of the seagrasses were higher in the area with low nutrients and lower in the high-nutrient site (Fig. 5). This agrees with the hypothesis that plants growing in nutrient-poor sites show higher C:N and C:P ratios than N:P ratios (Atkinson and Smith 1983, Duarte 1990). There were positive significant correlations between the concentrations of N and P content in both seagrass species at both sites, illustrating a close relationship between the two elements in plant metabolism (Duarte 1990). These results demonstrate that analysis of the nutrient contents of plant tissues may be more effective than water analysis for the early detection of changes in nutrient regimes in the seagrass meadows.

1000 1000 A Mjimwema B Mjimwema 800 Ocean Road 800 Ocean Road

600 600

400 400

C:P C:P ratio C:P C:P ratio

200 200

0 0

70 70 60 60 50 50 40 40

30 30

C:N ratioC:N C:N ratioC:N 20 20 10 10 0 0

50 50

40 40

30 30

20 20

N:P N:P ratio N:P N:P ratio 10 10

0 0 C. serrulata T. hemprichii C. serrulata T. hemprichii

Fig. 5. Elemental ratios for Thalassia hemprichii and Cymodocea serrulata leaves (A, left panel) and below ground tissues (B, right panel) at two sites with varying levels of nutrients; Mjimwema (nutrient poor site) and Ocean Road (nutrient rich site). Data are means ± SE, n = 18.

4.2 Ulva overgrowth affects seagrass photosynthesis

Seagrass photosynthesis depends upon light penetrating through the water column to reach the seagrass at the bottom. However, much light is lost before it reaches the seagrass. Shading by macroalgae blooms attributable to eutrophication has been implicated as a major cause of seagrass decline (Short et al. 1995). Results in this thesis (paper I) conform to the previous studies, by showing that shading by an algal thallus resulted in a significant reduction of available irradiance (Table 2). This indicates that, in the natural eutrophic environment where other factors contribute to light attenuation, the situation is likely to be intensified.

Table 2. Percentage reduction of light when passing through Ulva intestinalis thalli and absorption factor of Zostera marina in that light (Mean ± S.E). ND = not determined.

% light remaining Absorption factor Without Ulva 100 0.82 ± 0.08 Ulva 1 layer 44.2 ± 1.42 0.67 ± 0.04 Ulva 2 layers 24.6 ± 0.86 ND Ulva 3 Layers 15.9 ± 0.58 ND

In addition to reducing light reaching the seagrasses, Ulva thalli also affects the quality of that light by absorbing much of the useful spectrum, resulting in filtered light mainly in the green area of the spectrum, which is considered less efficient in photosynthesis. Interestingly, in this study there were no significant differences in maximum photosynthetic rates between seagrasses in normal or in Ulva-filtered light (Fig. 6). Although the light-capturing efficiency of seagrass was reduced under Ulva-filtered light (in the light limiting region), the relative quantum yield of the seagrass was always higher under the Ulva-filtered light. This finding illustrates that, provided the amount of light reaching the seagrass is adequate, changes in light quality caused by an overcast of green algae probably have little or no effect on the photosynthetic rate of the seagrass (paper I).

) Ik = 100 ± 55

-1 25

-2 Ik = 116 ± 59 20  = 0.26 ± 0.05

15 = 0.22 ± 0.03 10

( µmol electron m electron µmol (

max max Normal light

ETR 0 Ulva-filtered light

0 100 200 300 400 500

PAR ( µmol photons m-2 s-1 )

Fig. 6. Zostera marina. Electron transport rate in relation to irradiance levels under normal (filled circle) and Ulva-filtered light (open circle). Vertical bars represent the standard error, n = 40.

Changes in the chemical properties of seawater, whether as a result of biological processes through interactions among organisms, anthropogenic activities in the coastal areas, or a combination of factors, also influence organisms’ performance. In this study, the co-occurrence of macroalgae and seagrass resulted in changes in the chemistry of the seawater (paper I), by significantly increasing its pH, consequently affecting the amount of inorganic carbon available, and hence reducing photosynthetic rates in the seagrass. When Ulva was added to the seawater, water pH rose to 10.3; the -1 point at which the concentration of CO2 is reduced to less than 0.05µmol l hence, the observed reduction in photosynthesis rates (Fig. 7).

)

s 2 - 25

20 mol electrons m electrons mol

μ 15

( R² = 0.80 max

10 ETR

5 Normal light Ulva filtered light R² = 0.86 0 7.9 8.3 8.7 9.1 9.5 9.9 10.3 pH

Fig. 7. Photosynthetic rates of Zostera marina in relation to changes in seawater pH induced by addition of Ulva intestinalis thalli under normal (closed circles, solid trendline) and Ulva-filtered (open circles, dashed trendline) lights. Data points are averages of different measurements pooled together, n = 41.

4.3 Mussels enhance seagrass photosynthesis

Paper IV aimed at investigating the possible influence of CO2 released by mussels via respiration in the surrounding water on seagrass photosynthesis and the effects of pH changes induced by seagrass photosynthesis on mussels calcification. It was observed that electron transport rate of the seagrasses was positively affected by the presence of mussels (Fig. 8). The pH increased less in the treatments containing a mixture of seagrass and mussels than in those containing seagrass alone, implying that some CO2 was indeed released by the mussels. However, the small amounts of respiratory CO2 from the mussels seemed unlikely to account for the observed increase in seagrass photosynthesis. More likely, a stirring effect from mussels during filter feeding may have contributed to the observed changes by decreasing the boundary layer around the seagrass leaves, thus lessening limitation to Ci uptake in the seagrass, in particular during low tides when the mixing of water in the tidal flats is limited (Madsen et al. 2001).

450

400

* 350 *

rETR 300

Seagrass 250 Seagrass and 4 mussels Seagrass and 7 mussels

200 00:00 00:20 00:40 01:00 01:20 01:40 02:00 02:20

Time (h:m)

Fig. 8. Thalassia hemprichii: relative electron transport rate (rETR) in relation to incubation time among different cylinders with different treatments (data points are average values ± SE, n = 32). * indicates significant difference. The rETR increased with the increase in ambient light intensity (PAR) in all treatments.

Also, we could not find that the calcification rates of mussels were affected by the pH changes induced by seagrass photosynthesis (paper IV). However, it is possible that if predicted ocean acidification occurs, mussels could benefit through the counter-action of pH increase generated in the seagrass meadows.

5. Conclusion

The results presented in this thesis demonstrate that increased levels of nutrients in the water column affect the performance of seagrasses, but primarily indirectly.

 Co-occurrence of green macroalgae in seagrass meadows negatively affects the photosynthetic activity of seagrasses through competition for available CO2. On the other hand, photosynthetic capacity of seagrasses was boosted in the presence of mussels, while seagrass photosynthesis had no observable effects on mussel calcification.

 Nutrient content of the seagrass tissues reflect the nutrient concentrations in the water column of the studied sites. This suggests analysis of plant tissues as a reliable method for monitoring changes in the nutrient status of seagrass ecosystems in regions where such data are scarce.

 Photosynthetic capacity of seagrasses is affected by long term exposure to enhanced nutrient levels and the subsequent overgrowth of micro- and macro-epiphytes, that cause substantial reductions of light available for seagrass photosynthesis.

This thesis contributes to the understanding of multiple effects of nutrient over-enrichment on seagrass performance, with focus on the Western Indian Ocean region, where seagrass ecophysiological studies are still scarce. Nutrient over-enrichments induce multiple indirect effects on seagrass functioning, which act singly or in combination. Because the borderline between direct and indirect effects of nutrient over-enrichment in the field is challenging to define, major efforts should be directed towards minimizing the rates of nutrient loading in coastal environments and monitoring nutrient flux in coastal environments through analyses of water and plant tissues. These efforts must be made before remarkable harmful effects are evident, to reduce or prevent their impact and to predict their future responses to unanticipated natural impacts.

In the future....

As nutrient loading and CO2 emission is intensified through anthropogenic activities, it will be important to investigate the combined effects of increased nutrients and CO2 in seawater on the physiological responses of seagrasses. Changes in the chemical properties of seawater may influence the availability of nutrients.

To evaluate the functioning of the key enzymes involved in nutrient assimilation in seagrasses in response to the changing CO2 status of the marine environments. This could be addressed by measuring the activity of enzymes involved in nutrient assimilation in relation to varying levels of CO2 concentrations.

There exists limited research on seagrass physiological response in the Western Indian Ocean region; thus, there is a need to study other seagrass species within the region in regard to the interactive effects of nutrients and associated epiphytic loads on seagrass photosynthesis and consequent effects on the functioning of the below-ground parts.

The present study showed the importance of mussels and seagrass interaction in a short-term experiment. It will be important in future to continue to assess the long-term influences of mussels assemblages in the seagrass beds.

Acknowledgements

First and foremost, praise goes to God, for blessing me with the wellbeing, courage, strength, and endurance to finally finish this work. To my supervisor, Prof. Mats Björk: Thank you for bringing me into the world of sciences. Your constructive ideas, critical approaches to data interpretation, and tireless guidance were invaluable to the accomplishment of this work. To my co-supervisor, Dr. Thomas J. Lyimo: Thank you for introducing me to the world of marine sciences. Through you I found myself in the middle of intertidal seagrass meadows! I appreciate your support. I also extend my sincere gratitude to Prof. Birgitta Bergman for accepting me as a PhD student in the Physiology section, Botany Department, Stockholm University and for your valuable comments on my thesis.

My scholarship was provided by the Swedish government through the Swedish Agency for Research Cooperation (Sida/SAREC) marine bilateral programme, to which I am sincerely grateful. Also my sincere appreciation goes to the Director, Coordinator, and Heads at the University of Dar es Salaam, Institute of Marine Sciences, who made funds available whenever needed. Many thanks to the managements of the Western Indian Ocean Marine Science Association (WIOMSA) and the International Foundation for Sciences (IFS). Through your support I was able to attend different courses, workshops, and conferences, through which I learnt new things and met new people in my life. Without your support, I could not have managed.

To my colleagues, students, and friends at the University of Dar es Salaam Departments of Botany, Zoology and Wildlife Conservation, Aquatic Sciences, Molecular Biology and Biotechnology, and the Institute of Marine Sciences: Thank you. You have made my journey very enjoyable and colourful. To my fellow students, group mates, friends, group leaders, and everybody in the physiology section of the Botany Department, Stockholm University: Thank you very much for sharing all the moments that finally I accomplish this study. The working environment was very pleasant because of you.

Outside of academia, I wish to extend my sincere gratitude to my family, to my sisters, brothers, aunts, uncles, and in-laws for your love, prayers, and encouragements. You were always there for me. My sincere appreciation and special thanks goes to my sister Stella, who was a real mother to my daughter in my absence. She was so little when I left her, but she never felt my absence because you were always there for her. Mungu akubariki! To my daughter Doreen and your pal Elisha: You are so adorable I could feel it even when we were far apart. Thank you for your understanding and for your prayers! It is good that the journey is over and I can spend some more time with you. To my husband Denis: you’re the most important and unique gift in my life. Your love, passion, care, patience, enthusiasm, and encouragements defined my path. Thanks for being such a great father and companion to our daughter when I was away.

It is impossible to mention every person who contributed to the successful accomplishment of this study – that could be another thesis of its own! but I extend my sincere appreciation to every person (in or out of academia) who in one way or another facilitated the successful completion of this thesis. God bless you all!

References

Ackerman JD (2006) Sexual reproduction of seagrasses: pollination in the marine context. In: Larkum AWD, Orth RJ, Duarte CM (Eds.), Seagrasses: biology, ecology and conservation. Springer, the Netherlands, pp 89-109.

Alcoverro T, Romero J, Duarte CM, Lopez NI (1997) Spatial and temporal variations in nutrient limitation of seagrass Posidonia oceanica growth in the NW Mediterranean. Mar Ecol Prog Ser 146, 155-161.

Anderson DH, Robinson RJ (1946) Rapid electrometric determination of the alkalinity of seawater. Ind Engin Chem Anal Ed 18:767-769

Atkinson JB, Smith A (1983) C:N:P ratios of benthic marine plants. Limnol Oceanogr 28, 568-574.

Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol 59, 89–113

Beer S, Björk M (2000) Measuring rates of photosynthesis of two tropical seagrasses by pulse amplitude modulated (PAM) fluorometry. Aquat Bot 66, 69-76.

Beer S, Björk M, Gademann R, Ralph P (2001) Measurements of photosynthetic rates in seagrasses. In: Short FT, Coles RG (Eds.), Global Seagrass Research Methods. Elsevier, New York, pp 183–198.

Beer S, Björk M, Hellblom F, Axelsson L (2002) Inorganic carbon utilization in marine angiosperms (seagrasses). Funct Plant Biol 29, 349-354.

Beer S, Mtolera M, Lyimo T, Björk M (2006) The photosynthetic performance of the tropical seagrass Halophila ovalis in the upper intertidal. Aquat Bot 84, 367-371.

Beer S, Rehnberg J (1997) The acquisition of inorganic carbon by the seagrass Zostera marina. Aquat Bot 56, 277-283.

Björk M, Axelsson L, Beer S (2004) Why is Ulva intestinalis the only macroalga inhabiting isolated rockpools along the Swedish Atlantic coast? Mar Ecol Prog Ser 284, 109-116.

Björk M, Mohammad SM, Björklund M, Semesi A (1995) Coralline algae, important coral reef builders threatened by pollution. Ambio 24, 502-505.

Björk M, Weil A, Semesi S, Beer S (1997) Photosynthetic utilisation of inorganic carbon by seagrasses from Zanzibar, East Africa. Mar Biol 129, 363-366.

Borowitzka MA, Lavery PS, van Keulen M (2006) Epiphytes of seagrasses. In: Larkum AWD, Orth RJ, Duarte CM (Eds.), Seagrasses: biology, ecology and conservation. Springer, the Netherlands: pp 441-461.

Borum J, Greve TM (2004) The four European seagrass species. In: Borum J, Duarte CM, Krause-Jensen D, Greve TM (Eds.), European Seagrasses: an introduction to monitoring and management. The M & MS project. pp 1– 7.

Borum J, Sand-Jensen K (1996) Is total primary production in shallow coastal marine waters stimulated by nitrogen loading? Oikos 76, 406-410.

Brun FG, Hernandez I, Vergara JJ, Perez-Llorens JL (2003) Growth, carbon allocation and proteolytic activity in the seagrass Zostera noltii shaded by Ulva canopies. Funct Plant Biol 30, 551-560.

Bulthuis DA, Axelrad DM, Mickelson MJ (1992) Growth of the seagrass Heterozostera tasmanica limited by nitrogen in Port Phillip Bay, Australia. Mar Ecol Prog Ser 89, 269-275.

Burkholder JM, Tomasko DA, Touchette BW (2007) Seagrasses and eutrophication. J Exp Mar Biol Ecol 350, 46-72.

Burkholder JM, Mason KM, Glasgow HBJ (1992) Water-column nitrate enrichment promotes decline of eelgrass Zostera marina: Evidence from seasonal mesocosm experiments. Mar Ecol Prog Ser 81, 163–178

Chisholm JRM (2000) Calcification by crustose coralline algae on the northern Great Barrier Reef, Australia. Limnol Oceanogr 45, 1476-1484. de la Torre-Castro, M., Rönnbäck, P., 2004. Links between humans and seagrasses – an example from tropical East Africa. Ocean Coast Manage 47, 361-387.

Drechsler Z, Sharkia R, Cabantchik ZI, Beer S (1993) Bicarbonate uptake in the marine macroalga Ulva sp. is inhibited by classical probes of anion exchange by red blood cells. Planta 191, 34-40.

Duarte CM (1990) Seagrass nutrient content. Mar Ecol Prog Ser 67, 201-207.

Duarte CM, Chiscano CL (1999) Seagrass biomass and production: a reassessment. Aquat Bot 65, 159-174.

Duarte CM, Dennison WC, Orth RJW, Carruthers TJB (2008) The charisma of coastal ecosystems: addressing the imbalance. Estuar Coast 31, 233- 238.

Duarte CM, Gattuso JP (2010) Seagrass meadows. In: Encyclopedia of earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for Science and the Environment). http://www.eoearth.org/article/Seagrass_meadows.

Fabry VJ, Seibel BA, Feely RA, Orr JC (2008) Impacts of ocean acidification on marine fauna and ecosystem processes. Ices J Mar Sci 65, 414-432.

Falkowski PG, Raven JA (2007) Aquatic photosynthesis. Princeton University Press, New Jersey.

Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281, 237-240.

Fourqurean JW, Zieman JC, Powell GVN (1992) Phosphorus limitation of primary production in Florida Bay evidence from CNP ratios of the dominant seagrass Thalassia testudinum. Limnol Oceanogr 37, 162-171.

Frankovich TA, Fourqurean JW (1997) Seagrass epiphyte loads along a nutrient availability gradient, Florida Bay, USA. Mar Ecol Prog Ser 159, 37-50.

Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimi Biophys Acta 990, 87-92.

Gras AF, Koch MS, Madden CJ (2003) Phosphorus uptake kinetics of a dominant tropical seagrass Thalassia testudinum. Aquat Bot 76, 299-315.

Green EP, Short FT (2003) World atlas of seagrasses. California University Press, California.

Hamisi MI, Lyimo TJ, Muruke MHS, Bergman B (2009) Nitrogen fixation by epiphytic and epibenthic diazotrophs associated with seagrass meadows along the Tanzanian coast, Western Indian Ocean. Aquat Microb Ecol 57: 33-42

Hauxwell J, Cebrian J, Valiela I (2003) Eelgrass Zostera marina loss in temperate estuaries: relationship to land-derived nitrogen loads and effect of light limitation imposed by algae. Mar Ecol Prog Ser 247, 59-73.

Hays CG (2005) Effect of nutrient availability, grazer assemblage and seagrass source population on the interaction between Thalassia testudinum (turtle grass) and its algal epiphytes. J Exp Mar Biol Ecol 314, 53-68.

Heck KL, Valentine JF (2006) Plant-herbivore interactions in seagrass meadows. J Exp Mar Biol Ecol 330, 420-436.

Heiss WM, Smith AM, Probert PK (2000) Influence of the small intertidal seagrass Zostera novazelandica on linear water flow and sediment texture. New Zeal J Mar Fresh 34, 689-694.

Hemminga MA, Duarte CM (2000) Seagrass ecology. Cambridge University Press, Cambridge.

Holmer M, Olsen AB (2002) Role of decomposition of mangrove and seagrass detritus in sediment carbon and nitrogen cycling in a tropical mangrove forest. Mar Ecol Prog Ser 230, 87-101.

Invers O, Romero J, Perez M (1997) Effects of pH on seagrass photosynthesis: a laboratory and field assessment. Aquat Bot 59, 185-194.

Invers O, Zimmerman RC, Alberte RS, Perez M, Romero J (2001) Inorganic carbon sources for seagrass photosynthesis: an experimental evaluation of bicarbonate use in species inhabiting temperate waters. J Exp Mar Biol Ecol 265, 203-217.

Jassby AD, Platt T (1976) Mathematical formulation of relationship between photosynthesis and light for phytoplankton. Limnol Oceanogr 21, 540-547.

Johnson MW, Heck JKL, Fourqurean JW (2006) Nutrient content of seagrasses and epiphytes in the northern Gulf of Mexico: evidence of phosphorus and nitrogen limitation. Aquat Bot 85, 103-111.

Kautsky H, Appel W, Amann H (1960) Chlorophyllfluoreszenz und kohlenäureassimilation. Biochemisches zeiterschrift 322, 277-292.

Kilminster KL, Walker DI, Thompson PA, Raven JA (2006) Limited nutritional benefit to the seagrass Halophila ovalis, in culture, following sediment organic matter enrichment. Est Coast Shelf Sci 68, 675-685.

Kotta J, Møhlenberg F (2002) Grazing impact of Mytilus edulis L. and Dreissena polymorpha (Pallas) in the Gulf of Riga, Baltic Sea estimated from biodeposition rates of algal pigments. Ann Zool Fenn 39, 151-160.

Krause GH, Weiss E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Phys 42, 313-349.

Kuo J, den Hartog C (2006) Seagrass morphology, anatomy, and ultrastructure. In: Larkum AWD, Orth RJ, Duarte CM (Eds.), Seagrasses: biology, ecology and conservation. Springer, the Netherlands, pp51-87.

Lamonte M, Dunton KH (2006) Effects of drift macroalgae and light attenuation on chlorophyll fluorescence and sediment sulfides in the seagrass Thalassia testudinum. J Exp Mar Biol Ecol 334:174–186.

Larkum AWD, Drew EA, Ralph PJ (2006) Photosynthesis and metabolism of seagrasses at the cellular level. In Seagrasses: Biology, ecology and conservation, eds. W.D. Larkum, R.J. Orth, and C.M. Duarte, 323–345. Dordrecht, The Netherlands: Springer.

Lauringson V, Malton E, Kotta J, Kangur K, Orav-Kotta H, Kotta I (2007) Environmental factors influencing the biodeposition of the suspension feeding bivalve Dreissena polymorpha (Pallas): comparison of brackish and freshwater populations. Est Coast Shelf Sci 75, 459-467.

Lee KS, Park SR, Kim YK (2007) Effects of irradiance, temperature, and nutrients on growth dynamics of seagrasses: a review. J Exp Mar Biol Ecol 350, 144-175.

Liu SL, Wang WL, Dy DT, Fu CC (2005) The effect of ulvoid macroalgae on the inorganic carbon utilization by an intertidal seagrass Thalassia hemprichii. Bot Bulle Acad Sinica 46, 197-203.

Lourenço SO, Barbarino E, Nascimento A, Freitas JNP, Diniz GS (2006) Tissue nitrogen and phosphorus in seaweeds in a tropical eutrophic environment: What a long-term study tells us. J Appl Phycol 18: 389-98.

Martins I, Marques JC (2002) A model for the growth of opportunistic macroalgae (Enteromorpha sp.) in tidal estuaries. Est Coast Shelf Sci 55, 247-257.

Maxwell K, Johnson GL (2000) Chlorophyll fluorescence: a practical guide. J Exp Bot 51(345), 659-668.

Mazzella L, Alberte RS (1986) Light adaptation and the role of autotrophic epiphytes in primary production of the temperate seagrass, Zostera marina L. J Exp Mar Biol Ecol 100, 165-180. 40

McGlathery KJ (2001) Macroalgal blooms contribute to the decline of seagrass in nutrient-enriched coastal waters. J Phycol 37,453-456.

Mohammed MS, Mgaya YD (2001) Nutrients and their dynamics in the coral reefs off Zanzibar town. In: Richmond M, Francis J (eds), Marine science development in Tanzania and eastern Africa. Proceedings of the 20th Anniversary Conference on Advances in Marine Sciences in Tanzania. Institute of Marine Sciences. University of Dar es Salaam and Western Indian Ocean Marine Science Association, Zanzibar, Tanzania, pp 171- 183.

Moore KA, Wetzel RL (2000) Seasonal variations in eelgrass (Zostera marina L.) responses to nutrient enrichment and reduced light availability in experimental ecosystems. J Exp Mar Biol Ecol 244, 1-28.

Moriarty DJW, O’Donohue MJ (1993) Nitrogen-fixation in seagrass communities during summer in the Gulf of Carpentaria, Australia. Aust J Mar Freshw Res 44, 117-125.

Newell RIE (2004) Ecosystem influences of natural and cultivated populations of suspension-feeding bivalve molluscs: a review. J Shellfish Res 23:51-61.

Nielsen OI, Koch MS, Jensen HS, Madden CJ (2006) Thalassia testudinum phosphate uptake kinetics at low in situ concentrations using a IT radioisotope technique. Limnol Oceanogr 51, 208-217.

Oliviera EC, Österlund K, Mtolera MSP (2005). Marine plants of Tanzania. A field guide to seaweeds and seagrasses. Stockholm University, Sweden, 229-236 pp.

Orth RJ, Carruthers TJB, Dennison WC, Duarte CM, Fourqurean JW, Heck KL, Hughes AR, Kendrick GA, Kenworthy WJ, Olyarnik S, Short FT, Waycott M, Williams SL (2006) A global crisis for seagrass ecosystems. Bioscience 56, 987-996.

Palacios SL, Zimmerman RC (2007) Response of eelgrass Zostera marina to CO2 enrichment: possible impacts of climate change and potential for remediation of coastal habitats. Mar Ecol Prog Ser 344, 1-13.

Parsons RT, Maita Y, Lalli CM (1989) A manual of chemical and biological methods for seawater analysis. Pergamon Press, Oxford, pp 173.

Pedersen MF, Borum J, Fotel FL (1997) Phosphorus dynamics and limitation of fast- and slow-growing temperate seaweeds in Oslo Fjord, Norway. Mar Ecol Prog Ser 399, 103-115.

Pérez-Lloréns JL, Niell FX (1995) Short-term phosphate uptake kinetics in Zostera noltii Hornem: a comparison between excised leaves and sediment-rooted plants. Hydrobiologia 297, 17-27.

Peterson BJ, Heck KL (1999) The potential for suspension feeding bivalves to increase seagrass productivity. J Exp Mar Biol Ecol 240, 37-52.

Peterson BJ, Heck KL (2001a) An experimental test of the mechanism by which suspension feeding bivalves elevate seagrass productivity. Mar Ecol Prog Ser 218, 115-125.

Peterson BJ, Heck KL (2001b) Positive interactions between suspension- feeding bivalves and seagrass – a facultative mutualism. Mar Ecol Prog Ser 213, 143-155.

Ralph PJ, Gademann R (2005) Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat Bot 82, 222-237.

Richmond MD (2002). A field guide to the seashores of Eastern Africa and the Western Indian Ocean Islands. Sida/SAREC-UDSM, Dar es Salaam, Tanzania.

Riebesell U, Zondervan I, Rost B, Tortell PD, Zeebe RE, Morel FMM (2000) Reduced calcification in marine plankton in response to increased atmospheric CO2. Nature 407, 634-637.

Ries JB, Cohen AL, McCorkle DC (2009) Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology 37, 1131-1134.

Riley JP, Skirrow G (1965) Chemical oceanography. Academic Press, New York.

Romero J, Lee KS, Pérez M, Mateo MA, Alcoverro T (2006) Nutrient dynamics in seagrass ecosystems. In: Larkum AWD, Orth RJ, Duarte CM (Eds.), Seagrasses: biology, ecology and conservation. Springer, the Netherlands, pp 227–254.

Ruíz JM, Pérez M, Romero J, Tomas F (2009) The importance of herbivory in the decline of a seagrass (Posidonia oceanica) meadow near a fish farm: an experimental approach. Bot Mar 52, 449-458.

Sabine CL, Feely RA, Gruber N, Key RM, Lee K, Bullister JL, Wanninkhof R, Wong CS, Wallace DWR, Tilbrook B, Millero FJ, Peng TH, Kozyr A, Ono T, Rios AF (2004) The oceanic sink for anthropogenic CO2. Science 305, 367-371.

Semesi S, Beer S, Björk M (2009) Seagrass photosynthesis controls rates of calcification and photosynthesis of calcareous macroalgae in a tropical seagrass beds. Mar Ecol Prog Ser 382, 41-47.

Schneider K, Erez J (2006) The effect of carbonate chemistry on calcification and photosynthesis in the hermatypic coral Acropora eurystoma. Limnol Oceanogr 51(3), 1284-1293.

Schreiber U (2004) Pulse-amplitude (PAM) fluorometry and saturation pulse method. In: Papageorgiou G, Govindjee (Eds.), Chlorophyll fluorescence: a signature of photosynthesis. Advances in Photosynthesis and Respiration Series. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Schwarz AM, Bjork M, Buluda T, Mtolera H, Beer S (2000) Photosynthetic utilisation of carbon and light by two tropical seagrass species as measured in situ. Mar Biol 137, 755-761.

Short FT, Burdick DM, Kaldy JE (1995) Mesocosm experiments quantify the effects of eutrophication on eelgrass, Zostera marina. Limnol Oceanogr 40, 740-749.

Short FT, Carruthers T, Dennison W, Waycott M (2007) Global seagrass distribution and diversity: a bioregional model. J Exp Mar Biol Ecol 350, 3-20.

Short FT, Dennison WC, Capone DG (1990) Phosphorus-limited growth of the tropical seagrass Syringodium filiforme in carbonate sediments. Mar Ecol Prog Ser 62, 169-174.

Short FT, Neckles HA (1999) The effects of global climate change on seagrasses. Aquat Bot 63, 169-196.

Short FT, Wyllie-Echeverria S (1996) Natural and human-induced disturbance of seagrasses. Environ Conserv 23, 17-27.

Silva J, Santos R (2004) Can chlorophyll fluorescence be used to estimate photosynthetic production in the seagrass Zostera noltii? J Exp Mar Biol Ecol 307, 207-216.

Sjöö GL, Mörk E (2009) Tissue nutrient content in ulva spp. (chlorophyceae) as bioindicator for nutrient loading along the Coast of East Africa. Open Environ Biol Monit J 2, 11-17.

Smith SV, Kinsey DW (1978) Calcification and organic carbon metabolism as indicated by carbon dioxide. In: Stoddart DR (ed), Coral reef: research methods. UNESCO, Paris.

Smith SV, Key GS (1975) Carbon dioxide and metabolism in marine environments. Limnology and Oceanography 20, 493-495.

Touchette BW, Burkholder JM (2000a) Review of nitrogen and phosphorus metabolism in seagrasses. J Exp Mar Biol Ecol 250, 133-167.

Touchette BW, Burkholder JM (2000b) Overview of the physiological ecology of carbon metabolism in seagrasses. J Exp Mar Biol Ecol 250, 169-205.

Udy JW, Dennison WC (1997) Physiological responses of seagrasses used to identify anthropogenic nutrient inputs. Mar Freshw Res 48, 605-614.

Vergara JJ, Peralta G, Pérez-Lloréns JL, Hernández I (1997) Seasonal variation of photosynthetic performance and light attenuation in Ulva canopies from Palmones river estuary. J Phycol 33, 773-779.

Wall CC, Peterson BJ, Gobler CJ (2008) Facilitation of seagrass Zostera marina productivity by suspension-feeding bivalves. Mar Ecol Prog Ser 357, 165-174.

Waycott M, Duarte CM, Carruthers TJB, Orth RJ, Dennison WC, Olyarnik S, Calladine A, Fourqurean JW, Heck KL, Hughes AR, Kendrick GA, Kenworthy WJ, Short FT, Williams SL (2009) Accelerating loss of seagrasses across the globe threatens coastal ecosystems. Proc Natl Acad Sci U S A 106, 12377-12381.

Welsh DT, Bartoli M, Nizzoli D, Castaldelli G, Riou SA, Viaroli P (2000) Denitrification, nitrogen fixation, community primary productivity and inorganic N and oxygen fluxes in an intertidal Zostera noltii meadow. Mar Ecol Prog Ser 208, 65-77.

Welsh DT, Bourgues S, deWit R, Herbert RA (1996) Seasonal variations in nitrogen-fixation (acetylene reduction) and sulphate-reduction rates in the rhizosphere of Zostera noltii: Nitrogen fixation by sulphate reducing bacteria. Mar Biol 125, 619-628.