Seagrasses and eutrophication - DiVA Portal

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Seagrasses and eutrophication Interactions between seagrass photosynthesis, epiphytes, macroalgae and mussels Esther Francis Mvungi

Transcript of Seagrasses and eutrophication - DiVA Portal

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

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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.

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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

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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.

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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

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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

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ionic forms of carbon (carbonic acid, bicarbonate, and carbonate) in

equilibrium with CO2, as described by Falkowski and Raven (2007):

H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3

- ↔ 2H

+ + CO3

2-

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

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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

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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.

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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).

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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).

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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

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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, 39

o19' 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, 39

o21' 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).

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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, 39

o 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.

21

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.

22

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).

23

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).

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.

Site Species Shoot density

(no. shoots m-2

)

Canopy

height (cm)

Above ground

biomass (gDWm-2

)

Below ground

biomass (gDWm-2

)

Above to below-

ground (AG/BG)

biomass ratios

Dry weight of

epiphytes (gg-1

)

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

24

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.

A

Measurement days

Day 0 Day 4 Day 7 Day 11 Day 15 Day 21

Fv/F

m

0.0

0.2

0.4

0.6

0.8

1.0

0:0 µM

1:0.1 µM

5:0.5 µM

25:2.5 µM

125:12.5 µM

B

Measurement days

Day 0 Day 23 Day 52 Day 95

0.0

0.2

0.4

0.6

0.8

1.0

0:0 µM

1:0.1 µM

10:1 µM

100:10 µM

1000:100 µM

25

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.

A

0 200 400 600 800 1000 1200 1400 1600

ET

R (

um

ol ele

ctr

on m

-2 s

-1)

0

10

20

30

40

Un-epiphytised-ORD

Under the epiphytes-ORD

Un-epiphytised-MJI

Under the epiphytes-MJI

B

PAR (umol photons m-2

s-1

)

0 200 400 600 800 1000 1200 1400 1600

ET

R (

um

ol e

lectr

on m

-2 s

-1)

0

10

20

30

40

Uni-epiphytised-ORD

Under the epiphytes-ORD

Un-epiphytised-MJI

Under the epiphytes-MJI

26

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,

27

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.

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.

0

200

400

600

800

1000

C:P

ra

tio

Mjimwema

Ocean Road

A

0

10

20

30

40

50

60

70

C:N

ra

tio

0

10

20

30

40

50

C. serrulata T. hemprichii

N:P

ra

tio

0

200

400

600

800

1000

C:P

ra

tio

Mjimwema

Ocean Road

B

0

10

20

30

40

50

60

70

C:N

ra

tio

0

10

20

30

40

50

C. serrulata T. hemprichii

N:P

ra

tio

28

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.

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).

% 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

29

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

point at which the concentration of CO2 is reduced to less than 0.05µmol l-1

hence, the observed reduction in photosynthesis rates (Fig. 7).

PAR ( µmol photons m-2 s-1 )

0 100 200 300 400 500

ET

Rm

ax (

µm

ol e

lectr

on

m-2

s-1

)

0

5

10

15

20

25

30

Normal light

Ulva-filtered light

Ik = 100 ± 55

Ik = 116 ± 59

= 0.26 ± 0.05

= 0.22 ± 0.03

30

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).

R² = 0.86

R² = 0.80

0

5

10

15

20

25

30

7.9 8.3 8.7 9.1 9.5 9.9 10.3

ET

Rm

ax(μ

mol e

lect

rons

m-2

s-1)

pH

Normal light

Ulva filtered light

31

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.

Time (h:m)

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

rET

R

200

250

300

350

400

450

Seagrass

Seagrass and 4 mussels

Seagrass and 7 mussels

*

*

32

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.

33

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.

34

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.

35

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!

36

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