Abe2004e

Abe2004e
The microphytobenthos and its role in
aquatic food webs
Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Christian-Albrechts-Universität
zu Kiel
vorgelegt von
Nicole Aberle-Malzahn
Kiel 2004
Referentin: PD Dr. K.H. Wiltshire
Koreferent/in:
Tag der mündlichen Prüfung:
Zum Druck genehmigt:
Kiel, den
Der Dekan
‘THERE’S A UNIVERSE INSIDE THESE WATERS’
PATY MARSHALL-STACE, ‘MEZA GEBEL’ (1995)
CHAPTER 1
Chapter 1 General introduction .................................................................................................. 5
Chapter 2 Seasonality and diversity patterns of freshwater and marine microphytobenthic
communities ............................................................................................................................. 12
2.1
Introduction .............................................................................................................. 13
2.2
Material & Methods ................................................................................................. 14
2.3
Results ...................................................................................................................... 18
2.4
Discussion ................................................................................................................ 29
Chapter 3 ‘Spectral fingerprinting’ for specific algal groups on sediments in situ: a new sensor
.................................................................................................................................................. 38
3.1
Introduction .............................................................................................................. 39
3.2
Material & Methods ................................................................................................. 40
3.3
Results ...................................................................................................................... 45
3.4
Discussion ................................................................................................................ 51
Chapter 4 Microcosm experiments on grazing efficiency and selectivity of the freshwater
hydrobiid snail Potamopyrgus antipodarum preying upon microphytobenthic assemblages. 54
4.1
Introduction .............................................................................................................. 55
4.2
Material & Methods ................................................................................................. 56
4.3
Results ...................................................................................................................... 58
4.4
Discussion ................................................................................................................ 62
Chapter 5 Factors influencing microphytobenthos community structures beneath macrophyte
beds- a marine and freshwater mesocosm study. ..................................................................... 67
5.1
Introduction .............................................................................................................. 68
5.2
Material & Methods ................................................................................................. 69
5.3
Results ...................................................................................................................... 73
5.4
Discussion ................................................................................................................ 84
Chapter 6 Selectivity and competitive interactions between two benthic invertebrate grazers
(Asellus aquaticus and Potamopyrgus antipodarum)- an experimental study using 13C- and
15
N-labelled diatoms............................................................................................................... 102
6.1
Introduction ............................................................................................................ 103
6.2
Material & Methods ............................................................................................... 104
6.3
Results .................................................................................................................... 106
6.4
Discussion .............................................................................................................. 111
Chapter 7 General Discussion ................................................................................................ 118
Chapter 8 Summary................................................................................................................ 127
Chapter 9 Zusammenfassung ................................................................................................. 130
Chapter 10 Reference list ....................................................................................................... 133
4
CHAPTER 1
Chapter 1
General introduction
The littoral regions of lakes and coastal seas are one of the most productive ecosystems on
earth and their productivity by far exceeds that of the open oceans. The major primary
producers are macro- and microalgae colonizing all sorts of substrates and covering vast
areas within the euphotic zones of aquatic systems. Furthermore, since primary production is
the basis for secondary producers, the overall importance of littoral zones as complex
ecosystems cannot be underestimated. These areas provide extensive habitats and trophic
linkages for a large variety of organisms ranging from microfauna to wading birds and
demersal fish. Thus, their significance is not large merely from an ecological point of view but
also with regard to commercial aspects (fisheries, recreation areas, tourism). The
maintenance, protection and investigation of these zones should be of considerable public
interest.
The benthos in the euphotic zones comprises not only of macroscopical vegetated habitats
rather vast substrate areas are predominately colonized by photosynthetically active
microorganisms. This is especially true for sediment surfaces which appear to be bare
deserts without any obvious plant life, but on closer inspection the brownish or greenish
colouring of the surfaces turns out to be due to microorganisms assemblages, the so called
microphytobenthos. This term refers to the microscopic, unicellular eukaryotic algae
(Baccilariophyceae, Chlorophyceae and Dinophyceae) and the prokaryotic Cyanobacteria
which live on sediment surfaces. They grow in habitats ranging from intertidal sand and mud
flats, salt marshes, submerged aquatic vegetation beds to subtidal sediments. Although less
conspicuous than macroalgae or vascular plants, the microphytobenthos itself can contribute
significantly to primary production in littoral zones (Daehnick et al., 1992; Pinckney &
Zingmark, 1993; Colijn & De Jonge, 1984) and in many shallow aquatic systems the biomass
of benthic microalgae exceeds that of the phytoplankton in the overlying waters.
External factors influencing microphytobenthos growth
The main limiting factors of microphytobenthos are the availability of nutrients and light.
Thus, microphytobenthic assemblages are found at the uppermost surface layers of the
sediments right at the sediment-water interface. As the penetration of light is largely confined
to the upper 0.2-2 mm, the distribution of benthic microalgae is restricted to this relatively thin
surface layer (Wolff, 1979; MacIntyre et al., 1996). The layers in which light is good enough
allowing the microphytobenthos to photosynthesise vary both with the granulometry of the
sediment and its organic content. Many benthic microalgae are known for their mobility and
CHAPTER 1
they show diel rhythms of vertical migration, moving to and away from the surface in
response to a multitude of factors e.g. light, tide cycles, desiccation, predation or
resuspension (Admiraal et al., 1984; Pinckney & Zingmark, 1991; Paterson et al., 1998).
Although the velocities at which the cells migrate vertically are low, ranging from 10 to 27 mm
h-1 (Hopkins; 1963), the ability to move is important to the alga as the top layers of the
sediment represent a region with strong physical and chemical gradients. Within a few
millimetres of depth, the sediment properties go from fully oxygenated to anoxic conditions
and pH, sulphide, irradiance, and nutrients are known to show strong vertical variability
(Joergensen et al., 1983; Wiltshire, 1992; Wiltshire, 1993). But despite their variability over
vertical scales, there also appear to be considerable spatial fluctuations on a horizontal scale
(centimetres to meters). Possible causes for the patchy distribution are variations in the
texture and relief of the sediment surface (Joergensen & Revsbech, 1983; Jumars & Nowell,
1984; Gaetje, 1992) or microscale nutrient, irradiance, water content and salinity gradients
(Wolff, 1979). Because of their location at the sediment surface, the microphytobenthos plays
an important role in modulating nutrient fluxes at the sediment-water interface and this is
particularly important with regard to oxygen and nitrogen budgets of the sediments
(Sundbaeck et al., 1991; Wiltshire, 1993; Wiltshire et al., 1996). In general it is assumed that
growth of benthic microalgae is not limited by nutrients, since nutrient concentrations in the
pore water are generally high (Cadée & Hegemann, 1974; Admiraal, 1984). However, in the
thin layer of diatoms at the sediment surface biomass may be highly concentrated, and thus
nutrients may temporarily become depleted (Admiraal, 1977). When abundant, the
microphytobenthos can, furthermore, stabilize the sediment surface against resuspension
and erosion by secreting mucilaginous films and forming thin, brownish mats or carpets
(Paterson et al., 1990; Delgado et al., 1991; De Brouwer & Stal, 2001). These biofilms are
mainly formed by diatoms excreting Extracellular Polymeric Substances (EPS) whereas the
amount of excretion is directly related to the rate of primary production (Cadée & Hegemann,
1974).
Microphytobenthic community structures
The microphytobenthos includes representatives of several algal classes (Baccilariophyceae,
Chlorophyceae, Cyanobacteria, Dinophyceae). On sandy and muddy substrate, edaphic
microalgae living on a variety of benthic surfaces are often dominated by diatoms (Colijn &
De Jonge, 1984; Admiraal et al., 1984; De Jonge & Colijn, 1994; Agatz et al., 1999) whereas
coccal and filamentous green algae and Cyanobacteria are usually known to occur at some
seasonal stages (Yallop et al., 1994; Taylor & Paterson, 1998; Hillebrand & Kahlert, 2001;
Nozaki et al., 2003). Microphytobenthic diatom populations are usually composed of
pennate, prostrate forms, which are either epipsammic or epipelic (Daehnick et al., 1992;
6
CHAPTER 1
Moncreiff et al., 1992; Gaetje, 1992; Agatz et al., 1999; Mitbavkar & Anil, 2002). Epipsammic
diatoms are monoraphidean, araphidean, biraphidean and centric species of small size that
grow attached to sediment particles to which they are glued by mucilaginous pads or stalks.
The epipelic forms are biraphidean species which actively move through the sediment by
means of the mucilaginous secretion of their raphes (Round, 1971). However, the difference
between the two categories is not absolute as there are epipsammic diatoms that are
capable of movement, though generally much slower than epipelic species (Harper, 1969)
and furthermore, many diatom genera have representatives in either of these groups (e.g.
Nitzschia sp., Navicula sp., Amphora sp.) (Wolff, 1979). In general, the relative exposure to
wave action and currents is thought to favour the dominance of epipsammic biomass and
relative shelter the dominance of epipelic forms. In contrast, to epi- or periphyton
communities where a distinct three-dimensional layer is usually developed, these patterns
are missing on microphytobenthic biofilms and only few erect forms are present. Thus, the
microphytobenthos is characterized as a distinctly flat, two-dimensional community (Miller et
al., 1987).
Despite the typical characteristics of a microphytobenthic community, it has increasingly
been seen that benthic algae may not be strictly edaphic and that planktonic forms can
temporarily dwell on sediments (Drebes, 1974, Gaetje, 1992; De Jong & De Jonge, 1995).
The same algal classes are found in both the phytoplankton and the microphytobenthos and
the basis of separating these types are mainly due to morphological and preferred habitat
characteristics. However, the distinction between benthic and pelagic life styles can be fluid
especially in shallow water systems since, the exchange of organisms between the sediment
and the water column is common. The microphytobenthos can be resuspended by water
currents and wave action and thus they then dwell in the water column and contribute to the
planktonic community (Drebes, 1974; De Jonge & van Beusekom, 1995; De Jong & De
Jonge, 1995). On the other hand, phytoplanktonic organisms can sink to the bottom under
calm conditions and settle on the sediment in considerable amounts where they live on and
photosynthesise and become incorporated into the microphytobenthos (Potter et al., 1975;
Gaetje, 1992; Blomqvist, 1996). Thus, these bentho-pelagic forms must be taken into
consideration.
Microphytobenthic life cycles
The life cycles of benthic microalgae are complex and past research showed that some
broad generalizations can be made. The biomass in sheltered, muddy habitats is higher than
in exposed sandy habitats (Cadée & Hegemann, 1974; Colijn & Dijkema, 1981; Delgado,
1989; Sundbaeck et al. 1991). Variations in the biomass of microphytobenthos in adjacent
but distinct habitats can be as great as those over large geographic distances (Sullivan &
7
CHAPTER 1
Moncreiff, 1990; Pinckney & Zingmark, 1993; Moncreiff & Sullivan, 2001). In temperate
regions microphytobenthic biomass, primary production and chlorophyll contents show a
spring or summer maximum similar to biomass peaks of planktonic algae (Kann, 1940;
Admiraal & Peletier, 1980; Khondker & Dokulil, 1988; De Jonge & Colijn, 1994; Sundbaeck et
al., 2000; Nozaki et al., 2003). Due to seasonal variations in light intensities in the northern
hemisphere these blooms are of relatively short duration and with increasing latitude they
occur later in the year. Many studies on primary productivity and chlorophyll contents of
microphytobenthic assemblages have been conducted over the last 30 years. However, their
comparability is severely restricted by differences in methodologies (sediment volume,
sampling techniques, measurement techniques), habitats and geographical distance. In
response to these factors the chlorophyll contents observed ranged from less than 1 mg m–2
(Golfe de Fos, France; Plante-Cuny & Bodoy, 1987) to 560 mg m–2 (Ems-Dollard Estuary,
The Netherlands; Colijn & De Jonge, 1984) as well as primary production rates ranging from
less than 1 mg C m–2 d-1 at the same site in France (Golfe de Fos, France; Plante-Cuny &
Bodoy, 1987) to 115 mg C m–2 h-1 in the Ems-Dollard Estuary (Colijn & De Jonge, 1984).
The production variability can be explained by changes in irradiance and chlorophyll a
concentration ranges as well as by environmental factors (e.g. temperature, nutrient
contents).
Seasonal and temporal fluctuations not only occur in terms of total biomass but they also
have been demonstrated on a taxonomic level. The dominance of particular algal groups at
different times of the year have been shown by several authors and it was found that despite
a general dominance of diatoms, green algae and Cyanobacteria are known to occur in high
abundances during the summer period (Kann, 1940; Kann, 1993; Khodker & Dokulil, 1988;
Yallop et al., 1994; Taylor & Paterson, 1998; Hillebrand & Kahlert, 2002; Nozaki et al., 2003).
Furthermore, seasonal succession has also been demonstrated on genera and species
levels and thus, inter-annual taxonomic fluctuations are known to occur (De Jonge & Colijn,
1994; Khodker & Dokuli, 1988).
Food-web interactions and trophic significance
As pointed out before, sediment habitats represent complex aquatic ecosystems and, apart
from the sediment microflora, they are also inhabited by innumerable invertebrate species.
The sediment dwellers can be classified according to their size ranges as proposed by
Plante-Cuny & Plante (1984) into microfauna (ciliates), meiofauna (harpacticoid copepods,
nematodes,
ostracods)
and
macrofauna
(mainly
amphipods,
isopods,
gastropods,
polychaetes, mussels). Most of these taxa can be defined as deposit- and suspensionfeeders that consume, obligatory or optionally, herbivorous food items. Furthermore, smaller
benthic organisms may also play a governing role as trophic linkages to macrofaunal
8
CHAPTER 1
consumers or other predators (e.g. demersal fish, birds). Apart from detritus and bacteria, the
secondary production in shallow aquatic systems can be supported largely by the primary
productivity of benthic microalgae (Daehnick et al, 1992; Moncreiff et al., 1992; Miller et al.,
1996; Moncreiff & Sullivan, 2001). Past studies on grazer-microalgae interactions have
stressed the relative importance of the microflora as food source for benthic consumers
(Fenchel 1968; Fenchel, 1975 a; Sumner & McIntire, 1982; Plante-Cuny & Plante, 1984;
Underwood & Thomas, 1990; Hillebrand et al., 2002; McCormick & Stevenson, 1991).
Consequently, there is now a consensus of opinion that the microphytobenthos can be
considered as the major food source for herbivore invertebrates in the euphotic zone.
Additional support to these findings has been given by recent stable nitrogen, carbon and
sulphur isotope studies which have demonstrated very convincingly that benthic microalgae
are the basis for secondary production at the bottom of shallow freshwater and marine
aquatic systems (Sullivan & Moncreiff, 1990; Hecky & Hesslein, 1995; Moncreiff & Sullivan,
2001; Herman et al., 2000; James et al., 2000b). Due to its high abundance and productivity,
the microphytobenthos is considered to be a reliable and, furthermore, a highly nutritious
food source (Fry & Sherr, 1984; Kitting et al. 1984; Plante-Cuny & Plante, 1984; Decho &
Fleeger, 1988; Jernakoff et al. 1996; James et al., 2000b; Moncreiff & Sullivan, 2001). Thus,
there is sufficient evidence that the relative importance of labile fractions derived from the
renewable pool of microphytobenthos by far outweigh the significance of refractory detritus
material as a food source for benthic organisms.
State of the art
To date, most studies dealing with microphytobenthic assemblages in temperate regions
focused on intertidal areas in marine and estuarine habitats (Admiraal et al., 1984; Pinckney
& Zingmark, 1991; Paterson et al., 1998; Pinckney & Zingmark, 1993; Colijn & De Jonge,
1984; De Jonge & Colijn, 1994; Herman et al., 2000). The restriction of microphytobenthic
research to intertidal mud and sand flats of the northern hemisphere is most likely related to
their great significance in terms of spatial distribution and ecological relevance. In addition,
accessibility and sampling are facilitated in those regions due to low tides and thus intense
research can easily be conducted. In contrast, the potential importance of the
microphytobenthos in littoral zones and especially in freshwater lakes has received little
attention. Thus, its role in freshwater ecosystems still remains poorly investigated (Lowe,
1996). If benthic microalgae in freshwater habitats were addressed at all, these studies
focussed predominantly on epiphytic (Cholkny, 1927; Kann, 1940; Kann, 1993; Ho, 1979;
Cox, 1993) or periphytic communities (Kann, 1940; McCormick & Stevenson, 1991;
Hillebrand & Kahlert, 2001; Hillebrand et al. 2002) whereas studies on the sediment
microflora are rare (Miller et al., 1987; Khondker & Dokulil, 1988; Nozaki et al. 2003;
9
CHAPTER 1
Hillebrand & Kahlert, 2002). However, in the littoral zones of lakes not only do solid surfaces
or macrophyte leaves serve as substrate for benthic microalgae, but soft sediments are
widespread in lakes and their potential as habitat for sediment microalgae has mostly been
neglected. Accordingly, not much is known about microphytobenthic communities in lakes
and it can be speculated that its significance in whole lake ecosystems might be
underestimated.
Focus of the present study
This work aimed at elucidating the impact of grazer-microalgae interactions in benthic foodwebs. As a basis for forthcoming grazing experiments the assessment of microphytobenthic
communities in a marine and a freshwater habitat have been conducted. To show distribution
and seasonality pattern and to detect key species from each habitat were the primary focus
of these field assessments. Proceeding from this basis, several aspects of microalgal
consumption with an emphasis on feeding preferences, selectivity and competitive
interactions were worked out. In this respect the interplay between positive and negative
effects of grazer activity, the impact of active or passive selection and consumers versus
productivity aspects were of considerable interest. Thus, the present study should
demonstrate system-specific characteristics of microphytobenthic communities and to outline
their ecological role in shallow aquatic environments.
In the following chapters, a variety of studies are described that investigated various aspects
of the microphytobenthos in a number of different systems with an emphasis on community
structure and trophic linkages. In Chapter 2 the primary focus was on the assessment of
microphytobenthic communities with traditional methods. The purpose of these studies was,
to evaluate succession patterns at two ecologically relevant and contrasting sites. These
investigations ought to provide insights into the complex structure of the sediment microflora
of each site and to improve our knowledge on taxonomic composition, fluctuation and
seasonal variability. In this respect, the aspects of growth form, structure and morphological
habit were of special interest. Nevertheless, it should be pronounced that the prevailing
motive of assessing various microphytobenthic communities within this study was to detect
key genera in each habitat which could be of considerable significance in the benthic food
web. Thus, the field assessments enabled a classification of trophically relevant microalgal
genera and these investigations served as a basis for following studies on grazer-microalgae
interactions.
However, sampling sediment surfaces for microphytobenthic assessments with traditional
methods in inter- and subtidal areas rapidly revealed several drawbacks. Although the
technology of sampling and analysing microalgal populations in sediments has improved in
the last couple of years (Revsbech et al. 1981; Revsbech & Joergensen, 1983; Wiltshire et
10
CHAPTER 1
al. 1997; Paterson et al. 1998; Barranguet and Kromkamp 2000; Wiltshire 2000), the fact
remains that it is extremely difficult to adequately sample populations of microalgae on
sediments. In order to improve quantitative and qualitative assessments with high spatial and
temporal resolution, a new benthic sensor was devised in this study (Chapter 3). Apart from
monitoring the total chlorophyll concentrations at the sediment surface, the benthic
fluoroprobe did now enable a rapid evaluation of the community structure and distribution in
situ.
All experiments described in Chapter 4-6 aimed at throwing light on the various aspects of
grazer-microalgae interactions and to elucidate the impact of feeding preferences, active
versus passive selectivity and competitive interactions.
A first approach in order to study grazer-microalgae interactions was to focus on gastropod
grazers from natural Schöhsee sediments (Chapter 4). For this purpose small-scale
laboratory experiments with varying densities of the hydrobiid snail Potamopyrgus
antipodarum were conducted. The aim was to quantify grazing efficiencies, feeding
preferences and to evaluate the controversially discussed turning points between positive
and negative effects of grazer activity.
In Chapter 5 the emphasis was on the functional role of several herbivore species in
influencing ecosystem processes in vegetated subtidal habitats. Until relatively recently,
macrophyte beds and sediment communities have been universally studied as isolated
habitats and the coupling between the macrophyte communities and the sediment microflora
have mostly been neglected. However, recent studies showed that the microphytobenthos
contributes to large degrees to the food-web within macrophyte beds and, furthermore, the
necessity of considering this habitat as a whole was emphasised (Sullivan & Moncreiff, 1990;
Moncreiff et al, 1992; Moncreiff & Sullivan, 2001). By establishing treatments with all possible
combinations of grazers, their impact on the sediment microflora beneath and adjacent to
macrophyte beds was investigated. The aspects of constantly high nutrient loads, taxonomic
composition shifts and competitive interactions were of special interest.
In order to investigate feeding selectivity and interspecific competition between herbivore
grazers more closely, a stable isotope enrichment experiment was conducted (Chapter 6).
For this purpose two diatom species with different morphologies and size ranges were
labelled with
13
C and
15
N. Our aim in this study was to use this new tool to investigate the
aspect of assimilation and to distinguish between active selectivity (active choice of food
component) and passive feeding preferences (by mechanism of food intake or digestibility),
factors that are hardly to detect with traditional methods.
The general outcomes of these various studies are further discussed in Chapter 7, thereby
outlining system specific characteristics of the microphytobenthos, its trophic significance as
well as nutritional aspects.
11
CHAPTER 2
Chapter 2
Seasonality and diversity patterns of freshwater and
marine microphytobenthic communities
In this chapter the primary focus was to evaluate succession patterns of microphytobenthic
assemblages at two ecologically relevant and contrasting sites. Furthermore the purpose of
these studies was to provide insights into the complex structure of the sediment microflora of
each site and to improve the knowledge on taxonomic composition, fluctuation and seasonal
variability. The assessments should serve as a basis for forthcoming grazing experiments on
grazer-microalgae interactions.
12
CHAPTER 2
2.1 Introduction
The diversity and functional role of microphytobenthic communities has become a major
topic in benthic research the last two decades (Blanchard, 1990; Blanchard, 1991;
Sundbaeck & Joensson, 1988; Montagna et al., 1995; Plante-Cuny & Plante, 1984). The
term microphytobenthos refers to the microscopic, photosynthetic eukaryotic algae and
Cyanobacteria that live on sediment surfaces. These microorganisms inhabit the surface
layers of sediments on vertical and horizontal scales and therefore they play an important
role for nutrient and oxygen fluxes at the sediment water interface (Joergensen et al., 1983;
Asmus, 1984; Wiltshire et al.,1996). Their occurrence is limited through the depth penetration
of light (MacIntyre et al., 1996) and therefore they are highly related to environmental
parameters, e.g. grain sizes, nutrient supplies, mechanical stress. Their key function as
primary producers in littoral zones has been emphasized in many studies (Daehnick et al.,
1992; Pinckney & Zingmark, 1993; Colijn & DeJonge, 1984) and in addition to this their great
importance within the benthic food-web has also been pointed out (Sumner & McIntire, 1982;
Plante-Cuny & Plante, 1984; Underwood & Thomas, 1990; Hillebrand et al., 2002;
McCormick & Stevenson, 1991; Herman et al., 2000).
Microphytobenthic habitats are widespread: they occur from salt marshes, submerged
aquatic vegetation beds to intertidal and subtidal sediments including beaches (MacIntyre et
al., 1996). Additionally, since the taxonomic composition of microphytobenthic assemblages
is closely related to different nutrient levels, their overall importance as sensitive indicators of
water quality has been stressed (Lange-Bertalot, 1979; Kann, 1986).
Despite their potential importance in the littoral zones of freshwater lakes, microphytobenthos
has received relatively little attention from limnologists and its role in freshwater ecosystems
still remains poorly investigated. Consequently, not much is known about the composition,
fluctuation and seasonal occurrence of the sediment microflora (Lowe, 1996). Until now most
studies in freshwater habitats focused on epiphytic algae (Kann, 1940; Kann, 1993; Cholkny,
1927; Ho, 1979; Cox, 1993) whereas studies on microphytobenthic assemblages are
extremely rare (Kann, 1940; Miller et al., 1987; Khondker & Dokulil, 1988; Cyr, 1998; Nozaki
et al. 2003). However, the littoral zones of lakes not only consist of solid substrate (e.g.
rocks, wood) or macrophyte zones and soft sediments often represent the main substrate in
lakes for microphytobenthic communities. Lake epipelic and epipsammic algae often reach
high biomasses and productivity (Gruendling, 1971; Khondker & Dokulil, 1988; Cyr, 1998),
they regulate nutrient exchanges at the sediment–water interface (Carlton & Wetzel, 1987)
and are an important and high quality food source for benthic invertebrates (Admiraal et al.,
13
CHAPTER 2
1983; Montagna et al, 1995). Therefore, to comprehend a whole lake ecosystem, it is
imperative not to neglect microphytobenthic communities.
In order to understand more about the specific composition of an example lake
microphytobenthic community, a field experiment was conducted in the Schöhsee (Plön,
Germany) from spring to autumn 2001. Two different sites of contrasting sediment types
were chosen in order to compare microalgal communities from both sites and to evaluate
similarities. This study should provide data on abundance, diversity and seasonal variations
of benthic microalgae in the Schöhsee. The assessment of the microphytobenthic community
was to serve as a basis for forthcoming experiments on grazer-microalgae interactions in
order to investigate their role in freshwater sediments.
In addition to the assessment of the freshwater sediment microflora, a sampling campaign of
intertidal sediments at a marine site was conducted. Since past research has stressed the
importance of microphytobenthic assemblages especially in intertidal areas (Pinckney &
Zingmark, 1993; Colijn & De Jonge, 1984; Herman et al., 2000), a marine Wadden Sea site
was also chosen in this study (Dorum, Lower Saxony, Germany). The aim was to elucidate
distribution and seasonality pattern and to reveal temporal fluctuation in algal biomass,
abundance and composition.
2.2 Material & Methods
Schöhsee
Sampling sites
Investigations on natural microphytobenthic assemblages were conducted from May to
October 2001 in the Schöhsee. The Schöhsee has a surface area of 0.78 km² with a
shoreline of 4.7 km. The mean water depth is 10.9 m, with a maximum depth of 29.4 m. The
lake has a low catchment area and is categorized as an oligotrophic lake of low productivity.
Two different sites were chosen in order to investigate the influence of sediment
characteristics (muddy and sandy) on the structure of microalgal communities. Both sites
were 30m apart in the vicinity of the island “Kleiner Warder” and both had an experimental
area of 0.25 m². The sandy site was at 0.8 m water depth whereas the muddy site was
situated at 1.2 m water depth.
Experimental design
In order to apply precise sampling techniques and to keep disturbance of the sediment
surfaces to a minimum while sampling, we decided to deploy sediment caps filled with
natural sediments from the sites prior to the experiment. These caps were made from
cylindrical plastic tubes (∅ 14 mm, surface area 154 mm²) provided with a screw cap. A
14
CHAPTER 2
gauze with a mesh size of 500 µm was glued to the bottom of the caps in order to enable a
sufficient permeability (sketch 1). The caps were filled with autoclaved sediment from each
site respectively, closed with a lid and kept frozen. At the beginning of the experiment 36
caps were inserted by SCUBA into the sediment under frozen conditions at each site,
afterwards the uppermost surface layer of the caps was adjusted to be flush with the surface
of the surrounding sediments and finally the lids were removed. The first sampling took place
four weeks after the field deployment and a monthly sampling interval was chosen. Each
month six caps were chosen randomly from each site, closed under water and transferred to
a tray in order to keep the samples in an upright position. Immediately after sampling the
caps were returned to the water surface and preserved with liquid nitrogen.
screw cap
sediment
plastic tube
25 mm
gauze (500µm)
∅ 14 mm
Sketch 1: Sediment cap made from cylindrical plastic tubes provided
with a screw cap for closing in situ.
Sample preservation
The original Cryolander sampling procedure described by Wiltshire et al. (1997), was used in
a modified way for these Schöhsee sediments. Since the device is not applicable under
water, it was necessary to modify the techniques slightly. The Cryolander consists of a brass
tube (1mm thick) which is 50 mm in diameter and 80 mm in height. In order to preserve the
uppermost surface layer of the caps immediately after the return to the water surface, the
Cryolander was placed on top of the sediment surface of each tube and subsequently liquid
nitrogen (3-5 ml) was gently dribbled on to the absorbent cotton in it. The cotton is at ambient
temperature, and this causes the liquid nitrogen to vaporize. This vapour freezes the
sediment surface immediately without distortion even on a micrometer scale. Once the
15
CHAPTER 2
surface is frozen, the liquid nitrogen was then poured onto it evenly through the Cryolander
mesh. As the liquid nitrogen continues to freeze the sediment, the depth of frozen sediment
increases rapidly until an approximately 2 cm thick layer is frozen. The samples can then be
stored in liquid nitrogen for future use.
Sample processing
The frozen samples were cut into 0.5 cm thin discs in the laboratory. Subsequently the
sediment disc was placed on the stage of a freezing microtome (Leica CM 1900) using a
freezing medium ensuring that the sediment surface was absolutely horizontal. The surface
was then cut into slices at 250 µm intervals down to a depth of 500 µm; the surface layer
from 0-250 µm and the deeper layer from 250-500 µm. A description of the micro-slicing
technique is given in Wiltshire (2000). For cell counts and taxa composition these sediment
sections were fixed with Lugol’s iodide solution, transferred to a Sedgewick-Rafter counting
chamber and counted under an inverted light microscope. The results from the surface and
the sub-surface layer were pooled for taxonomic composition thus the data presented here
comprises of algal cells from 0-500 µm sediment depth. Chlorophyll sample processing and
HPLC-analysis followed the instructions given by Wiltshire (2000). For this purpose the
sediment layers were freeze-dried and suspended in nanograde acetone and frozen at –70
°C for a minimum of 72 hours. Afterwards, the extracts were sonified for 90 minutes, filtered
through 0.2 µm pore-size cellulose filters and then injected by an autosampler straight into
the HPLC-system (Waters Alliance 910). The pigments were identified and quantified using a
diode-array detector (Waters Alliance 910). Chlorophyll measurements were conducted by
extracting pigments from the different sediment layers with acetone (100%) and measured
using high performance liquid chromatography (HPLC, Waters Alliance 910). A detailed
description of the extraction procedure and the measurement conditions is given in Wiltshire
(2000).
Statistical analysis
To test for significant differences in total cell numbers and chlorophyll a contents at both
sediment types a full-factorial ANOVA and a Tukeys HsD-Test were used. For comparisons
of seasonality patterns within every single sediment type a MANOVA and an Duncan-test
were used. Diversity indices were calculated and multivariate analyses were carried out
using PRIMER 5.2 ( 2001 Primer-E Ltd.) and STATISTICA. Diversity was measured by the
Shannon-Weaver function (H’; loge) (Shannon and Weaver, 1963) and Evenness was
calculated by using Pielou’s Evenness (Pielou, 1969). The similarity between samples was
calculated using Cluster Analysis, based on clustering of untransformed data.
16
CHAPTER 2
Dorum (Wadden Sea)
Sampling site
The sediment samples were obtained from a site near Dorum-Neufeld north of the Weser
estuary (southern German Bight) in the Lower Saxony Wadden Sea National Park (53° 44’
00’’ N, 8° 30’ 20’’ E) on the North Sea coast of Germany. Chlorophyll samples were taken
from March to September 2002 (August excluded) on a monthly interval at low tide, whereas
samples for cell numbers and taxonomic composition were retrieved from March to July only.
According to Wickham et al. (2000), the sediment in this area is a fine sand (87% of the
particles in Wentworth class 4, or 0.125 to 0.0625 mm diameter) with an average water
content of 31% (salinity 36 PSU) and an organic content of 2% (dry weight).
Sample preservation
In contrast to the Schöhsee experiment, it was not necessary to use sediment caps as
sampling devices this time. Since the Dorum-site was situated on an intertidal sand flat it was
possible to use the Cryolander in its original way. The sediments were cryolanded in situ at
monthly intervals and samples were always obtained at low tide from the same site. In order
to sample the uppermost surface-layer as accurate as possible, the Cryolander was placed
on the sediment surface and some liquid nitrogen was gently dribbled on to the absorbent
cotton above (for descriptions see section “sample preservation” of Schöhsee sediments).
Sample processing
The cryolanded sediment discs (∅ 50 mm) were cut up in the laboratory into six equal
sediment squares (app. 1 cm² each) whereas three of each blocks were used for chlorophyll
a determination or light-microscope analyses.
Taxonomic analysis, microsclicing of the sediments and chlorophyll determination followed
the same methods as described for Schöhsee sediments.
Statistical analysis
To test for significant changes in chlorophyll concentration an ANOVA was used, whereas
the factor month served as independent variable and chlorophyll a content as dependent
variable. Differences between month were detected with the aid of a Duncan post-hoc test. In
addition, the same statistical procedure was performed for total cell numbers. Diversity
indices were calculated and multivariate analyses were carried out using PRIMER 5.2 (
2001 Primer-E Ltd.) and STATISTICA. Diversity was measured by the Shannon-Wiener
function (H’; loge) (Shannon & Weaver, 1963) and Evenness was calculated by using
Pielou’s Evenness (Pielou, 1969). The similarity between samples was calculated using
Cluster Analysis, based on group average clustering of untransformed data.
17
CHAPTER 2
2.3 Results
Schöhsee
Chlorophyll a content
Total chlorophyll a contents at the muddy site showed fairly uniform values throughout the
whole sampling period (June-October) and no significant differences between months were
detected (p>0.05; figure 1). In addition, no significant differences were found between the
chlorophyll a contents of surface and subsurface sediments (p>0.05). Maximum chlorophyll a
concentrations at the sediment surface occurred in June (0.41 µg cm-2 ± 0.13) and lowest
values in August (0.16 µg cm-2 ± 0.1). The chlorophyll a concentrations at the sandy site
showed higher variations. Significantly higher chlorophyll contents at the sediment surface
were detected in July and September (p< 0.05) when compared to June and October when
chlorophyll a concentrations were fairly low. In July and September the concentrations
reached a maxima of 0.71 µg cm-2 ± 0.36 and 0.74 µg cm-2 ± 0.26. A minima of 0.24 µg cm-2
± 0.01 and 0.24 µg cm-2 ± 0.03 were found in June and October. No significant difference
between the chlorophyll a contents of surface and sub-surface sediments layers was
detected. When comparing the chlorophyll a contents of surface sediments at both sites a
significant difference between sandy and muddy substrate was seen (p=0.02). The
chlorophyll a concentrations on the sandy sediment were significantly higher than on the
muddy sediment. In contrast, these differences disappeared with increasing sediment depth.
SD= 0.36
SD= 0.27
chlorophyll a (µg cm-2)
0,8
mud
sand
0,6
0,4
0,2
0,0
June s
June d
July s
July d
August s
August d
September s
September d
October s
October d
Figure 1: Chlorophyll a concentrations (µg cm-2) on mud and on sand in the Schöhsee sampled from
June to October 2001. Bars present mean values and standard deviations (SD) are given. Different
sediment layers are indicated as s (surface layer; 0-250 µm) and d (deep layer; 250-500 µm).
18
CHAPTER 2
Total cell numbers
The total cell numbers at both sites were highest in May and June and in October 2001
whereas a decline during the summer period (July to September) could be detected (figure
2). In general the highest algal abundances were in the surface layer (0-250µm) of each site.
A decrease in cell numbers occurred with increasing sediment depth (250-500µm). At the
beginning of the sampling period (May) the muddy site showed values of 266 cells cm-2 ± 36
and the sandy site of 165 cells cm-2 ± 24 at the sediment surface (0-250µm) whereas the
subsurface layer (250-500µm) showed values ranging from 153 cells cm-2 ± 27 (sand) and 53
cells cm-2 ± 11 (mud). Lowest cell numbers occurred in September, were only 25 cells cm-2 ±
15 were found at the sediment surface of muddy sediments and 35 cells cm-2 ± 19 at the
surface of the sandy substrate. A slight increase in total cell numbers was found in the
surface layer in October (128 cells cm-2 ± 76, mud; 47 cells cm-2 ± 28, sand). When
comparing the total cell numbers of both sites a significant difference in algal abundance
occurred only in May (p= 0.019). All other sampling periods showed no significant difference
in algal abundance between the muddy and the sandy site (p>0.05).
mud
sand
total cell numbers * cm
-2
300
200
100
0
May s
May d
June s
June d
July s
July d
August s
August d
September s September d
October s
October d
Figure 2: Total cell numbers (cells cm-2) on mud and on sand in the Schöhsee sampled from May to
October 2001. Bars present mean values and standard deviations (SD) are given. Different sediment layers
are indicated as s (surface layer; 0-250 µm) and d (deep layer; 250-500 µm).
When comparing the seasonality patterns for the sediment types, significant differences
between months were found for the sandy site surface but not for the subsurface layer. The
cell numbers of the sediment surfaces showed significant differences between the samples
taken in May and June compared to surface sediments sampled from July to October
(p<0.05). In contrast no significant differences were detected for the subsurface layer of the
sandy site. The May cell numbers for the muddy sediment surface showed significant
differences compared to all other months (p<0.05). In addition, the June algal abundance in
19
CHAPTER 2
the surface layer were significantly different to May (p= 0.0235), July (p= 0.0341) and
September (p= 0.0161). The surface sediments sampled in September and October were
significantly different from one another (p= 0.0404). In contrast to the sandy site, major
variations also occurred in the subsurface layers of muddy sediments. Algal abundance
beneath the surface showed significantly higher cell numbers in May compared to samples
taken from June to October (p<0.05). In addition, June subsurface abundance data were
significantly different to July (p= 0.0149) and again the September samples compared to
October (p= 0.0360).
Taxonomic composition
Both sites showed no significant taxonomic composition differences (p=0.58). The sandy as
well as the muddy sediments were colonized by similar algal assemblages and both sites
showed the same seasonality patterns.
The similarities between months on the muddy site showed a clustering pattern of algal
assemblages sampled in May and June (figure 3).
0
similarity (%)
20
40
60
80
M9
M7
M6
M6
M6
M8
M5
M5
M5
M9
M9
M7
M8
M8
M7
M 10
M 10
M 10
100
month
Figure 3: Similarities (%) in taxonomic composition between months on mud (M) in the Schöhsee
sampled from May (M5) to October (M10).Absolute cell numbers in the top 500 µm of the sediments
are considered.
20
CHAPTER 2
100
Melosira
Pinnularia
Diploneis
Stauroneis
Cymbella
Navicula
Cyclotella
Nitzschia
Synedra
Placoneis
Surirella
Fragilaria
Gyrosigma
Amphora
Caloneis
Chlorophyta
Pediastrum
taxonomic composition (%)
80
60
40
20
0
May
June
July
August
September October
month
Figure 4: Taxonomic composition on mud in the Schöhsee sampled from May to October 2001. Relative
abundances of different taxonomic groups are calculated as % of the total algal cells.
0
similarity (%)
20
40
60
80
S9
S9
S 10
S7
S8
S7
S9
S 10
S 10
S6
S6
S7
S6
S5
S5
S5
S8
100
month
Figure 5: Similarities (%) in taxonomic composition between months on sand (S) in the Schöhsee
sampled from May (S5) to October (S10). Absolute cell numbers in the top 500 µm of the sediments
are considered.
21
CHAPTER 2
Both months showed the dominance of Fragilaria sp. (17-19%), Navicula sp. (12-19%),
Nitzschia sp. (5-13%), Stauroneis sp. (8-9%) and Pinnularia sp. (4-13%) (figure 4).
The chain-forming bentho-pelagic Melosira sp. comprised 5-6% of the total algal community.
The genus Synedra sp. was present in both months but showed a strong dominance only in
June (35%). In addition, filamentous green algae comprised 5% to the total algal community
in May and the coccal green algae Pediastrum sp. 1 %. The numbers decreased in June
dramatically. For all other diatom taxa percentages of 1-4% of the total were found. During
summertime there was a clear change in taxonomic composition when compared to the
spring period and samples from July to September showed clear similarities. From July
onwards the algal community changed to a Stauroneis sp.-dominated population which
contributed from 28 to 43% to the total algal community. Other dominant taxa were: Synedra
sp. (11-15%), Navicula sp. (11-19%) and Pinnularia sp. (11-13%). In addition, the taxon
Gyrosigma sp. was highly abundant in July (20%). In October these distribution patterns
changed and a clear dominance of Nitzschia sp. was seen (35%). Other abundant taxa in
October were: Diploneis sp. (15%), Stauroneis sp. (16%) and Pinnularia sp. (11%). From
July to October no green algae were found.
On sandy substrate similar distribution and seasonality patterns were found as for muddy
sediments. Cluster analysis revealed similarities between May and June samples (figure 5).
taxonomic composition (%)
100
Melosira
Pinnularia
Diploneis
Stauroneis
Cymbella
Navicula
Cyclotella
Nitzschia
Synedra
Placoneis
Surirella
Fragilaria
Gyrosigma
Amphora
Caloneis
Pediastrum
80
60
40
20
0
May
June
July
August
September October
month
Figure 6: Taxonomic composition on sand in the Schöhsee sampled from May to October 2001. Relative
abundances of different taxonomic groups are calculated as % of the total algal cells.
22
CHAPTER 2
Both months showed high percentages of Synedra sp. (23-30%), Fragilaria sp. (14-17%) and
Navicula sp. (13-14%) (figure 6).
In June Stauroneis sp. had increased to 16% of the total algal community and the genus
Amphora appeared (5%). In addition, the green algae Pediastrum sp. comprised up to 5 % of
the total algal community in May and up to 6 % in June. With the start of the summer period
the samples changed in composition. In July the algal community still showed similar
patterns as in May and June but cell numbers of Stauroneis sp. (30%) and Amphora sp.
(10%) increased whereas percentages of Navicula sp., Synedra sp. and Fragilaria sp.
decreased. During the summer (July- September) the sediments were similar and Stauroneis
sp. (30-47%) and Synedra sp. (11-23%) dominated. The genus Amphora sp. (6-10%),
Navicula sp. (5-15%) and Pinnularia sp. (8-9%) contributed minor percentages to the total
algal community. In October equal parts of the microphytobenthic assemblage were
presented by Navicula sp. (32%) and Stauroneis sp. (31%) and Synedra-cells decreased in
number (9%). Pinnularia sp. made up 10% of the autumn community and Gyrosigma sp.
appeared (7%). No green algal taxa were found during the summer and autumn period.
Diversity and Evenness
The diversity index H’ showed similar diversity patterns for both the sandy and the muddy
substrate. Highest diversities were shown for microphytobenthic communities in May and
June with H’ ranging between of 2.0-2.2 on mud and 2.1-2.2 on sand (figure 7). During the
summer period a continuous decrease in diversity was been reaching a minimum of 1.59 ±
0.16 (mud) and 1.60 ± 0.20 (sand) in August. A slight diversity increase was seen for
September and October with H’ ranging between of 1.71-1.80 on mud and 1.61-1.72 on
sand. Pielou’s Evenness index E showed no clear trend for seasonality and a high variability
between months. On muddy and on sandy substrates E was lowest in August (0.71 ± 0.04
and 0.71 ± 0.11). A maximum for E was found on muddy sediments in September (0.85 ±
0.09).
23
CHAPTER 2
2,6
H' (Diversity) mud
E (Evenness) mud
H' (Diversity) sand
E (Evenness) sand
2,4
Diversity indices H' & E
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
May
June
July
August
September October
month
Figure 7: Diversity indices H’ (Diversity) and E (Evenness) on mud and on sand in the Schöhsee
sampled from May to October 2001. Mean values and standard deviations (SD) are given.
24
CHAPTER 2
Dorum
Chlorophyll a content
During the whole sampling period from March to September the chlorophyll a contents of the
Dorum sediments were highly variable. In general the concentrations in surface layer were
slightly higher than at depth, although these differences were not significant (figure 8;
p>0.05). Lowest contents at the sediment surface (0-250 µm) occurred in May (0.27 µg cm-2
± 0.04) and highest in June (0.69 µg cm-2 ± 0.23). Apart from April, the June surface values
were significantly higher than all other surface sediments sampled (p<0.05). In contrast,
chlorophyll values from subsurface layers (250-500 µm) were more evenly distributed and
significantly lower values were found only in March, May and September were achieved in
comparison to June-samples.
1,2
chlorophyll a (µg cm-2)
1,0
0,8
0,6
0,4
0,2
0,0
March s
March d
April s
April d
May s
May d
June s
June d
July s
July d
August s August d September sSeptember d
Figure 8: Chlorophyll a concentrations (µg cm-2) on intertidal sediments (Dorum) sampled from March to
September 2002. Bars present mean values and standard deviations are given. Different sediment layers
are indicated as s (surface layer; 0-250 µm) and d (deep layer; 250-500 µm).
Total cell numbers
Highest cell numbers were found for surface sediments in April (20781 cells cm-2 ± 11608). A
minimum of 4142 cells cm-2 ± 2029 was found in May (figure 9). When compared to algal
abundances of surface sediments in March and April, the May values were significantly lower
(p<0.05). In addition, cell numbers from 250-500 µm in March showed remarkably high
numbers (19854 cells cm-² ± 17344) and they were significantly higher than sub-surface May
cell numbers.
25
CHAPTER 2
total cell numbers * cm
-2
40000
30000
20000
10000
0
March s
March d
April s
April d
May s
May d
June s
June d
July s
July d
Figure 9: Total cell numbers (cells cm-2) on intertidal sediments (Dorum) sampled from March to July
2002. Bars present mean values and standard deviations are given. Different sediment layers are indicated
as s (surface layer; 0-250 µm) and d (deep layer; 250-500 µm).
Taxonomic composition
During the sampling season from March to July, all months showed distinct differences in
algal taxonomic composition. However, cluster analyses showed similarities between the
March and April as well as the June and July community (figure 10). In contrast, the algal
assemblages in May were distinctly different. The taxonomic composition of algal
assemblages showed an overall dominance of Navicula sp. in March (91%), whereas the
remaining algae consisted predominantly of Amphora sp. (3%) and Stauroneis sp. (3%)
(figure 11). In April Navicula sp. comprised of more than 50% of the total algal community
and the benthopelagic Cylindrotheca closterium made up 27%. Minor contributions (3-5%)
consisted of Amphora sp., Stauroneis sp. and some undetermined diatom species. When
compared to the community patterns observed in April, Navicula sp. was also found in May
(63%) and, the cyanobacterium Merismopedia sp. also appeared (10%). The remaining
proportion was made of Diploneis sp., Amphora sp., Stauroneis sp., Cylindrotheca closterium
and undetermined diatom species. In June and July the relative contribution of Merismopedia
sp. increased considerably making up 32% and 80% respectively. Simultaneously the
proportions of Navicula sp. declined to 41% (June) and 12% in July. The number of
taxonomic groups contributing to the total algal community remained almost constant even
though.
26
CHAPTER 2
20
similarity (%)
40
60
80
5C
5A
5A
5C
3C
5B
3C
5A
3C
5B
6C
5B
6A
7A
6B
6B
5C
6B
6C
6C
6A
6A
7C
7C
7A
7A
7B
7C
7B
7B
3B
4B
3B
3B
3A
3A
3A
4C
4C
4B
4B
4A
4A
4A
4C
100
month
Figure 10: Similarities (%) in taxonomic composition between months of intertidal
sediments (Dorum) sampled from March (3) to July (7). Sub-samples from each Cryolander
are indicated as A-C. Absolute cell numbers in the top 500 µm of the sediments are
considered.
100
Navicula
Achnanthes
Diploneis
Diatoms (undetermined)
Amphora
Amphiphora
Nitzschia
Gyrosigma
Licmophora
Mastogloia
Stauroneis
Cocconeis
Caloneis
Epithemnia
Cymbella
Cylindrotheca closterium
Merismopedia
taxonomic composition (%)
80
60
40
20
0
March
April
May
June
July
month
Figure 11: Taxonomic composition on intertidal sediments (Dorum) sampled from March to July 2002.
Relative abundances of different taxonomic groups are calculated as % of the total algal cells.
27
CHAPTER 2
Diversity and Evenness
The diversity index H’ and the evenness E showed a steady increase from March to June
followed by an abrupt decline in July (figure 12). A minimum of 0.5 ± 0.3 for H’ and 0.3 ± 0.1
for E was seen in March and these values were significantly lower than from April to June
1,8
H' (Diversity)
E (Evenness)
1,6
Diversity indices H' & E
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
March
April
May
June
July
month
Figure 12: Diversity indices H’ (Diversity) and E (Evenness) on intertidal sediments (Dorum)
sampled from March to July 2002. Mean values and standard deviations (SD) are given.
(p<0.05). When compared to the sampling period from March to May, the diversity index H’
increased significantly in June to values of 1.5 ± 0.1 (p<0.05). A similar trend was recorded
for Pielou’s evenness. However, in this case, the increase in June was only significant in
comparison to March and April data (p<0.05). The sediment surfaces sampled in July
showed a sharp decline to March values in diversity and evenness. These values were
significantly lower than H’- and E-values detected from April to June (p<0.05). The
differences in diversity resulted mainly from the relative levels of dominance rather than from
changes in the number of forms.
28
CHAPTER 2
2.4 Discussion
Schöhsee
Chlorophyll a contents
The total chlorophyll a contents of the uppermost surface layers (0-500 µm) at both sites
showed similar concentrations ranging from 0.2 (mud, August) to 1.1 µg chlorophyll a cm-2
(sand, September). When compared to former studies on lake sediments these values are at
the lower end of concentrations measured at lake “Neusiedlersee”, “Mikolajskie”, “Biwa” and
at three lakes in Southern Ontario (Wasmund, 1984; Khondker & Dokulil, 1988; Cyr, 1998;
Nozaki et al., 2003). In cases where much higher chlorophyll a concentrations occurred, they
were usually correlated with mass occurrence of special algal taxa depending on light and
nutrient availability at different times of the year. Nozaki et al. (2003), for example found that
the development of filamentous green algae contributed to a sharp increase in algal biomass
from April to June in lake Biwa and that these mass occurrences were the result of
eutrophication. The Schöhsee, with its low catchment area, is categorized as an oligotrophic
lake. Mass occurrences of green or blue-green benthic microalgae were not detected in this
study, neither at the muddy nor at the sandy site. The absence of these algal groups might
be responsible for the low chlorophyll a concentrations detected at the sediment surfaces of
Schöhsee. It can be speculated that low nutrient availabilities may have caused these low
chlorophyll a contents at the sediment surface. This might be a typical feature of
unproductive lake systems.
The vertical distribution of chlorophyll a contents showed different patterns for the muddy and
the sandy site. On the muddy substrate the surface layer chlorophyll concentrations were
similar to the sub-surface layer values. No significant decline with increasing sediment depth
could be detected. In contrast, total chlorophyll a concentrations on the sandy substrate in
general showed higher values at the sediment surface than at deeper sediment layers,
although this pattern changed in October. In general total chlorophyll a concentrations are
known to decline with increasing sediment depth (Wasmund, 1984) and therefore highest
chlorophyll a concentrations are usually found at the surficial millimetre of sediments (Gaetje,
1992; Wiltshire 2000). However, chlorophyll distribution patterns are highly variable as they
are influenced by many external factors, e.g. hydrography, grazers, sediment grain size,
organic contents as well as physiological features of microalgal cells and their migrating
behaviour (Admiraal et al., 1984; Wasmund, 1984; Pinckney & Zingmark, 1991; Gaetje,
1992; MacIntyre et al., 1996).
But when comparing chlorophyll data from different studies, it is necessary to take into
account the different sediment volumes sampled as all these studies used different sampling
techniques. In contrast to our study, the previously conducted studies used the first top
29
CHAPTER 2
centimetre of the sediment surface in order to detect total chlorophyll a contents. Thus, the
amount of pigments measured also includes chlorophyll a from deeper sediment layers.
Wasmund (1989) for example found that appreciable amounts of intact chlorophyll could be
found down to 10 cm sediment depths and consequently these pigments were situated below
the depth to which light penetrates. However, a significant portion of chlorophyll found at
deeper sediment layers originates from settled planktonic material (Stevenson et al., 1985;
Wasmund, 1989) which is not distinguishable from benthic microalgae. Therefore, detecting
chlorophyll from surface sediments at a centimetre scale may lead to an overestimation of
chlorophyll a derived from benthic microalgae alone. Compounding this is the fact that the
chlorophyll a contents of algae are highly variable depending on season, physiological status
and physical conditions. As a consequence chlorophyll a measurements can only be
indicative of biomass (Wolff, 1979; De Jonge & Colijn, 1994).
Cell numbers
Cell numbers at both sites reached a maximum in spring followed by a summer decline and
higher numbers again in autumn. The spring maximum is consistent with seasonality
patterns investigated for different lake systems all over the world (Kann, 1940; Khondker &
Dokulil, 1988; Nozaki et al., 2003). High productivities of microalgae are known to be directly
linked to environmental parameters (light, nutrient, temperature). As nutrient and light
availabilities are generally high at this time of the year, spring biomass maxima of
microphytobenthic algae are an expected phenomena. During summer, nutrients usually get
depleted and macrophyte growth increases in the littoral zones and reduces light availability
to a large extent. Thus, a decline of microalgal abundances must be expected. However, in
the Schöhsee do not only macrophytes like Phragmites sp. and Potamogeton sp. affect the
light regime at the sediment surfaces, but large Alder populations (Alnus sp.) also contribute
to the shading of the shoreline. Evidence for decreasing light intensities at the experimental
sites in summer is found in the development adjacent populations of Chara aspera which is
known to be highly adapted to low light regimes (Kann, 1940).
These patterns are supported by seasonal variations in lake “Biwa” (Nozaki et al., 2003) and
from several northern German lakes (Kann, 1940), where abundances declined during the
summer period either. Thus, these studies are in good correspondence to seasonal algal
abundance observed here. In contrast, studies from “Neusiedlersee” showed reasonable
abundances of epipelic algae during summer.
Similar to the lakes “Neusiedlersee” (Khondker & Dokuli, 1988) and “Biwa” (Nozaki et al.,
2003), autumn abundances were low for the Schöhsee. As nutrient and light availabilities are
parameters that show large variations between lakes and even within a single lake system,
no general seasonality patterns for all lakes can be proposed. Each lake system has its
30
CHAPTER 2
unique characteristics and especially the different nutrient levels, latitudes and physical
parameters must be considered when comparing seasonality patterns.
Cell numbers at both sites showed highest cell numbers in the uppermost surface layer and
a decrease with increasing sediment depth. This is not astonishing as the light availability is
highest at the sediment surface and photosynthesis is restricted to a very thin layer at the
sediment surface (Wasmund, 1984; Carlton & Wetzel, 1987). Therefore, in the sublittoral
zones, algal abundances are usually expected to be highest at the uppermost sediment
surface.
When comparing total chlorophyll contents and cell numbers no positive correlation was
found. Usually both parameters are used to describe biomass characteristics of algal
communities and often good correlations are found (Karlstroem & Backlund, 1977; Khondker
& Dokuli, 1988; Mitbavkar & Anil, 2002). However, as already pointed out before, the total
chlorophyll a content of algal communities is highly variable depending on e.g. the
physiological status of algal cells, cell sizes and light intensities (Wolff, 1979) and thus both
parameters do not always correlate.
Taxonomic composition & diversity
The taxonomic composition of benthic microalgae in the Schöhsee was mainly restricted to
diatom assemblages. In contrast to other lake sediments, where green algae or
Cyanobacteria are known to be abundant in some seasons, diatoms where by far the most
important taxonomic group found associated with Schöhsee sediments. Green coccal and
filamentous algae were only detected in spring in low percentages and these then completely
disappeared from July to October.
Mass occurrences of filamentous green algae or Cyanobacteria, as described for several
lake systems, are known to be directly linked to high water column nutrient loadings (Kann,
1940; Kann 1993; Hillebrand & Kahlert, 2001; Nozaki et al., 2003). The Schöhsee, with its
oligotrophic and unproductive character, seems to favour diatom-dominated sediment
communities. A possible explanation could be that that diatoms are not as dependent on
water column nutrients as filamentous algae (Kann, 1940; Kann, 1993) since they have
highly effective mechanisms in getting access to nutrients at the sediment-water interface
(Admiraal, 1984; Sundbaeck et al., 1991; Wiltshire, 1993; Hillebrand & Kahlert; 2002). In
addition, diatoms are known for their mobility and they show diel rhythms of vertical migration
in response to a multitude of factors e.g. light, hydrodynamics, tides, nutrient and perhaps as
a strategy protecting them from grazing or erosion (Admiraal, 1984; Pinckney & Zingmark,
1991; Paterson et al., 1998). These characteristics might promote a higher competitiveness
of diatoms in unproductive lakes. The assumption is supported by the fact, that green algae
occurred only in spring when enough nutrients were potentially still available, but as soon as
31
CHAPTER 2
the nutrient levels declined the community was dominated by diatoms again. This
phenomenon is in good agreement with studies conducted by Hillebrand & Kahlert (2001)
and Nozaki et al. (2003), who found high chlorophyte numbers in spring.
In addition to nutrient availabilities, different microalgal taxa have different light demands and
consequently light conditions can regulate colonization patterns of microalgal assemblages.
As described by Kann (1940), benthic diatoms have a highly adaptive photosynthetic
pigment apparatus and they are well adapted to low-light regimes. Depending on the light
intensities, seasonal shifts in the xanthophyll cycle have been shown (Wiltshire et al., 1997)
and thus their viability at different light regimes is most likely related to their adaptive
potential. In contrast to chlorophyll a, these pigments have more efficient photosynthetic
yields and light absorption capacities and therefore it enables diatoms to grow at low-light
conditions. The two investigation sites of this study, were situated in the upper sublittoral
zone with a north-easterly orientation. The plots were close to the shore and, as already
pointed out before, the shoreline was characterized by large Alder-populations and wast
Chara aspera-meadows indicating low-light conditions. It can be concluded that the diatoms
probably were better adapted to these environmental factors as they have a higher resilience
to low light conditions. The colonization patterns can not be generalized for the whole littoral
zone of the Schöhsee as different littoral zones probably had varying environmental
conditions.
The diversities detected for Schöhsee sediments at both sites showed relatively high values
which is similar to data from “Neusiedlersee” (Khondker & Dokulil, 1988). Most of the
dominant taxa can be categorized as pennate, prostrate forms, which were either
epipsammic or epipelic. Epipsammic diatoms grow attached to sediment particles whereby
epipelic forms actively move through the sediments by means of their raphes (Round, 1971).
Prostrate forms are typical for variable environments (sand, mud) where disturbance,
predominantly through wave action or current, plays an important role in structuring the algal
community. On highly exposed substrates, however, algal assemblages are found to be
dominated by epipsammic forms (Wolff, 1979). Thus, unstable sediments are usually
colonized by prostrate diatoms, forming distinctly flat, two-dimensional communities (Miller et
al., 1987). In contrast with epi- or periphyton communities, where a third, vertical dimension
usually develops over time, this did not occur with these microphytobenthic biofilms and only
few erect forms were present.
In our study the sandy as well as the muddy sediments were colonized by similar algal
assemblages and both sites showed the same seasonality patterns. A clear succession from
spring to summer was observed, as a shift from a Navicula-, Fragilaria- and Synedradominated population in spring to a Stauroneis-dominated community in summer was
detected. Furthermore, the autumn community also showed clear changes and this was
32
CHAPTER 2
especially distinct on muddy substrate where the algal population changed to a Nitzschiadominated community in October. In addition, the genus Amphora sp. and Gyrosigma sp.
gained considerably in importance in summer and autumn.
It can be summarized that the microphytobenthic communities at the Schöhsee-sites were
dominated both by epipelic and epipsammic diatoms (e.g. Navicula sp., Nitzschia sp.,
Pinnularia sp., Stauroneis sp.) thus indicating an intermediate degree of exposition. This is in
good correspondence with studies conducted by Miller et al. (1987). Wasmund (1984) also
found that these diatom taxa are abundant on epipelic or epipsammic substrate. However,
some typical forms like Cocconeis sp. or Achnanthes sp., which are usually found closely
attached to sediment particles were missing at our sites. This might be considered
characteristic for Schöhsee sediments. In addition, it has to be pointed out that only one
erect form was present in considerable amounts throughout the season and this was the
genus Synedra. This microalga has the ability to stick to surfaces by forming mucilage pads
and apparently this feature made it possible for the algae to grow well even on unstable
substrates. Only two other erect forms were found periodically- the chain-forming diatoms
Fragilaria sp. and Melosira sp.. The vegetative cells of both diatoms occurred mainly in
spring at the sediment surfaces and as these taxa are known to have bentho-pelagic life
cycles, it can be assumed that they had settled from the water column and inhabited, for a
short time period, the surface of the sediments. They also could have germinated from
resting stages in the sediment.
Conclusions Schöhsee
The microphytobenthic assemblages in the Schöhsee were characterized by relatively low
chlorophyll contents and cell numbers. The low productivity was most likely related to the
oligotrophic character of the Schöhsee and by reduced light conditions in the near-shore
sublittoral. The algal assemblages at both sites showed distinct seasonality and succession
patterns with clear shifts in community composition in spring, summer and autumn. In
general, the sediment microflora consisted predominately of prostrate diatoms forming a
distinctly flat, two-dimensional community. Cyanobacteria, green algae and erect diatoms
occurred rarely and in low abundances.
Dorum
Chlorophyll a contents
At our Wadden Sea site Dorum, the chlorophyll concentrations at the sediment surface (0500 µm) showed distinct variations during the sampling period that ranged from 0.5 to 1.3 µg
cm-2. Compared to other intertidal regions like the Ems-Dollard estuary or intertidal sand flats
in Sylt, where ranges of 2.5-25.0 µg cm-2 were recorded (Colijn & De Jonge, 1984; De Jonge
33
CHAPTER 2
& Colijn, 1994; Agatz et al., 1999), our contents in Dorum seem fairly low. But since the
previously mentioned studies used different analytical techniques and, in addition, they
considered the uppermost 1 cm of the sediment surface for chlorophyll determination,
comparisons are not really possible. Similar surface chlorophyll concentrations to our study
were achieved from several studies sampling the top 1000 µm of surface layer, showing
ranges of 0.1 to 4.0 µg cm-2 in the Westerschelde estuary (Barranguet & Kromkamp, 2000;
Middelburg et al., 2000), 0.5 to 8.0 µg cm-2 on a sandy tidal flat in Sylt (German Wadden Sea;
Riethmueller et al., 2000) and Gaetje (1992) gives values of 1.3 to 5.0 µg cm-2 for sandy and
muddy substrates in the Elbe Estuary.
In general, pigment concentrations are known to decrease with increasing sediment depth
(Wasmund, 1984; De Jonge & Colijn, 1994) and highest chlorophyll concentrations are
known to occur in the uppermost 1000 µm of the sediment surface (Gaetje, 1992; Wiltshire,
2000). These distribution patterns result from the fact, that light availability is highest at the
sediment surface and photosynthesis is restricted to a very thin layer at the sediment surface
(Wasmund, 1984; Carlton & Wetzel, 1987). However, the thickness of the euphotic sediment
layer varies between 2 and 5 mm, depending on sediment characteristics e.g. grain size
(Wolff, 1979), and therefore the occurrence of benthic microalgae is highly related to the
depth penetration of light into the sediment. The sediment characteristics of the Dorum tidal
flat obviously favoured a homogeneous distribution in the top 500 µm of the sediment surface
and this is still within the depth range of 1000 µm given by Gaetje (1992) and Wiltshire
(2000) were highest chlorophyll concentrations are known to occur.
Cell numbers
The microalgae cell numbers showed high seasonal variation. A maximum was found at the
sediment surface in April and a minimum in May. When compared to cell counts from other
regions in the German Wadden Sea area, the Dorum cell numbers are at the lower range of
data (Gaetje, 1992; Agatz et al. 1999; Riethmueller et al. 2000). However this is likely to be a
result of different sampling techniques and the sampled sediment volumes since the
previously mentioned studies sampled not only the top millimetre of the sediment surface
and thus, the amount of pigments measured also includes chlorophyll a from deeper
sediment layers. Pronounced seasonality is a typical feature of microphytobenthic
communities in intertidal areas and several studies have shown that seasonal variations are
mainly driven by temperature and irradiance (Admiraal & Peletier, 1980; Blanchard & CariouLe Gall, 1994; Sundbaeck et al., 2000). In temperate regions intertidal benthic microalgae
show biomass peaks similar to the water column in spring due to an increase in sediment
surface temperature. The Dorum site showed a biomass maximum in spring as well as slight
decreases in summer. This data fits well with seasonality patterns observed by several
34
CHAPTER 2
authors (Admiraal & Peletier, 1980; Gaetje, 1992; De Jonge & Colijn, 1994; Sundbaeck et al.,
2000).
Taxonomic composition & diversity
The taxonomic composition of the microphytobenthic community showed three different
successional phases that occurred during the sampling period from May to July 2002. The
first phase indicates a spring bloom that was predominately dominated by Navicula-species.
This small, prostrate form is widespread on intertidal flats and this genus is known to
contribute substantially to microphytobenthic communities (Gaetje, 1992; Agatz et al. 1999;
Riethmueller et al. 2000, Mitbavkar & Anil, 2002; Hagerthey et al., 2002). But in April
proportions of Navicula sp. decreased and this reduction was directly related to the
occurrence of the diatom species C. closterium which contributed to considerable amounts to
the total algal community in April. Gaetje (1992) found this particular diatom species to out
compete Navicula species in case of high grazing pressure while assuming that the largesized and needle-shaped morphology of C. closterium favoured its grazing-resistance. Since
Navicula-species are known to be a preferred food item for several meiofauna organisms
(Admiraal et al., 1983), it seems likely that the observed sudden biomass depression of
Navicula-cells in this study were most likely the result of efficient and highly selective grazing.
The importance of C. closterium for intertidal sand- and mud flats has been emphasized by
several authors, as this species has been found to be one of the most important
exopolysaccharides producers and thus make the special matrix which increases sediment
stability (Alcoverro et al., 2000; Staats et al., 2000; Underwood & Provot, 2000). In May,
however, the relative proportion of C. closterium decreased and apart from high abundances
of Navicula sp., other taxonomic groups gained considerable importance. From cluster
analysis it was shown that the diatom composition in May mainly reflected the spring and
summer populations and thus, this second seasonal stage could be characterized as
transition phase. This taxonomic composition was underpinned by the appearance of
Merismopedia sp., a Cyanobacterium that can be found regularly on sediment surfaces
forming mucus filament networks (Gaetje, 1992; Agatz et al. 1999; Riethmueller et al., 2000).
Since Merismopedia sp. is known to occur on nutrient-poor intertidal flats (Agatz et al., 1999),
the occurrence of the cyanobacterium was most likely related to diminished nutrient supplies
resulting from the previous diatom spring bloom. The assumption, that not only algal
composition but also total algal biomass could be affected by potential nutrient declines at
the sediment-water interface, was supported by low chlorophyll concentrations and
decreasing cell numbers in May. This is in good correspondence to investigations conducted
by De Jonge & Colijn (1994) who detected gradual or sudden decreases in
microphytobenthic biomass right after a spring bloom event. However, not only external
35
CHAPTER 2
parameters such as nutrient and light availabilities are potential causes for biomass declines
during the summer period since the impact of grazer presence in reducing algal biomass to
considerable degrees has been stressed in several studies (Plante-Cuny & Plante, 1984;
Underwood & Thomas, 1990; Hillebrand et al., 2002; McCormick & Stevenson, 1991). Most
benthic consumers show high abundances, reproduction and growth rates in summer and
thus, grazing losses often exceed microalgal production at this time of the year. These
findings are supported by several studies which showed that such summer depressions of
microphytobenthic biomass are directly related to high grazer efficiencies (Colijn & Dijkema,
1981; Gaetje, 1992).
Thereafter, the transition phase was followed by a third vegetation phase, the summer
period, which was clearly dominated by Merismopedia sp. and which displaced the relative
proportions of the remaining diatom taxa. The occasionally high abundances of
Cyanobacteria on the Dorum sandflat is not astonishing as previous studies have shown the
overall importance of Cyanobacteria in intertidal, microphytobenthic assemblages (Yallop et
al., 1994; Taylor & Paterson, 1998). It can be summarized that the habit of the
microphytobenthos in the intertidal of the Wadden Sea site was characterized by a distinct,
flat, two-dimensional community where other forms, such as stalked or chained diatoms,
were missing. Thus also here, a typical microphytobenthic assemblage as predicted by Miller
et al. (1987) was found.
The seasonal distribution of taxonomic groups showed a steady increase of diversity and
evenness over the course of the sampling period and an abrupt decrease in summer. The
actual low values in March and July, however, indicate associations influenced by a
predominance of a few taxa rather than from changes in the number of taxonomic groups. In
this regard, the low diversity was obviously related to mass occurrences of the diatom
Navicula sp. (March) and the Cyanobacterium Merismopedia sp. (July). When opposed to
diversity variables observed by Hillebrand & Sommer (1997) and Agatz et al. (1999), the
mean diversities in Dorum were within similar ranges. Furthermore, as pointed out by Agatz
et al. (1999), the herein detected diversity values might indicate an intertidal area of
moderate nutrient supplies.
Conclusions Dorum
Chlorophyll concentrations and cell numbers of the intertidal flat near Dorum showed
considerable temporal and seasonal variations. In good correspondence to biomass
changes, these fluctuations were underlined by distinct taxonomic compositions which
enabled the classification of a spring, a transition and a summer phase. Clear community
and diversity shifts where predominantly induced by mass occurrences of Navicula sp. and
Merismopedia sp. The composition of sediment microflora in Dorum represented a typical
36
CHAPTER 2
microphytobenthic community with its distinct flat and two-dimensional habit and the absence
of stalked or chained forms throughout the season.
37
CHAPTER 3
Chapter 3
‘Spectral fingerprinting’ for specific algal groups on
sediments in situ: a new sensor
This chapter presents a new benthic sensor for the differentiation of algal groups on
sediments in situ. This instrument was developed in order to improve quantitative and
qualitative assessments with high spatial and temporal resolution. This non-retrospect
approach was successfully applied in this study and results from the differentiation on
microphytobenthos populations in the field and in mesocosm experiments are compared with
algal biomass and pigment estimations. The potential role of this sensor for ground truth
investigations on the large-scale spatial and temporal variation of algal populations in
sediments is discussed.
CHAPTER 3
3.1 Introduction
Microphytobenthos of marine and freshwater sediments is a diverse assemblage of pro- and
eukaryotic autotrophic microalgae. The qualitative and quantitative assessment of this
important algal association, which is the main primary producer in shallow, especially
intertidal and littoral, coastal ecosystems (Admiraal & Peletier, 1980; Colijn & DeJonge,
1984) is a major scientific challenge. The determination of algal biomass has always been
problematic. Since 1890, when Haeckel, who considered phytoplankton counting a task
which could not be accomplished without ‘ruin of mind and body’, not much has changed and
this is even more true for algal biomass on sediments. Until the early nineties
microphytobenthos was a poorly studied subject primarily because the methods available to
us were few and difficult. As the technology of sampling and analysing microalgal
populations in sediments has improved (Revsbech et al., 1981; Revsbech & Joergensen,
1983; Wiltshire et al., 1997; Paterson et al., 1998; Barranguet & Kromkamp, 2000; Wiltshire,
2000), studies on microphytobenthos populations have become increasingly popular.
However, the fact remains that it is extremely difficult to adequately sample populations of
microalgae on sediments and the requirement of differentiating algal populations over large
areas for ground-truthing in remote sensing studies is usually statistically difficult to achieve
because the sediments are so patchy. Even the improved current methods, although quite
accurate, involve rather time consuming enumeration to species or major taxonomic groups
using counting chamber methods (Utermoehl, 1958) or High Performance Liquid
Chromatography analyses (Wiltshire & Schroeder, 1994) of the sediments using microtome
methods (Wiltshire, 2000). Perhaps the greatest problem with these methods is that they are
usually retrospect and not suited to instant assays in situ. Aspects such as patchiness and
algal migration are often not detected.
Fluorescence-emission measured around 685 nm is widely accepted as a measure of
chlorophyll contents of algae in aquatic systems. Indeed, depth profiling of chlorophyll
fluorescence in water bodies has been carried out since the early seventies (Kiefer, 1973;
Cullen et al., 1997). Since then some attempts have been made to distinguish different algal
groups in phytoplankton communities using their fluorescence properties (Yentsch &
Yentsch, 1979; Yentsch & Phinney, 1985; Kolbowski & Schreiber, 1995). Some of these
fluorescence methods have been adapted for sediments. Gorbunov et al. (2000) used FRR
(Fast-Repetition Rate) fluorometry to estimate photochemical yield and other photosynthetic
parameters of microphytobenthos in situ. Barranguet & Kromkamp (2000), Serodio et al.
(2001) and Glud et al. (2002) used PAM (pulse amplitude modulation)-technique (Schreiber
et al., 1986) to estimate primary productivity and electron transport rates of benthic samples.
However, none of these methods allowed in situ algal group differentiation.
39
CHAPTER 3
Based on our earlier work with a phytoplankton sensor (Beutler et al., 2002a) we set out to
devise a new benthic method for the quantitative and qualitative assessment in situ of
diverse populations of microphytobenthos with high spatial and temporal resolution, enabling
rapid evaluation of the community structure and distribution.
3.2 Material & Methods
Measurement principles
The colour of a photosynthetic organism is influenced by the pigments of the photosynthetic
apparatus. Furthermore, the colour of algae is a useful taxonomic criterion. Various
taxonomic groups differ significantly in their fluorescence excitation spectrum. Here, we
designate algal groups characterised by similar fluorescence excitation spectra as distinct
'spectral signature groups'. We are able to distinguish four spectral groups (1) green: algae
containing chlorophyll a/b, 2) blue: algae containing phycobilisomes rich in phycocyanin, 3)
brown: algae containing chlorophyll a/c and green light absorbing xanthophylls and 4) mixed:
algae containing chlorophyll a/c and phycoerythrin.
Figure 1: Schematic representation of the benthofluorometric measuring principle.
Our concept is based on the fact that fluorescence is emitted mainly by the chlorophyll a of
the photosystem II (PS II) antenna system, which consists of the evolutionarily conserved
chlorophyll a core antenna and species-dependent peripheral antennae. This association
results in spectral differences in the fluorescence excitation spectra. Using this method for
phytoplankton, Beutler et al.(1998; 2001; 2002a,b) were able to distinguish between four
algal groups within in situ fluorescence profiles and could correlate the biomass
concentrations of different spectral groups of algae. In Beutler et al. (2002b) the chlorophyll
40
CHAPTER 3
profiles were corrected for the influence of yellow substances. These determinations are
based on the concept that six spectral excitation ranges can be used to differentiate groups
of microalgae in situ within a few seconds. In addition, since sediments contain a lot of yellow
substances which can affect the optical differentiation of the algae, the device was equipped
with a correcting UV-LED for yellow substances.
Submersible sediment instrument
Because the sediments of interest are often underwater or, as in the intertidal, intermittently
underwater, it was important to build an underwater device. The optics and electronics are
mounted in a waterproof stainless-steel housing (l = 45 cm, ∅ = 14 cm) with a sealed optical
fibre bundle (5 m long; ∅ = 0.9 cm; Zeutec, Germany) extending out to a small light-proof
measuring chamber which is placed on the sediment and ensures a constant distance from
the sediment surface to the detector bundle. A schematic representation of the measuring
principle is depicted in figure 1. A diagram of the structure of the instrument is given in figure
2 a+b.
Figure 2: a) The fluorometer components: (1) microcontroller, (2) six light-emitting diodes, (3) shortpass filter to block red and IR emission, (4) focussing lens (f = 25 mm), (5) beamsplitter, (6) focussing
lens, (7) band-pass filter (see B), (8) integrated photomultiplier, (9) 12-bit AD-converter (conversion
rate of 100 kHz), (10) fibre bundle and (11) benthic sample. b) Photo of the fluorometer housed in a
water resistant cylindrical case. The fluorometer is connected with a 5 m long optical fibre to the
measuring head. The special disc-shaped measuring chamber that is placed on top of the sediment and
used as a connecting device for the fibre bundle is shown in the front.
Algal chlorophyll a and yellow substances are excited using light from six LEDs with the
following emission wavelengths: 370 nm (UV-A), 470 nm (blue), 525 nm (dark green), 570
nm (light green), 590 nm (yellow/orange) and 610 nm (red). The excitation light is guided
41
CHAPTER 3
through the beam splitter and the fibre bundle shown in figure 2 a. The exact specifications of
the light-emitting diodes used for the excitation of the pigment complexes are as follows
(centre wavelength and light intensity are given in parenthesis): 470 nm, Oshino OL-ESB
41510 (470 nm, 3 µE m2 s1, Oshino Lamps, Tokio, Japan), 2 × 590 nm Oshino OL Hewlett
Packard HLMP-DL08 (590 nm, 6 µE m2 s1), 610 nm, Oshino OL-SUA 14180 (610 nm, 3
µEm2 s1 Oshino Lamps). The LED light passes through a short-pass filter (50% transmission
at 615 nm DT cyan special, Balzers, Liechtenstein) and a focusing lens. The five lightemitting diodes (LEDs) are switched on sequentially at a frequency of 5 kHz. The measuring
pulse duration is 0.1 ms. Light intensities were determined at the position of the algal filter
with the PhAR sensor Hansatech QRT 1 (Hansatech, UK). Chlorophyll a fluorescence with
wavelengths between 690nm and 710nm is detected using a photomultiplier(H6779-01,
Hamamatsu, Hamamatsu City, Japan) behind a band pass filter (bbe-fk1, bbe Moldaenke,
Kiel, Germany). The photomultiplier signal is digitised by an AD converter (12-bit AD
converter, conversion rate: 100 kHz) and processed by the same microcontroller (MM-1035CAQ 18, Phytec, Mainz, Germany) used for controlling the LEDs.
Data can be stored in the probe or transferred directly to a PC, or for field measurements, a
handheld data logger. High sensitivity and dynamic range are extremely important as the
light is transmitted to and from the sediment surface via a sealed optical fibre enabling
measurement of fluorescence excitation spectra at low chlorophyll concentrations. During
measurement the probe can either be in water or, as in the intertidal, in air. For large-scale
spatial assessments of the benthic microflora, for example in the intertidal, the probe can
additionally be equipped with a backpacking device, allowing the user to carry the
fluorometer easily leaving the users hands free for the fibre bundle and the measuring
chamber. The spectra are recorded automatically with an integration-time of a second.
42
CHAPTER 3
Determination of chlorophyll concentrations on surfaces using
algal cultures
The basic running parameters of the benthic fluorometer were initially set against bench-top
multialgal fluorometer. It was, as described above, precalibrated for algal group
differentiation using suspensions of the standard calibration microalgae used by the
company bbe Moldaenke in their fluorometer calibrations. These were for the green spectral
group: Chlorella vulgaris; blue spectral group: Synechococcus leopoliensis; and for the
brown spectral group: Cyclotella meneghiniana. The mixed group (cryptophytes) were
excluded in this investigation because of their rarity in the benthic samples. These algae
were measured against a standardised bbe Moldaenke bench-top multialgal fluorometer,
filtered onto filters and measured by the probe. The factors used in the algorithms are given
in table 1. For general information on calibrating a multialgal fluorometer see also details in
Beutler et al. 2001; 2002 (a) and (b). After these initial settings, the benthic probe was
adjusted for concentration gradients by measuring different amounts (1-20 ml) of algal
suspensions filtered onto GFF-filters (Whatmann). The filters were subsequently measured
by placing the optical measuring fibre of the new instrument at a specific distance (5 mm)
above the surface. Using a lightproof measuring chamber placed on the sediment, with a
fixed height, constant distance between the fibre and the sediment surface was provided.
From the known filtered volume and a known filter surface area (4.2 cm2) the chlorophyll
quantities on the filter were calculated (µg cm-2) and set against the fluorescence response of
the instrument. See examples in figure 3.
Fluorescence at an excitation at 470 nm
800
600
Green 470nm
Bluegreen 470nm
Diatoms 470nm
400
200
0
0
2
4
6
8
10
Chl-a-concentration [µg/cm²]
Figure 3: Fluorescence intensities of three spectral algal groups at various concentrations at an excitation
wavelength of 470nm
43
CHAPTER 3
The filtrate was also measured to check that all the algae were retained on the filters. In the
measurement procedure described above, relative intensities aλk were determined by
measuring benthic samples with the benthic probe containing one algal group. The estimated
aλk are shown in table 1.
Table 1 The estimated aλk coefficients (1); 1= green: algae containing chlorophyll a/b, 2= blue: algae
containing phycobilisomes rich in phycocyanin and 3= brown: algae containing chlorophyll a/c and green
light absorbing xanthophylls. aλk are given in relative fluorescence intensities per chlorophyll density of
the samples (µg cm-²) at excitation wavelength λ .
aλk =1
aλk =2
aλk =3
370
nm
1
12.9
48.8
470
nm
76.1
-1.8
90.6
525
nm
252.5
118.1
706.3
570
nm
7.9
22.1
23.5
590
610
nm
nm
268.0 215.1
483.2 507.0
344.1 280.8
After the measurement with the probe, these filters were then extracted in 100 % acetone
and the chlorophyll concentrations measured in the HPLC; method as described in Wiltshire
(2000).
The determination of the distribution of the spectral algal groups is based on the premise that
the measured excitation spectrum at a fixed emission wavelength is a superposition of the
signals from the individual cells and yellow substances (see Beutler et al 2002a for details).
For the total fluorescence intensity at a single excitation intensity we get equation (1)
F(λML.) = ∑k =1 to n CCHLa.k fλk IML(λML)
(1)
where: CChl.k is the concentration of Chl a which is contained in cells of the k'th algal group
(or yellow substances). IML: the intensity of the measuring light (in µE m-2 s-1);
Χ 2= ∑λML (Fmeasured(λML.) - ∑k =1 to n CCHLa.k fk(λML.) IML(λML))
(2)
with Fmeasured(λML.): the measured fluorescence intensity of the sample at wavelength λML
To obtain the algal concentration CChl.k equation (2) was minimized by the use of the fit
procedure of Beutler et al (2002 a). The method was found to be sufficiently linear in the
44
CHAPTER 3
laboratory at chlorophyll densities below 5 µg cm-2, with errors due to self shading of below 5
%.
3.3 Results
In order to evaluate the applicability of the new benthic probe to natural situations, in
particular in view of the precalibration with merely three laboratory algae, we carried out a
series of tests. The first involved isolating and measuring a diverse array of benthic
microalgae from sediments and culturing these under standard laboratory conditions. The
second was to test the measurement efficiency on benthic algal assemblages and to detect
succession patterns under controlled laboratory conditions and the third was to evaluate the
probes performance on natural intertidal and littoral sediments.
3,5
3,0
3,5
d)
Stauroneis
HPLC
benthic fluorometer
3,0
3,0
Scenedesmus
2,5
HPLC
benthic fluorometer
2,5
chlorophyll-a (µg cm-²)
2,5
chlorophyll-a (µg cm-²)
g)
Diatom Mix
HPLC
benthic fluorometer
2,0
1,5
1,0
chl. a-content (µg/cm²)
a)
2,0
1,5
1,0
0,5
0,5
0,0
0,0
2,0
1,5
1,0
0,5
0,0
63 µl cm-²
125 µl cm-²
312 µl cm-²
63 µl cm-²
625 µl cm-²
1,6
HPLC
benthic fluorometer
1,2
1,0
0,8
0,6
0,4
63 µl cm-²
125 µl cm-² 312 µl cm-² 625 µl cm-² 938 µl cm-²
concentration of algal solution on filter
3,5
3,0
chlorophyll-a (µg cm-²)
chlorophyll-a (µg cm-²)
1,4
e)
Navicula
1250 µl cm-²
3,5
HPLC
benthic fluorometer
h)
Microcystis
3,0
2,5
chlorophyll-a (µg cm-²)
1,8
625 µl cm-²
concentration of algal solution on filter
concentration of algal solution on filter
b)
125 µl cm-²
2,0
1,5
1,0
HPLC
benthic fluorometer
Staurastrum
2,5
2,0
1,5
1,0
0,2
0,5
0,5
0,0
0,0
63 µl cm-²
125 µl cm-²
312 µl cm-²
4
f)
1,0
2
1
0
125 µl cm-²
312 µl cm-²
625 µl cm-²
concentration of algal solution on filter
HPLC
benthic fluorometer
Synechococcus
i)
8
chlorophyll-a (µg cm-²)
HPLC
benthic fluorometer
63 µl cm-²
10
1,2
Nitzschia
chlorophyll-a (µg cm-²)
chlorophyll-a (µg cm-²)
3
125 µl cm-² 312 µl cm-² 625 µl cm-² 1250 µl cm-²
concentration of algal solution on filter
concentration of algal solution on filter
c)
0,0
63 µl cm-²
625 µl cm-²
0,8
0,6
0,4
0,2
HPLC
benthic fluorometer
Micratinium
6
4
2
0
0,0
63 µl cm-²
125 µl cm-²
312 µl cm-²
concentration of algal solution on filter
63 µl cm-²
125 µl cm-²
312 µl cm-²
625 µl cm-²
concentration of algal solution on filter
63 µl cm-²
125 µl cm-²
312 µl cm-²
625 µl cm-²
concentration of algal solution on filter
Figure 4: (a-i): Chlorophyll a concentrations (µg cm-2) of different microalgal culture suspensions
measured with HPLC and the benthic fluorometer respectively. Microalgal cultures measured were:
Navicula sp., Nitzschia sp., Stauroneis sp. and a mixed diatom solution (brown group); Scenedesmus sp.,
Staurastrum sp., Micractinium sp. (green group) and Microcystis sp., Synechococcus sp. (blue group).
45
CHAPTER 3
Assessment of precalibration of the fluorometer with microalgae
Culture suspensions of benthic microalgae, Navicula sp., Nitzschia sp., Stauroneis sp.
(brown group); Scenedesmus sp., Staurastrum sp., Micractinium sp. (green group);
Microcystis sp., Synechococcus sp. (blue group) in different concentrations (63-625 µl cm-2),
were filtered onto Whatmann GFF filters and placed under the lightproof probe cuvette as
described above. After the measurement with the probe these filters were then extracted in
100 % acetone and the chlorophyll concentrations measured in the HPLC using the methods
of Wiltshire (1998). Examples of the relationships between the HPLC data and the probe are
depicted in figure 4 (a-i).
It is clear from the results that the precalibration of the probe was not optimal. The examples
show, when tested in an ANOVA, that for all the algae, the slopes of the chlorophyll
relationships of the methods were all significantly different (table 2). A good example is the
relationship between the data of various diatoms (brown group) shown in figure 4 a-d. Other
data showed that at times the HPLC values were higher than those values measured and
fitted using the initial algorithms of the probe (e.g. figures 4 d-f). Normally the HPLC values
were lower. The data also showed that at higher chlorophyll concentrations (above 2 µg cm-2)
the relationship between device and HPLC-derived values became unreliable.
Table 2: Assessment of precalibration of the fluorometer with microalgae. Algal culture, colour type,
slope, intercept and r² of the chlorophyll concentrations of single culture.
type
slope
intercept
r²
Mixed diatom culture
diatom
0,54
0,003
0,99
Synechococcus sp.
blue-green
0,55
0,06
0,97
Microcystis sp.
blue-green
0,58
0,24
0,95
Staurastrum sp.
green
0,89
0,25
0,99
Scenedesmus sp.
green
1,62
0,25
0,94
Stauroneis sp.
diatom
2,9
-0,26
0,90
Navicula sp.
diatom
3,35
0,00007
0,98
Micractinium sp.
green
3,86
2,24
0,96
Nitzschia sp.
diatom
7,85
-0,02
0,99
The result of such problems was that this information relayed back to the company bbe and
used to fine tune the calibration of the probe to the actual chlorophyll concentrations in the
algal layer on the filter. The new factors then used in the algorithms are given in table 3.
46
CHAPTER 3
Table 3: The estimated aλk coefficients (1); 1= green: algae containing chlorophyll a/b, 2= blue: algae
containing phycobilisomes rich in phycocyanin and 3= brown: algae containing chlorophyll a/c and green
light absorbing xanthophylls. aλk are given in relative fluorescence intensities per chlorophyll density of
the samples (µg cm-²) at excitation wavelength λ .
Aλk =1
Aλk =2
Aλk =3
370
nm
94.4
7
99
470
nm
15.2
4
13
525
nm
57.6
40
98
570
nm
0.4
1.4
0.5
590
nm
82
360
84
610
nm
71.2
345
62
Experimental cultivation of mixed microphytobenthic mats
In order to test whether the benthic fluoroprobe could be used to detect changes in
microphytobenthos populations over time, mixed microphytobenthic mats were grown
including defined numbers of rooting plants and grazing invertebrates under freshwater and
marine conditions in the laboratory. Natural sediments from the field were sieved into
experimental units and incubated under controlled conditions (16 hours light/8 hours dark
cycle with constant water flow) for 21 days. The fluorescence measurements were conducted
by laying the special disc-shaped measuring chamber on top of the sediment and, after a
short dark-adaptation time, measuring the chlorophyll a concentrations per spectral algal
group using the probe. In addition, surface sediments were sampled from the same units in
order to measure the chlorophyll a concentrations at the sediment surface (top 0-240 µm) via
HPLC. Measurements were made at the outset of the experiment, after seven and 21 days in
order to determine if the probe could be implemented for differentiating temporal
microphytobenthos population shifts in both freshwater and marine benthic systems.
marine mesocosms
freshwater mesocosms
a)
b)
benthic fluorometer (µg chlorophyll a * cm )
6,5
-2
-2
benthic fluoroprobe (µg chlorophyll a * cm )
0,9
0,8
0,7
0,6
r²=0,39
0,5
0,4
0,3
0,2
6,0
5,5
5,0
r²=0.01
4,5
4,0
3,5
0,0
0,2
0,4
0,6
HPLC (µg chlorophyll a * cm-2)
0,8
1,0
0,4
0,6
0,8
1,0
1,2
1,4
1,6
HPLC (µg chlrorophyll a * cm-2)
Figure 5: Correlation of chlorophyll a concentrations (µg cm-2) obtained from HPLC- and fluorometric
measurements in the marine (a) and the freshwater (b) mesocosms. Linear regression lines are given.
47
CHAPTER 3
The correlation between HPLC- and fluorescence measurements is given in figure 5 (a+b).
Within the marine units the chlorophyll a concentrations detected ranged from 0.06 to 1.22
µg cm-² (HPLC) and 0.24 to 0.83 µg cm-² (benthic fluorometer) (figure 5a) and an acceptable
correlation between both methods was achieved. The freshwater sediment, however,
showed much higher concentrations and the correlation between both methods was weak.
Chlorophyll a concentrations in the freshwater units ranged from 0.53 to 1.53 µg cm-² (HPLC)
and 3.71 to 5.95 µg cm-² (benthic fluorometer) (figure 5b). The measured concentration
ranges are rather high considering the fact that at values above 2 µg cm-2, the correlation
with quantitative HPLC values seemed dubious in our extended algal test described above
(see figure 4).
The population differentiation of the microphytobenthos showed that the chlorophyll contents
of the marine sediments initially comprised mainly of diatom (99%) and only 1% was
represented by Cyanobacteria (figure 6a). In the presence of grazers the community shifted
to a three-constituent-community after seven days comprising of Cyanobacteria, Chlorophyta
and diatoms (figure 6b) and this pattern changed only slightly after 21 days of grazer
presence (figure 6c). In the freshwater units, however, no green algae were detected and the
sediment microflora comprised of diatoms and Cyanobacteria. At the beginning of the
incubation diatoms made up 86% of the total algal community and Cyanobacteria 14%
(figure 6d). In the presence of grazers the proportion of Cyanobacteria increased to 26%
(day 7; figure 6e) and 21% (day 21; figure 6f). The algal differentiation detected
fluorometrically was checked for accuracy under the microscope and cell count analysis
showed very similar taxonomic compositions as detected with the probe. In the marine units
the algal assemblage was dominated by prostrate (Diploneis sp., Nitzschia sp.) and chainforming diatoms (Melosira nummuloides). Filamentous Cyanobacteria and green algae
showed lower proportions. The freshwater incubations showed a dominance of the chainforming diatom Fragilaria sp. and the cyanobacterium Merismopedia sp..
Thus, these experiments with natural microphytobenthic communities served to show the
benthic fluorometer proved to be very useful in following temporal changes in both marine
and freshwater mats. Increases and decreases of algal chlorophyll as well as differentiation
into the various algal groups on small time scales were be easily detectable using the device.
48
CHAPTER 3
grazer day 21
grazer day 7
start
a)
c)
b)
marine mesocosms
grazer day 7
start
e)
d)
diatom
cyanobacteria
green algae
grazer day 21
f)
freshwater mesocosms
Figure 6: (a-f): Major taxonomic components of the microphytobenthos in the marine (a-c) and the
freshwater (d-f) mesocosms detected with the probe. Relative proportions of different algal groups are
calculated as % of the total chlorophyll a contents of the microphytobenthos. The relative contributions
are show for the starts, after 7 days and after 21 days of incubation.
Natural intertidal and littoral sediments
The sediment fluorometer was tested on natural emerged intertidal sediments at
neighbouring sites in the German Wadden Sea (Dorum; Lower Saxony). Based on the colour
benthic fluorometer (µg chlorophyll a * cm-2)
3,5
r²=0,52
3,0
2,5
2,0
1,5
1,0
0,5
0,0
0
2
4
6
8
10
-2
HPLC (µg
chlorophyll a *(µg
cmcm) -2) obtained from
Figure 7: Correlation of chlorophyll
a concentrations
HPLC- and fluorometric measurements at an intertidal flat (Dorum, Wadden Sea).
Linear regression line is given.
49
CHAPTER 3
0,35
-2
chlorophyll a (µg cm )
0,30
0,25
0,20
0,15
0,10
0,05
0,00
15:00:00
low tide
16:00:00
17:00:00
18:00:00
19:00:00
20:00:00
time
Figure 8: Migration patterns detected from chlorophyll a measurements (µg cm-2) with the
benthic fluoroprobe in the Elbe Estuary (Belum) around sunset. Measurements started 2.5
hours before and ended 1 hour after low tide. The vertical line indicates the exact time of
low tide.
intensity, optically different sites were chosen. The sites were coloured light brown to dark
brown and they all were situated within an area of 20 m². The total chlorophyll a
concentrations at each site were first detected with the benthic fluorometer. These
measurements were verified with HPLC, using samples taken with the Cryolander method
(Wiltshire et al.,1997) and the micro-sliced surface layer (the top 0-240µm), see methods as
described by Wiltshire (2000). When measured with the fluorometer the sites were shown to
be very similar in their algal make-up as a dominance of diatoms was observed for all
sediment surfaces and this was verified by the pigments found in the chromatograms of the
HPLC analyses. The correlations of the HPLC values with the probe are given in figure 7 (r²=
0.52). Chlorophyll a concentrations in the field ranged from 0.11 to 8.56 µg cm-² (HPLC) and
0.28 to 3.11 µg cm-² (benthic fluorometer). The extreme patchy nature of the sediments at
this location was remarkable since all sites were situated at short distance to one another.
Such patchy distributions of benthic microalgae at small horizontal scales are well known
from intertidal as well as subtidal areas. Such distinct variations are most likely related to the
micro texture of the sediment surface (Joergensen et al., 1983; Jumars & Nowell, 1984) or to
microscale nutrient, irradiance and salinity gradients (Wolff, 1979). It is important that this
patchiness is detectable easily especially for ground truthing purposes.
Perhaps one of the most difficult phenomena to measure in situ is the migration of benthic
algae to and away from sediment surfaces related to light, tides, rain etc. (Paterson et al.,
50
CHAPTER 3
1998; Underwood et al., 1999). We used this phenomenon as a challenge to the new
fluorometer.
Depicted in figure 8 is the change in chlorophyll concentrations at the surface of a sediment
measured over the course of four hours during sunset at an intertidal flat in the Wadden Sea
(Belum; Lower Saxony, Elbe estuary). The measurements were started 2.5 hours before low
tide. At the beginning of the measurements concentrations of 0.11 µg cm-² ± 0.03 were
detected which increased continuously over exposure time to values of 0.25 µg cm-² ± 0.08
at just around low tide. Right before the tide came in, the concentrations at the sediment
surface decreased to values of 0.14 µg cm-² ± 0.04 and this phenomenon was related to the
migration behaviour of benthic microalgae, a means of escaping erosion by tidal movement.
Thus, the migration of the algae to and from the sediment surface from deeper layers was
successfully monitored using the new probe. Thus, the probe could be used for this form of
temporal resolution and indicates that it will be useful at differentiating successional switches
in algal groups at the sediment surfaces over the course of the daily light rhythms.
3.4 Discussion
It was our aim in this work to apply the concepts of multialgal fluorometry (Beutler et al.,
2002b) to a benthic probe. Taking into account the variety of sediments measured under
laboratory and under field conditions, the applicability of the probe in determining algal
populations on sediments in situ was successfully tested. However, we also found that
calibrating such a device is far from trivial and should ideally be an ongoing process. It could
be conceived that a data bank of measurements (should) be automatically set up in the
instrument software with exact chlorophyll concentrations (measured by HPLC) in the
uppermost 0-200 µm of sediments and ideally also with cell counts, whereby the weighting
factors for the algorithms be revaluated to suit a scientists system. The data also shows that
at higher chlorophyll concentrations (above 2 µg cm-²) which was less than assumed after
the initial laboratory calibrations, in the surface layers the device becomes unreliable. It
should be investigated if this problem could be alleviated by using a linear fit at lower
concentrations and an exponential fit at higher concentrations.
The preliminary calibration of such a device should be with mean factors for as many benthic
algal mats on and as many different substrates as possible. It does not suffice to calibrate it
with the usual algal standards or against a standardized instrument as is often carried out for
pelagic multialgal fluorometers. Under no circumstances should the device be calibrated
using wet chemical analytical techniques for chlorophyll estimation as these methods are,
particularly for sedimentary systems, extremely prone to error (see Wiltshire, 2000).
Our approach can be used to monitor algal assemblage composition on sediments and it is
an ideal tool for investigations on large-scale spatial and temporal variation of algal
51
CHAPTER 3
populations in sediments. It was, until now, not possible to carry out such detailed
investigations of algal assemblage structures in surface sediments within a reasonable time
frame. All measurements on sediments, apart from PAM measurements (Barranguet &
Kromkamp, 2000; Serodio et al., 2001; Glud et al., 2002), are retrospect and even PAM
measurements do not allow differentiation of algal populations. The results, particularly of
long-term sediment mesocosm experiments, show that the domination of algae in sediment
assemblages change rapidly (weeks) and that they not only comprise diatoms, which is often
assumed. Past research showed that strong seasonality patterns occur in microphytobenthic
communities and that, under certain circumstances, Cyanobacteria and chlorophytes can
contribute to large amounts to the sediment microflora (Agatz et al., 1999; Riethmueller et al.,
2000). Thus, the current resolution of three algal groups is useful and it enables in situ
differentiations of algal assemblages. However, the accuracy of the algal group differentiation
is probably limited by the species-dependent variability within each individual algal group and
by the influence of environmental factors on the fluorescence yield.
Apart from the advantages of in situ differentiation and determination of total algal
biomasses, we with this device could rapidly discern differences without having to wait for the
analyses. The latter is always a nuisance, as it doesn’t allow for experimental rethinking.
Furthermore, with its in situ practicality the sensor was well suited to monitoring migration
events of microalgae to and from the sediment surface, from deeper layers. This has been
done a few times using reflectance measurements (Paterson et al., 1998) and fluorescence
measurements (Mazel, 1997). However the methods used were cumbersome. Our probe will
also be useful when it comes to differentiating different tidal and diurnal succession of
populations, i.e. the replacement of diatoms at a sediment surface during the course of
exposure by green algae or Euglenids (Paterson et al., 1998). The sensor could be used for
long-term measurements (monitoring) of chlorophyll a concentrations related to different
spectral groups of algae in sediments over large spatial and temporal scales. This would be
of considerable use in ground-truth measurements in remote sensing.
Conclusions
Our new method represents a unique approach to the qualitative and quantitative
assessment of microphytobenthos in situ, with high spatial and temporal resolution, enabling
a rapid evaluation of the community structure and its distribution. In addition, the new method
can serve as a tool for long-term experimental investigations. In our case a marine and
freshwater mesocosm experiment served as an ideal experimental unit to test this technique
under laboratory conditions prior to field deployments. We, thus, believe that our approach
will become an important new tool in aquatic benthic ecology and in the management of
benthic aquatic resources. We also envisage that the device could be implemented on
52
CHAPTER 3
Landers in the submerged intertidal or in shallow lake systems were benthic
microphytobenthic communities are a rather underestimated but highly productive
community. Further developments and measurement refinements will permit a more detailed
classification of algal groups in future.
53
CHAPTER 4
Chapter 4
Microcosm experiments on grazing efficiency and
selectivity of the freshwater hydrobiid snail
Potamopyrgus antipodarum preying upon
microphytobenthic assemblages.
In this chapter I focused on grazer effects of the freshwater hydrobiid snail Potamopyrgus
antipodarum preying upon microphytobenthos. To quantify grazing efficiencies, feeding
preferences and to evaluate the controversially discussed turning points between positive
and negative effects of grazer activity were of special interest in this study.
54
CHAPTER 4
4.1 Introduction
Benthic microalgae contribute significantly to the primary production of shallow aquatic
systems and serve as an ideal diet for a large variety of small-sized grazing biota (protists,
meio- and macrofauna). Microalgae therefore play an important role in benthic food webs,
either as epiphytes or as sediment algae. Studies of grazing interactions between microalgae
and invertebrates have played a major role in aquatic ecology for decades (Asmus & Asmus,
1985; Blanchard, 1991; Miller et al., 1996; Montagna et al., 1995; James et al., 2000a).
Numerous invetigations have been conducted in order to obtain an insight into food-web
structures in freshwater systems. Most of these studies have focused on the role of grazermicroalgae interactions in pelagic ecosystems. In contrast, benthic food-webs are still poorly
understood. This is especially true for sediment microflora assemblages. In addition,
although having been often studied in marine systems we know virtually nothing about
freshwater microphytobenthic assemblages (Lowe, 1996; Cyr, 1998; Nozaki et al. 2003).
In our study we focused on the impact the gastropod grazer Potamopyrgus antipodarum on
natural microphytobenthic sediment communities from lake Schöhsee (Plön, Germany) as
these are one of the dominant grazers in this lake. In order to quantify their grazing efficiency
and to investigate feeding preferences, small-scale laboratory experiments with varying
grazer densities and different incubation times were carried out.
Past investigations on grazer-microalgae interactions have provided contradictory results as
far as the positive and negative effects of grazing invertebrates on microphytobenthic
communities are concerned (Underwood & Thomas, 1990). The literature to date is
controversial and can be divided into three categories: reduction or increase of cell numbers
and biomass, chlorophyll densities as well as abundances and diversity of particular species
or of whole algal communities. In some cases a negative effect on microphytobenthos could
be detected as a decrease of chlorophyll content (Cattaneo, 1983; Lamberti & Resh, 1983)
or of algal cell numbers or of biomass (Castenholz, 1961; Kesler, 1981; Lamberti & Resh,
1983; Underwood & Thomas, 1990; Hillebrand et al., 2002). In contrast, other studies
showed positive, beneficial effects of grazing activities which resulted in increased diversitiy
of algal assemblages (Sumner & McIntire, 1982; Eichenberger & Schlatterer, 1978) or an
increase in abundance of particular algal taxa (Hunter, 1980; Jacoby, 1987; Lamberti &
Resh, 1983). The fertilizing effect on microalgal assemblages resulting from grazer activities
can be explained by an increased nutrient supply. This can be triggered by several
mechanisms (McCormick & Stevenson, 1991). Grazers may increase nutrient diffusion while
physically destroying the structure of the uppermost surface layer and thus facilitating
nutrient release from deeper sediment layers (Wetzel, 1996). They can also cause this
release by removing senescent cells (Hillebrand & Kahlert, 2001), by sloppy feeding and
55
CHAPTER 4
adding nutrients via their excretion products (Kahlert & Baunsgaard, 1999; Mulholland et al.,
1991). Moreover this, the diverse array of effects is dependent on specific grazer types and
densities which can be directly related to the feeding mode of the animals. Grazing
invertebrates preying on benthic microalgae comprise a large group of organisms ranging
from the very small protists to larger-sized gastropods and crustaceans. Apart from their size
ranges, they differ considerably in their feeding habits since they are characterized by
specialized mouthparts and feeding techniques. Crustaceans like isopods or amphipods, for
example, have specific mandibles which allow them to shred larger food items and also to
choose between algae and detritus (Moore, 1975; Friberg & Jacobsen, 1994; Constantini &
Rossi, 1998; Duffy & Hay, 2001). Gastropods are known to be very efficient grazers on
biofilms, since they are able to reduce algal biomass with their radulas on a larger spatial
scale although rather unselectively (Nicotri, 1977; Hunter, 1980; McCormick & Stevenson,
1991; Sommer, 1997). However, feeding specializations not only exist between, but also
within these groups. For example nematodes are known to have very specialized mouthparts
ranging from a pipette-like mouth specialized to suck in bacteria and small microalgae to a
toothed pharynx in order to crack larger food items (Wieser, 1953; Jensen, 1987).
The primarily aim of this study was to quantify the gastropods grazing efficiency and to detect
selectivity patterns. Moreover, the points at which positive and negative effects of grazer
activity were switched and also correlations between biovolume, morphology or motility of
special algal taxa in case of feeding preferences were of particular interest to us in this study.
4.2 Material & Methods
Experimental design
Laboratory grazing experiments with varying grazer densities and at different incubation
times were conducted in July and November 2001 with natural sediments from a freshwater
lake (Schöhsee, Plön, Germany). Rectangular tanks with a surface area of 106 cm2 (8.5 x
12.5 cm) served as experimental units. The sediments used, contained natural
microphytobenthic communities taken from a sandy site in the littoral zones of the Schöhsee
at 0.5 m depth. Prior to the experiments the sediments were sieved with a 1000 µm sieve in
order to remove macrofauna. The aquaria were then filled with this sieved sediment and
water from the Schöhsee and left undisturbed for 24 hours. In the Schöhsee P. antipodarum
is one of the most important benthic grazers reaching abundances of 200 individuals m-2.
Therefore this species was chosen as the consumer for the experimental units. The
individuals were hand-picked from natural sediments, sorted by size class into petri-dishes
and stored overnight at 17°C without any food source. As a grazer fraction individuals with a
shell length of 3 mm were used. Three different experimental considerations existed: start
(t0), grazer treatments (+GR) and grazer-free controls (ctrl). Each treatment was replicated
56
CHAPTER 4
three times. The experimental set-up followed a randomised design. During the first
experiment (Experiment I, July 2001) an incubation time of 24 hours was chosen whereas
the second experiment (Experiment II, November 2001) was conducted at two incubation
intervals (24h and 48h) in order to achieve a higher temporal resolution of grazer effects. The
light regime was kept constant at a 12 hours day-12 hours night-cycle. At the beginning of
the experiment the initial starts (t0) were sampled and grazers were subsequently added to
the grazer units. Grazers were added at relatively high densities (approximately four to eight
times the maximum field densities) to ensure that short-term grazer effects would be
detected. A grazer density of seven P. antipodarum (700 individuals m-2) was chosen during
the first experiment in July 2001 and 15 individuals (1500 individuals m-2) were used during
the November experiment.
Sample processing
The determination of microalgal distribution, cell density and chlorophyll a content was
carried out using one Cryolander-core with a surface area of 18 cm2 (∅ 4.8 cm) which was
sampled from each treatment following the method described by Wiltshire et al. (1997). Each
cryo-preserved sediment disc was cut into six equal subsamples whereas three of each of
the subsamples were used either for chlorophyll a measurements or for light-microscope
analyses. The sediment surface of each sample was sliced at 60 µm intervals with a
Cryomicrotom (Leica CM 1900) down to a depth of 480 µm and this sediment horizon was
also used for further analyses. Chlorophyll sample processing and HPLC analyses followed
the method of Wiltshire (2000). For cell counts and microalgae determination the prepared
sediment layers were preserved with Lugol’s solution, inserted into a Sedgewick-Rafter
counting chamber and counted under an inverted light microscope.
Grazing rates were calculated from the equation: λ = µ - r whereas λ is the grazing rate (hr-1),
µ the gross growth rate and r the net gross rate. The standard deviations are indicated by the
symbol ± in the text.
To test for significant differences in chlorophyll contents, a full-factorial ANOVA and Tukeys
HSD-Test were used. In the case of total cell numbers a full-factorial ANOVA and a Duncan
Post hoc-Test were applied. Diversity indices were calculated by using PRIMER 5.2 ( 2001
Primer-E Ltd.). Diversity (H’) was measured by the Shannon-Weaver function: H’= -∑ (Pi *
loge Pi) whereas Pi= ni/n (ni is the number of individuals from one particular species, n the
total number of individuals of all species (Shannon and Weaver, 1963). The Evenness (E)
was calculated by using Pielou’s Evenness: E= H’ * log S whereas S is the total number
species (Pielou, 1969).
57
CHAPTER 4
4.3 Results
Total chlorophyll a contents
Experiment I
During the first grazing experiment the highest total chlorophyll a contents of 0.65 µg cm-2 ±
0.14 and 0.34 µg cm-2 ± 0.04 were measured at the beginning of the experiment (t0; 0-240
µm and 240-480 µm respectively) (figure 1A). The control units showed a significant decline
in chlorophyll a content at the sediment surface when compared to t0 (p= 0.0002). No
significant difference could be detected for surface chlorophyll concentrations of the starts
and the grazer treatments. Chlorophyll a contents of the deeper sediment horizons remained
constant within all the three different treatments and showed a significant decrease with
increasing sediment depth in the start samples (p= 0.0004).
B
Experiment I
0,8
-2
-2
chlorophyll a (µg cm )
1,0
chlorophyll a (µg cm )
A
0,6
0,4
0,8
Experiment II
day 1
day 2
0,6
0,4
0,2
0,2
0,0
0,0
t0 (s)
t0 (d)
ctrl (s)
ctrl (d) +GR (s) +GR (d)
t0 (s)
t0 (d)
ctrl (s)
ctrl (d) +GR (s) +GR (d)
ctrl (s)
ctrl (d) +GR (s) +GR (d)
Figure 1: A) Chlorophyll a concentrations (µg cm-2) at the top surface layer (s; 0-240µm) and the deeper
layer (d; 240-480µm) in the start (t0), control (ctrl) and grazer treatments (+GR) of Experiment I after one
day of incubation. B) Chlorophyll a concentrations (µg cm-2) at the top surface layer (s; 0-240µm) and the
deeper layer (d; 240-480µm) in the start (t0), control (ctrl) and grazer treatments (+GR) of Experiment II
after day 1 and day 2 of incubation.
Experiment II
At the beginning of the second grazing experiment the initial values of chlorophyll a were
similar to those of Experiment I with values of 0.51 µg cm-2 ± 0.18 (figure 1B). After the first
and the second day of the experiment the values of the control units remained constant when
compared to t0. A slight decrease in chlorophyll a contents was detected for the grazer
treatments with chlorophyll contents reaching values of 0.26 µg cm-2 (day 1) and 0.39 µg cm-2
(day 2). However this decline was marginally not significant.
58
CHAPTER 4
Total cell numbers
Experiment I
Total cell numbers at the sediment surface (0-240µm) showed similar values at the beginning
of the first grazing experiment (72.9 cells cm-2 ± 25.7) when compared to the control units
(70.8 cells cm-2 ± 30.5) (figure 2A). In the presence of grazers a significant decline of cell
numbers could be detected within the uppermost surface layer (26.3 cells cm-2 ± 2.3) when
compared to t0 and control treatments (p<0.05). The cell numbers at 240-480 µm sediment
depth showed similar patterns and reflected the same trends for surface sediments.
A
200
B
200
Experiment I
Experiment II
day 1
day 2
-2
150
total cell numbers * cm
total cell numbers * cm
-2
SD= 38.4
100
50
0
150
100
50
day 1
day 2
0
t0 (s)
t0 (d)
ctrl (s)
ctrl (d) +GR (s) +GR (d)
t0 (s)
t0 (d)
ctrl (s)
ctrl (d) +GR (s) +GR (d)
ctrl (s)
ctrl (d) +GR (s) +GR (d)
Figure 2: A) Total cell numbers (per cm²) at the top surface layer (s; 0-240µm) and the deeper layer (d;
240-480µm) in the start (t0), control (ctrl) and grazer treatments (+GR) of Experiment I after one day of
incubation. B) Total cell numbers (per cm²) at the top surface layer (s; 0-240µm) and the deeper layer (d;
240-480µm) in the start (t0), control (ctrl) and grazer treatments (+GR) of Experiment II after day 1 and
day 2 of incubation.
Experiment II
Unlike in the first grazing experiment in comparison to cell numbers at the beginning of the
experiment, a significant increase in cell numbers in both sediment layers was detected in
the control and the grazer treatments (day 1; p<0.05; figure 2B). In addition, the surface
layers of the grazer treatments showed significantly higher cell numbers after 24 hours when
compared to the control units (p= 0.002). On the second day of the experiment the cell
numbers in the absolute surface layer of the control treatments had increased significantly
when compared to the first day of incubation (p=0.0004). A maximum of 163.1 cells cm-2 ±
38.4 was reached. In contrast, cell numbers at the sediment surface of the grazer treatments
decreased significantly after two days (p=0.0001) to fairly low numbers (47.7 cells cm-2 ±
28.4).
59
CHAPTER 4
Taxa composition
The analyses of the algal assemblages revealed five major taxonomic groups in both grazing
experiments: Pinnularia sp., Stauroneis sp., Cymbella sp., Synedra sp. and Amphora sp..
The genus Caloneis sp. was abundant during the second grazing experiment. Both
experiments showed great differences as far as grazing effects and the influence of grazers
on different taxonomic groups are concerned.
Experiment I
During the first grazer experiment all algal taxa showed a decrease in cell numbers when
grazers were present. In some cases the reduction of cell numbers within the grazer
treatments was more than 50% compared to the controls (Stauroneis sp., Synedra sp.,
Caloneis sp., Amphora sp.; figure 3A). When comparing the initial cell numbers with the
numbers detected within the control treatments it becomes clear that only few genera
managed to increase their cell numbers within one day of incubation (Stauroneis sp.,
Diploneis sp., Amphora sp.). All other genera showed declining values when compared to the
start samples. In addition, the alga Surirella sp., present in the start and in the control
treatments at low abundances, disappeared completely in the presence of grazers.
Experiment II
During the second grazing experiment, with 15 grazers present, contrasting trends were
seen. All major algal groups showed highest cell numbers within the grazer treatments after
one day when compared to the control treatments or the start values (especially the genera
Pinnularia sp., Stauroneis sp., Cymbella sp., Synedra sp., Diploneis sp. and Amphora sp.;
figure 3B). These patterns changed after two days and decreasing cell numbers within the
grazer treatments were detected. However, when compared to the first grazing experiment
B 200
Experiment I
150
cell numbers * cm-2
cell numbers * cm-2
A 200
100
50
0
Experiment II
day 1
day 2
150
100
50
Pinnularia
Stauroneis
Cymbella
Synedra
Placoneis
Surirella
Caloneis
Diploneis
Amphora
Navicula
Nitzschia
Epithemnia
0
t0
ctrl
+GR
t0
ctrl
+GR
ctrl
+GR
Figure 3: A) Taxonomic composition of the microphytobenthos in Experiment I. The proportions of each
genera are given as relative cell numbers * cm-² in the start (t0), control (ctrl) and grazer treatments (+GR)
after one day of incubation. B) Taxonomic composition of the microphytobenthos in Experiment II. The
proportions of each genera are given as cell numbers * cm-² in the start (t0), control (ctrl) and grazer
treatments (+GR) after day 1 and day 2 of incubation.
60
CHAPTER 4
the overall reduction of cell numbers was less clear for the second grazing experiment, even
though the grazer abundance was higher. The control treatments showed increasing cell
numbers for both incubation days and the highest cell numbers were detected after the
second day. In this regard, Stauroneis was by far the fastest growing genera.
Grazing rates
The grazing rates calculated for every single algal group were higher for the first grazing
experiment (Experiment I, day 1) compared to the second experiment (figure 4). During the
first incubation all nine algal groups were grazed by P. antipodarum whereby the highest
grazing rates were detected for Surirella sp., Stauroneis sp., Placoneis sp., Caloneis sp. and
Amphora sp.. In contrast to the first grazing experiment, the second experiment showed
different temporal trends and grazer efficiencies. Even though grazer abundance was higher
within the second experiment, negative grazing rates were calculated for all taxonomic
groups after the first day of incubation. This amounted to an overall positive effect on the
0,08
Pinnularia
Stauroneis
Cymbella
Synedra
Placoneis
Surirella
Caloneis
Diploneis
Amphora
Navicula
Nitzschia
Ephitemnia
grazing rate * hr-1
0,06
0,04
0,02
0,00
-0,02
-0,04
-0,06
Experiment I day 1
Experiment II day 1
Experiment II day 2
Figure 4: Grazing rates λ (hr-1) detected in the grazer treatments (+GR) of Experiment I (day 1) and
Experiment II (day 1+2).
total algal community within the grazer treatments. Diploneis sp. and Navicula sp. were the
genera that benefited most. After two days of incubation a shift from negative to positive
grazing rates was observed for all taxonomic groups. The whole algal community was thus
reduced by grazer activity.
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CHAPTER 4
Diversity and Evenness
The diversity index H’ showed slightly higher values at the beginning of the first grazing
experiment (H’= 1.8 ± 0.1) when compared to the second experiment (H’= 1.6 ± 0.2). During
the first experiment the diversities decreased in the control and the grazer treatments and
reached similar values of 1.6 ± 0.1 and 1.7 ± 0.2 (figure 5A). Different diversity patterns were
observed during the second experiment, where diversities first increased after the first day of
incubation in the grazer and the control treatments and then declined by day 2 (figure 5B).
Despite the different trends in diversity, Pielou’s Evenness showed very similar values both
for the first and the second grazing experiment. The evenness at the beginning of the
experiments were almost identical with values of 0.9 ± 0.03 (Experiment I) and 0.8 ± 0.1
(Experiment II). All through the experiments, the evenness remained constant and no
differences between treatments were seen.
A
2,2
B
Experiment I
Experiment II
day 1
day 2
2,2
Diversity indices H' & E
2,0
Diversity indices H' & E
2,4
1,8
1,6
1,4
1,2
1,0
0,8
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,6
0,4
t0
H' (Diversity)
E (Evenness)
ctrl
+GR
t0
ctrl
+GR
ctrl
+GR
H' (Diversity)
E (Evenness)
Figure 5: A) Diversity indices H’ (Diversity) and E (Evenness) in the start (t0), control (ctrl) and grazer
treatments (+GR) in Experiment I after one day of incubation. B) Diversity indices H’ (Diversity) and E
(Evenness) in the start (t0), control (ctrl) and grazer treatments (+GR) in Experiment II after day 1 and
day 2 of incubation.
4.4 Discussion
Although past research has provided extensive information on grazer-microalgae
interactions, the interplay between positive and negative effects still remains one of the most
discussed research topics in benthic ecology (Underwood & Thomas, 1990). Since grazer
effects are complex and variable, depending on grazer type, abundance as well as on
external parameters, the ecological role of grazer activity cannot be generalized.
62
CHAPTER 4
In our study, the chlorophyll concentrations at the beginning of each experiment were similar
and therefore the initial conditions were comparable. However, in the presence of grazers
only slight changes in chlorophyll contents at the sediment surfaces were found. This is in
contrast to a variety of grazer studies that demontrated clear decreases of chlorophyll a in
the presence of gastropod grazers (Cattaneo, 1983; Lamberti & Resh, 1983; Jacoby, 1987;
Hill et al, 1992). However, despite these differences, some studies support our findings.
Hillebrand & Kahlert (2002), e.g., found contradictory grazer effects on sediment chlorophyll
a contents for two freshwater sites in Sweden. Interestingly also, in contast to the freshwater
habitat lake “Erken”, the chlorophyll a concentrations at their brackish site “Väddö” were
completely unaffected by grazer presence even though gastropod abundances were higher
than for the brackish habitat. Hunter (1980) has also described increasing chlorophyll a
contents in the presence of gastropod grazers and concluded that grazing proportionally
increases primary productivity. Our results show that the chlorophyll contents at the sediment
surface remained almost uneffected by grazing. This is quite astonishing since clear feeding
tracks from the gastropods’ radula were visible on the sediment surface and therefore
decreasing chlorophyll concentrations could have been expected. Chlorophyll contents are
known to provide only rough estimates for the determination of grazing activity since the
amount of chlorophyll in the algal cells is known to be highly variable and dependent on a
variety of parameters, e.g. light and physiological status (Wolff, 1979; De Jonge & Colijn,
1994). Furthermore, the accuracy of chlorophyll determination depends also on the methods
used, since spectrophotometric measurements don’t differentiate for breakdown products
while HPLC-analysis does. Thus, detecting grazer effects from chlorophyll a measurements
only, may lead to an underestimation of algal consumption and a misjudgement of the actual
grazer efficiency.
In contrast to chlorophyll a contents, the total cell numbers at the sediment surface fluctuated
depending on grazer densities and incubation time. A significant decline of cell numbers in
the presence of grazers occurred during the first experiment after an incubation time of 24
hours, whereas the second experiment revealed a sharp increase in cell numbers over the
same time period. After two days this pattern changed and a significant grazer effect on cell
numbers was detected. These contradictory effects seem common in studies on gastropod
grazing. Most studies report a high grazing efficiency of gastropods resulting mostly in a
significant decrease in cell numbers and biomass (Nicotri, 1977; Sumner & McIntire, 1982;
Lamberti & Resh, 1983; Underwood & Thomas, 1990; McCormick & Stevenson, 1991;
Hillebrand & Kahlert, 2001). However, grazing may benefit both the algal community as a
whole or individual species, as the presence of herbivore invertebrates may increase the
diversity and the abundance of certain species (Nicotri, 1977; Hunter, 1980; Sumner &
McIntire, 1982; Jacoby, 1987; McCormick & Stevenson, 1991). The reasons for these
63
CHAPTER 4
diverse effects are still a main focus of studies in benthic food web ecology and it is often
assumed that these patterns are mainly caused by selective or differential grazing. However,
other mechanisms such as those involved with the exchange of nutrients between the
invertebrates and the algae must also be considered. It has been shown that grazing
pressure on microalgal assemblages can result in increased nutrient content or productivity
of algal cells (Lamberti & Resh, 1983; McCormick & Stevenson, 1991; Hillebrand & Kahlert,
2001). This positive effect of consumers on their prey can be the result of the excretion of
nutrients, the removal of senescent cells, or increased uptake of nutrients by the remaining
cells (Lamberti et al. 1987; McCormick and Stevenson, 1991; Hillebrand & Kahlert, 2001).
The hydrobiid snail P. antipodarum is known to be an effective grazer on benthic microalgal
assemblages (Fenchel, 1975a; James et al., 2000a, b; Dorgelo & Leonards, 2001;
Broekhuizen et al. 2002). In the Schöhsee P. antipodarum is one of the most important
benthic grazers reaching abundances of 200 individuals m-². The natural densities are low
compared to reports from James et al. (2000a) where P. antipodarum-densities of up to 1000
m-² were found (Lake Coldridge, New Zealand). In our experiments the grazers were added
at relatively high densities to ensure the detection of short-term grazer effects, but not
unnaturally, so the densities were similar to those reported by James et al. (2000a).
In general, the grazing efficiency is considered to be closely related to consumer density.
Steinman et al. (1987) and McCormick & Stevenson (1991), e.g., reported strong densitydependent effects of grazer abundances on algal biomass, taxonomic composition and
physiognomic properties in the presence of gastropod and caddisfly predators. Our results
do not show such clear density-related trends but rather indicate a switching of positive and
negative effects on algal assemblages in relation to grazer numbers and incubation time. At
low grazer densities, an immediate reduction of cell numbers to more than 50% of the
available biomass was detected. However, an increase in grazer densities initially showed an
inverse correlation with algal abundances and a fertilizing effect on microalgal assemblages
was revealed. This positive trend on the total algal community disappeared over the duration
of the experiment and effective grazing could be detected after two days of incubation. As
already mentioned before, our results are in good correspondence to grazing patterns found
by Hillebrand & Kahlert (2002) since they detected no direct correlation to grazer density
either. Thus, the relation between grazing efficiency and consumer densities seems to be a
highly complex interplay and the turning points between the positive and negative effects are
hardly to define.
In our study, both grazing experiments showed very similar taxonomic compositions and
diversities. The algal assemblages were dominated by the same five major taxonomic groups
within both grazing experiments and only the genus Caloneis sp. showed higher densities
during the second grazing experiment. The diversity indices H’ and E showed no significant
64
CHAPTER 4
differences for the different treatments and both experimental approaches. As already
mentioned before, intermediate grazer abundances are known to favour nutrient availabilities
at the sediment-water interface to some extent. High nutrient supplies on the other hand are
known to function as triggers for decreasing diversity in microalgal communities (Sullivan,
1976; Carrick et al.1988; Hillebrand & Sommer, 1997) and therefore our constant diversity
variables seem contradictory. However, there are a variety of other studies which have
postulated both increases or decreases of algal diversity in the presence of grazers (Sumner
& McIntire, 1982; Jacoby, 1987; McCormick & Stevenson, 1989; Underwood & Thomas,
1990). Thus diversity shifts caused by grazer activity seem complex. Our results do not
indicate grazer-related diversity changes. Although the algal community as a whole was
obviously not effected by grazing pressure, individual taxa showed positive or negative
trends when consumers were present.
Taxonomic compositions of algal assemblages are often considered to be a function of
grazer selectivity. Selective removal of algal cells is assumed to be facilitated by
morphological and size-dependent features (Nicotri, 1977; Hunter, 1980; McCormick &
Stevenson, 1991). McCormick & Stevenson (1989), e.g. reported that stalked and loosely
attached forms were more susceptible to snail grazing than firmly attached, prostrate algae.
In addition, they indicated that grazers stimulate the growth of understory species by
removing overlying cell assemblages. However, our experiments revealed that sometimes
the same taxonomic groups benefited from grazing activity while in other cases they were
strongly preyed upon. For example, the large-sized genera Stauroneis sp., Amphora sp.,
Synedra sp. and Cymbella sp. were highly reduced in their abundances during the first
experiment while in the second grazing experiment it was shown that after one day the same
genera benefited most from grazing activity.
These distinct grazing differences compared to other studies may originate from the
differences in microalgal assemblages investigated. All of the studies in the literature focused
on periphyton communities. Microphytobenthic assemblages on unstable substrates such as
mud and sand, have been neglected. In contrast, to epi- or periphyton communities where a
distinct three-dimensional layer is usually developed, these patterns are missing on
microphytobenthic biofilms and only few erect forms are present. The microphytobenthos is
usually characterized by prostrate diatoms, forming distinctly flat, two-dimensional
communities (Miller et al., 1987). Therefore, it is not astonishing that most of the dominant
taxa in our experiments fall into the category of prostrate forms, living closely attached to
sediment particles. These were in particular Pinnularia sp., Stauroneis sp., Amphora sp., and
Cymbella sp., the major taxonomic groups that were mainly affected through grazer activity.
In addition, it has to be pointed out, that stalked or erect forms were a minor component of
the algal assemblages and most of the characteristic taxa forming three-dimensional
65
CHAPTER 4
communities were completely missing (e.g. Gomphonema sp., Diatoma sp., Fragilaria sp.,
Melosira sp.). The only erect form that was present in considerable amounts was the genus
Synedra sp.. This microalgae has the ability to stick to surfaces by forming mucilage pads
and therefore it is the only stalked form that could have shown a higher susceptibility to snail
grazing due to its morphological habits. When comparing the grazing efficiency on prostrate
forms versus stalked forms in our experiment, it becomes apparent that both forms were
preyed upon similarly. Furthermore, Synedra sp. was reduced only slightly, especially during
the second grazing experiment. However there is one feature that connects all major
taxonomic groups and this is their size range. Each of these algal groups is characterized by
relatively high biovolumes when opposed e.g. to Navicula- or Nitzschia-species. This
facilitates their potential as food sources and makes them an easy prey for consumers. This
is especially evident in case of the gastropods’ bulldozer-like feeding mode which
characterizes them as rather unselective grazers but with a large spatial efficiency (Sommer,
1997).
Conclusions
In this study we have shown that the gastropod P. antipodarum can have both positive
(fertilizing) and negative effects on microphytobenthic algal biomass. The diversity of the
algae remained unaffected by these snails alluding to their unselectivity and the fact that
microalgal morphology and growth form was unimportant. This is in contrast to periphyton
communities where morphological habits play a major role in grazer-microalgae interactions
and thus these results provide evidence for predominately size- and density-dependent
grazing patterns in microphytobenthic communities dominated by P. antipodarum.
66
CHAPTER 5
Chapter 5
Factors influencing microphytobenthos community
structures beneath macrophyte beds- a marine and
freshwater mesocosm study.
This chapter focuses on the impact of the sediment microflora beneath and adjacent to
macrophyte beds and on the functional role of several herbivore species in influencing
ecosystem processes in vegetated subtidal habitats. The aspects of constantly high nutrient
loads, taxonomic composition shifts and competitive interactions are discussed.
67
CHAPTER 5
5.1 Introduction
Macrophyte beds in shallow aquatic areas constitute extremely productive ecosystems. The
main contributors to the high productivity are vascular plants and, associated with them,
microscopic algae living closely attached to macrophyte leaves. In addition, the sediment
microflora covering the sediments beneath and adjacent to the macrophyte beds play an
important role. Apart from their major importance as primary producers these systems are
characterized by a high biodiversity as they function as a habitat for many invertebrate and
small vertebrate organisms. This plants and microalgae association plays an important role
within the benthic food web and as sediment stabilizers in highly dynamic systems. Recent
studies indicate that epiphytic and microphytobenthic microalgae were by far the most
important primary productivity component within macrophyte beds contributing up to 87% of
the total primary production of the system (Moncreiff et al, 1992; Moncreiff & Sullivan, 2001).
Research has shown that the plant-associated microflora can be considered the major food
source in this community. Unlike macrophyte leaves and detritus, they represent a reliable
and highly nutritious diet (Fry & Sherr, 1984; Kitting et al. 1984; Plante-Cuny & Plante, 1984;
Decho & Fleeger, 1988; Jernakoff et al. 1996; Moncreiff & Sullivan, 2001). Apparently, the
main function of the vascular plant is to provide shelter and structure for associated
organisms as well as to provide a source of detritus, while hardly contributing to the food web
itself.
Until relatively recently, macrophyte beds and sediment communities have been universally
studied as isolated habitats and the coupling between the plant-epiphyte community and the
sediment microflora have mostly been neglected. However, studies have shown the relative
importance of considering this habitat as a whole, instead of ignoring the microphytobenthos
beneath and adjacent to macrophyte beds (Sullivan & Moncreiff, 1990; Moncreiff et al, 1992;
Moncreiff & Sullivan, 2001).
From studies on macrophyte-epiphyte communities it is known that grazers activity maintains
an intact macrophyte habitat (Orth & Van Montfrans, 1984; van Montfrans et al., 1984;
Neckles et al, 1993; Jernakoff et al., 1996; Valentine & Heck, 1999; Heck et al., 2000). As
long as light and nutrients are available in sufficient quantities, microalgae are considered to
be more competitive than vascular plants and an increase in epiphytes can impede the
growth of the macrophyte hosts (Worm & Sommer, 2000). Consequently, grazing by macroor meiofaunal invertebrates plays an important role in regulating microalgal growth and thus
contribute to the stability of the system (Orth & van Montfrans, 1984; van Montfrans et al.,
1984; Neckles et al, 1993). Similar patterns have been shown in studies dealing with
periphyton and microphytobenthic communities (Sumner & McIntire, 1982; Lamberti & Resh,
1983; McCormick & Stevenson, 1991; Kahlert & Baunsgaard, 1999). Most of the former
experimental studies have considered the grazer community to be a relatively homogeneous
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CHAPTER 5
functional group grazing unselectively on microalgae and detritus (Edgar 1990; Jernakoff et
al, 1996) thus, there are strong seasonal and spatial variations in grazer assemblages in the
field. Conversely other investigations showed differences in feeding preferences and
selectivity by grazing organisms and governing roles of particular taxa have also been
detected (Duffy & Hay, 1994; Brendelberger, 1995; Jernakoff & Nielsen, 1997; Sommer,
1997; Duffy & Hay, 2000).
The aim of our study was to simulate freshwater and marine macrophyte bed assemblages
under controlled conditions in order to study the impact of different macrograzers on the
macro-and microfloral community. We wished to evaluate the importance of the sand
microflora beneath and adjacent to the macrophyte beds in particular. To this end, we used
freshwater and marine mesocosm experiments to test the influence of the most common
grazing invertebrates on microphytobenthic communities within macrophyte beds.
5.2 Material & Methods
Experimental design
In summer 2002 marine and freshwater laboratory mesocosm experiments were set up. The
experiments were run using nine plastic tanks (117 x 93 x 60 cm; see sketch 1).
Sketch 1: Experimental set-up of the mesocosms. Circles present the six sub-units from each tank.
Arrows show the water-flow direction and the irrigation system of the sub-units.
Each tank was split into six smaller mesocosm units, using cylindrical transparent plastics
(BP Chemicals; ∅ 30 cm, height: 60 cm; see sketch 2). The two experiments were conducted
with 54 mesocosms for each experiment. Plastic trays (Gies, ∅ 30 cm) were set up at the
69
CHAPTER 5
bottom of each treatment, filled with 2000 µm-sieved sediment from the field in order to
eliminate the presence of macroinvertebrate grazers. After the sediment had settled for 24h
the experimental units were planted with freshly harvested and washed macrophytes. The
marine experiments were planted with the seagrass Zostera marina and set up with
sediments from patches within the same seagrass meadow from the Baltic site
“Falkensteiner-Strand” in the Kiel Fjord. For the freshwater experiments we used the
macrophyte Potamogeton perfoliatus and sediments from the Schluensee. Each unit was
stocked with the same number of macrophytes as was known for the natural abundances at
each site. Z. marina was planted at abundances of 20-23 shoots per unit (total plant length:
10 m) and P. perfoliatus with a total number of 5-6 plants per unit (total plant length: 1 m).
After having planted the mesocosms the macrophyte-sediment-community was left
undisturbed for four days.
Sketch 2:.Experimental set-up (side view) of the mesocosm units with grazer presence. Arrows present
the water-flow direction.
Littorina littorea (Gastropoda), Idotea balthica (Isopoda) and Gammarus salinus (Amphipoda)
were used as grazing organisms in the marine experiment. In the freshwater experiment we
introduced Radix ovata (Gastropoda), Asellus aquaticus (Isopoda) and Gammarus pulex
(Amphipoda) into the experimental units. In addition to the starts and the control treatments,
seven different grazer-treatments were chosen (see table 1) in order to investigate the
influence of the three different macroinvertebrate grazers.
70
CHAPTER 5
Table 1: List of treatments and grazer combinations in the marine and the freshwater mesocosm
experiments
treatment grazer combinations
Marine mesocosm experiment
start
control
I
G
L
IG
IL
GL
IGL
Freshwater mesocosm experiment start
control
A
G
R
AG
AR
GR
AGR
Idotea balthica
Gammarus salinus
Littorina littorea
Idotea balthica, Gammarus salinus
Idotea balthica, Littorina littorea
Gammarus salinus, Littorina littorea
Idotea balthica, Gammarus salinus,Littorina littorea
Asellus aquaticus
Gammarus pulex
Radix ovata
Asellus aquaticus, Gammarus pulex
Asellus aquaticus, Radix ovata
Gammarus pulex, Radix ovata
Asellus aquaticus, Gammarus pulex, Radix ovata
The aim of this study was to induce effects of several grazer species on benthic algal
biomass and community structure. The focus herein was to detect varying grazing patterns
considering interspecific competition and grazer exclusion.
Grazer abundances introduced into the grazer treatments were related to natural
abundances found by Jaschinksi & Gohse-Reimann (pers. com.) for each natural site. The
initial grazer biomass was 40 mg organic carbon in the marine treatments and 10 mg organic
carbon in the freshwater mesocosms which is well within the range of field abundance. The
number of grazers in the single grazer treatments, corresponding to 40 or 10 mg organic
carbon, were 18 I. balthica, 25 G. oceanicus and 6 L. littorea in the marine experiment and
18 A. aquaticus, 18 G. pulex and 3 R. ovata in the freshwater treatments. Mixed-grazer
treatments were stocked using a supplementary design whereby biomass of total numbers of
grazers were kept constant and only fractions of total grazer numbers were used to achieve
a total estimate of 40 or 10 mg organic carbon. Each treatment was replicated in three
independent mesocosms in a randomised design. The marine as well as the freshwater
experiments were sampled at the beginning of the experiment (time 0) and after seven and
21 days of incubation. The marine experiment was started on the 17th of July 2002 when
grazers were introduced to the tanks and the last samples were taken after 21 days on the
8th of August 2002. The freshwater experiment was run straight after the marine experiment
had finished and ran from the 22nd of August to the 12th of September 2002. The mesocosms
were supplied independently with a constant flow of either sand-filtered brackish water from
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the Kiel Fjord (salinity: 14.7 PSU ± 0.7) or, in case of the freshwater mesocosms, with tap
water. Water flowed out of each tank continuously through a hole, 2 cm in diameter, that was
covered with a 1-mm plastic mesh. The light regime was adapted to summer conditions with
a 16h day and 8h night cycle. Light intensities from the lamps above tanks ranged within
treatments between 86.0 µmol s-1 m-2 ± 19.5 at the water surface, 7.2 µmol s-1 m-2 ± 5.6 at the
sediment surface of the marine mesocosms and 33.4 µmol s-1 m-2 ± 12.0 in the freshwater
units. The temperature in the climate room was 17°C whereas water temperature in the
mesocosms was slightly higher due to a heating-effect of the lamps; 18.3 °C ± 0.5 in the
marine mesocosms and 18.0°C ± 0.2 in the freshwater treatments. Nutrients from the inflow
to the experimental units were determined daily using an auto-sampler from the Institute of
Marine Science/Kiel using the methods of Grasshoff et al. (1983). In addition, pore water
nutrient analyses from the sediment were performed at the end of each experiment. The
nutrient data was provided by Jaschinski (unpublished data).
Sampling & sample processing
In order to sample sediment surfaces for chlorophyll a and cell counts two plastic tubes (Vol.
50 ml) were inserted into the freshly-sieved sediments of each tank prior to the regeneration
period of the sediment-macrophyte regeneration time at the beginning of each experiment.
The tubes (∅ 2.3 cm; height: 2.5 cm) had been cut off at the bottom and covered with a 500
µm plastic mesh in order to guarantee a transfer within the sediment column (see Chapter 2).
The top of the tube was placed level with the surface of the sediment. Prior to the sampling,
the tubes were closed in-situ with a screw-lid and could therefore be retrieved easily.
Subsequently the sediment samples were preserved by using a slightly modified Cryolandertechnique as described in Wiltshire et al. (1997). The Cryolander was placed on top of the
sediment surface of each tube and some liquid nitrogen was dribbled onto the absorbent
cotton in it. The liquid nitrogen was instantly vaporised due to the cottons’ ambient
temperature and the vapour froze the sediment surface without any distortion. Afterwards the
entire sample was dropped into liquid nitrogen and subsequently stored at –80°C until further
processing. The microslicing of the sediment surface and further pigment analysis was
carried out using method Wiltshire (2000). Frozen samples were cut into 0.5 cm thin discs,
placed on the stage of a freezing microtome (Leica CM 3050S) by using frozen oil and cut
into slices at 125 µm intervals at an area of 1 cm². The sediment layers were cut to a
sediment depth of 0-500 µm and subsequently frozen in liquid nitrogen and then freeze-dried
overnight. The pigment analysis of freeze dried sediment samples was conducted by using a
Waters 910-HPLC-system. Details on extraction, gradients, flow rate etc. are given by
Wiltshire (2000).
72
CHAPTER 5
Statistics
To test for significant differences in chlorophyll a contents and cell numbers a full-factorial
ANOVA and an LSD Post hoc-Test were applied. Diversity variables H’ and E were
calculated with PRIMER 5.2 ( 2001 Primer-E Ltd.) whereas diversity was based on the
Shannon-Weaver function (H’; loge) (Shannon and Weaver, 1963) and Evenness (E) on
Pielou’s Evenness (Pielou, 1969). The tests for significant diversity differences were
performed by applying an ANOVA and LSD post hoc-Tests.
5.3 Results
Marine mesocosms
Chlorophyll a content
In the Z. marina beds of the marine mesocosm experiment the chlorophyll a content at the
sediment surface showed slightly higher concentration during the short-term incubation when
compared to the long-term period. After seven days the lowest chlorophyll a concentrations
were detected in the treatments with IGL (0.37 µg cm-2 ± 0.36) whereas maximum contents
were in the combined-grazer treatment IG (1.70 µg cm-2 ± 0.40) (figure 1A). Significantly
lower chlorophyll a concentrations after seven days occurred in the presence of all three
grazers when compared to the G- and the IG-units (p<0.05). However, no significant
differences were detected between the grazer treatments and the starts (t0) and the controls
(ctrl) (p>0.05). As the experiment progressed, some of the grazer units showed decreased
chlorophyll contents (figure 1B). For example by day seven to day 21 with G. salinus as a
single grazer and I. balthica in combination with G. salinus (IG) and L. littorea (IL) (p<0.05).
B
2,5
2,0
chlorophyll a (µg cm-2)
chlorophyll a (µg cm-2)
A
1,5
1,0
0,5
2,5
2,0
1,5
1,0
0,5
0,0
0,0
t0
ctrl
I
G
L
IG
treatment
IL
GL
IGL
t0
ctrl
I
G
L
IG
IL
GL
IGL
treatment
Figure 1: A, B: Chlorophyll a concentrations (µg cm-2) at the top 500 µm of the sediment surface in the
marine mesocosms after 7 (A) and after 21 days (B) of incubation. The x-axis presents the start values (t0)
and the different treatments: control (ctrl), single-grazer treatments (I, G, L), the combined grazer
treatments (IG, IL, GL) and the all-three grazer-treatment (IGL). Bars present mean values and standard
deviations are given.
73
CHAPTER 5
Cell numbers
In contrast to the total chlorophyll contents, the cell numbers at the sediment surface gave
contradictory results. In the first seven days of the experiment very low cell numbers were
found and no significant differences between the different treatments could be detected
(figure 2A; p>0.05). After seven days a minimum of 492 cells cm-2 (IGL) and a maximum of
2775 cells cm-2 (IG) were counted. At the end of the three week incubation period, a
significant increase in cell number was found in the controls and in the majority of grazer
treatments (figure 2B; treatments: ctrl, I, L, GL, IGL) and as much as 10 to15 times higher
cell numbers were determined. The lowest cell numbers of 4719 cells cm-2 were found in the
combined presence of I. balthica and L. littorea (IL) and this treatment therefore differed
significantly from the I- and the IGL-units.
B 20000
20000
total cell numbers * cm-2
total cell numbers * cm
-2
A
15000
10000
5000
SD= 15026
SD= 20784
15000
10000
5000
0
0
t0
ctrl
I
G
L
IG
treatment
IL
GL
IGL
t0
ctrl
I
G
L
IG
IL
GL
IGL
treatment
Figure 2: A, B: Total cell numbers (cells cm-2) at the top 500 µm of the sediment surface in the marine
mesocosms after 7 (A) and after 21 days (B) of incubation. The x-axis presents the start values (t0) and the
different treatments: control (ctrl), single-grazer treatments (I, G, L), the combined grazer treatments (IG,
IL, GL) and the all-three grazer-treatment (IGL). Bars present mean values and standard deviations are
given
Major taxonomic groups
At the beginning of the marine mesocosm experiment the microphytobenthic community was
dominated by diatoms (>90%). Over 70% of these diatoms were of the prostrate type and
only 16% belonged to chain forming or stalked diatom genera (1%) (figure 3A). 8% of the
algae were Cyanobacteria and only 0.5% chlorophytes. After one week of incubation this
pattern changed slightly in the control units. Most of the grazer treatments however showed
distinct composition changes when compared to the start (t0) and the control-units (ctrl). One
thing which was remarkable after the first week was the simultaneous increase in stalked
diatoms and chlorophytes in the controls and in the grazer treatments respectively (I, IG, IL).
Diatom chains were reduced in almost every treatment, in the controls as well as in the
74
CHAPTER 5
grazer units. The strongest composition changes in comparison to start- and control-samples
were detected for treatments with the single grazers G. salinus and L. littorea. These
invertebrates obviously allowed the dominance of prostrate diatoms as this group made up
94-99% of the total algal community of these units. In contrast, the combined grazer
treatments IG and IL showed that prostrate forms made up 25 and 34% of the
microphytobenthos
of
these
units.
Increasing
shares
of
stalked
diatoms
and/or
Cyanobacteria and chlorophytes could be detected for the treatments I, IG, IL, GL and IGL.
In the marine mesocosms the long incubation period showed distinct composition changes
and an overall dominance of chain forming diatoms appeared (67-87%; figure 3B).
A
B
100
80
algal groups (%)
algal groups (%)
80
100
60
40
20
60
40
20
0
0
t0
ctrl
I
G
L
IG
IL
GL
IGL
t0
ctrl
I
G
treatment
prostrate diatoms
stalked diatoms
diatom chains
Cyanobacteria
Chlorophyta
L
IG
IL
GL
IGL
treatment
prostrate diatoms
stalked diatoms
diatom chains
Cyanobacteria
Chlorophyta
Figure 3: A, B: Algal group composition at the top 500 µm of the sediment surface in the marine
mesocosms after 7 (A) and after 21 days (B) of incubation. Relative proportions of the different taxonomic
groups are calculated as % of the total algal cells. The x-axis presents the start values (t0) and the different
treatments: control (ctrl), single-grazer treatments (I, G, L), the combined grazer treatments (IG, IL, GL)
and the all-three grazer-treatment (IGL).
75
CHAPTER 5
Detailed taxonomic composition
Despite the similarities of the algal communities in all of the marine mesocosms after one
week, the detailed taxonomic composition still showed differences between the starts (t0),
the controls (ctrl) and the grazer treatments. When comparing the initial community structure
(t0) with the controls (ctrl), lower proportions of Fragilaria sp., Amphora sp. and Navicula sp.
were detected in the controls concurrent with an increase of Diploneis sp., Synedra sp. and
chlorophytes (figure 4A). In the presence of the G. salinus and L. littorea as single grazers,
the microphytobenthos showed distinct taxonomic differences when compared to treatments
with I. balthica. In contrast to the I-treatments, G- and L-treatments showed a sharp decline
of Synedra sp., Cyanobacteria and Chlorophyta and only very few Melosira nummuloides
were found, whereas the proportions of Nitzschia sp. and Diploneis sp. increased. In
addition, similar trends were found for the combination of these both grazers (GL), although
Synedra sp. showed much higher proportions this time and M. nummuloides increased. The
combined grazer treatments IG and IL were characterized by similar community structures
with high total shares of Synedra sp., Cyanobacteria and Chlorophyta and the reduction of
Pinnularia sp.. In the presence of all three grazers (IGL) the sediment had a composition that
was dominated by Diploneis sp., Navicula sp., Amphora sp., Nitzschia sp. and Cyanobacteria
and low numbers of M. nummuloides. Complete extinction of chlorophytes and the stalked
diatom Synedra sp. occurred.
A
B
80
taxonomic composition (%)
taxonomic composition (%)
100
60
40
20
100
80
60
40
20
0
0
t0
ctrl
I
G
L
IG
treatment
IL
GL
IGL
t0
ctrl
I
G
L
IG
IL
GL
IGL
Pinnularia sp.
Melosira nummuloides
Stauroneis sp.
Cymbella sp.
Navicula sp.
Synedra sp.
Nitzschia sp.
Diploneis sp.
Amphora sp.
Gyrosigma sp.
Fragilaria sp.
Caloneis sp.
Placoneis sp.
Diatoma sp.
Nitzschia longissima
Chlorophyta (filaments)
Cyanobacteria (filaments)
treatment
Figure 4: A, B: Taxonomic composition at the top 500 µm of the sediment surface in the marine
mesocosms after 7 (A) and after 21 days (B) of incubation. Relative proportions of the different genera are
calculated as % of the total algal cells. The x-axis presents the start values (t0) and the different
treatments: control (ctrl), single-grazer treatments (I, G, L), the combined grazer treatments (IG, IL, GL)
and the all-three grazer-treatment (IGL).
After three weeks the microalgal assemblage changed completely and no similarity between
the initial community structure and the control- and grazer treatments could be found. In this
case M. nummuloides, a chain-forming diatom that was not present initially, dominated the
76
CHAPTER 5
community in numbers between 43% (L) and 72% (control) (figure 4B). In addition, the
species Nitzschia longissima appeared during the long-term-incubation for the first time and
Fragilaria sp. recurred. The genera Navicula, Pinnularia, Nitzschia, Amphora and Cymbella.
which initially colonized the sediment surface, were reduced after three weeks and some of
them were almost extinct. One grazer treatment that showed a slightly different distribution in
comparison to the majority of grazer units, was the treatment with L. littorea as single grazer.
Here low M. nummuloides as well as low Cyanobacteria proportions and a complete
reduction of Chlorophyta were seen. Higher percentages of Diploneis sp. and Nitzschia
longissima were detected.
A
B
2,0
diversity indices H' & E
diversity indices H' & E
2,0
1,5
1,0
0,5
0,0
1,5
1,0
0,5
0,0
t0
ctrl
I
G
L
IG
treatment
IL
GL
IGL
t0
ctrl
I
G
L
IG
IL
GL
IGL
treatment
Figure 5: A,EB:(Evenness)
Diversity indices H’ (Diversity) and E (Evenness) at the
top 500 µm of the sediment surface
E (Evenness)
H ' (Diversity)
H ' mesocosms
(Diversity)
in the marine
after 7 (A) and after 21 days (B) of incubation.
The x-axis presents the start
values (t0) and the different treatments: control (ctrl), single-grazer treatments (I, G, L), the combined
grazer treatments (IG, IL, GL) and the all-three grazer-treatment (IGL). Mean values and standard
deviations are given.
Diversity & evenness
Diversity (H’) and evenness (E) evaluations gave similar values at the beginning of the
experiment (t0) when compared to control- and grazer-treatments after one week of
incubation (figure 5A). The diversity in the L. littorea-units declined slightly although these
differences were not significant (p>0.05). In addition, no changes for evenness could be
detected for either the start- or for the control values or for grazer treatments. As the
experiment proceeded both indices showed higher variances and, in general, diversity and
evenness declined (figure 5B). After 21 days diversity and evenness in the control treatments
had declined significantly when compared to the first incubation period (p<0.05). The
diversity in the G- and IL-units were significantly higher than in the presence of all three
grazers (IGL) and evenness was higher at the beginning of the experiment (t0) as well as in
the IL- treatment in comparison to the IGL-units (p<0.05). Diversity and evenness of the
microphytobenthic community both showed a clear decrease with regard to total cell
77
CHAPTER 5
numbers (figure 6). The declining trend of H’ with increasing cell numbers showed a steeper
gradient (y= -0.36 + 2.7).
diversity indices H' + E
2,5
2,0
y = -0,3596x + 2,6941
r² = 0,2928
1,5
1,0
0,5
y = -0,2005x + 1,3186
r² = 0,4549
0,0
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
log total cell numbers * cm-2
Evenness (E)
Diversity (H')
regression line
Figure 6: Correlation between the total algal biomass (log total cell numbers *cm-2) and the
diversity indices H’ (Diversity) and E (Evenness) respectively at the top 500 µm of the sediment
surface in the marine mesocosm treatments. Solid lines are linear regressions.
Freshwater mesocosms
Chlorophyll a content
The chlorophyll a contents at the sediment surface of the freshwater units showed similar
concentration ranges both after one and after three weeks of incubation. During the shortterm incubation period the total chlorophyll a values ranged from 1.1 µg cm-2 ± 0.2 in the AGtreatment to 3.1 µg mesocosms cm-2 ± 1.2 in the presence of A. aquaticus (figure 7A;
p<0.05). The chlorophyll a contents in the AG-units were also significantly lower compared to
all single-grazer units (A,G,R), the combined-grazer treatment AR and the start samples (t0)
(p<0.05). Significantly higher concentrations were shown for A. aquaticus-units (A) in
78
CHAPTER 5
comparison with combined-grazer treatments AG and GR as well as the treatments with all
three grazers present (AGR) (p<0.05). After 21 days of incubation the chlorophyll a
concentrations changed only slightly. A significant decrease was found in the presence of A.
aquaticus and an increase in the AG-units (figure 7B). For the combined-grazer treatment
AG highest chlorophyll a concentrations were found (2.6 µg cm-2 ± 0.5). These values were
significantly higher than data obtained after three weeks from the G-, AR- and AGRtreatments (p<0.05).
B
5
4
5
4
-2
chlorophyll a (µg cm )
chlorophyll a (µg cm-2)
A
3
2
3
2
1
1
0
0
t0
ctrl
A
G
R
AG
AR
GR AGR
t0
ctrl
A
treatment
G
R
AG
AR
GR AGR
treatment
Figure 7: A, B: Chlorophyll a concentrations (µg cm-2) at the top 500 µm of the sediment surface in the
freshwater after 7 (A) and after 21 days (B) of incubation. The x-axis presents the start values (t0) and the
different treatments: control (ctrl), single-grazer treatments (A, G, R), the combined grazer treatments
(AG, AR, GR) and the all-three grazer-treatment (AGR). Bars present mean values and standard
deviations are given.
Cell numbers
The number of algal cells showed lower abundances after the first week and at the end of the
three weeks period an increase in cell numbers was found. As far as total cell numbers
during the first incubation period are concerned, no significant differences between the
various treatments could be detected (figure 8A; p>0.05).
After three weeks, cell numbers increased significantly within the single-grazer treatments A
and G as well as in the presence of all three grazers (AGR) (figure 8B; p<0.05). When
compared to initial cell numbers (t0), most of the long-term treatments showed distinct
increases in algal abundance (A, G, R, AG, AGR). The single-grazer units A and G showed
significant differences compared to the controls (p<0.05). A minimum of 70075 cells cm-2 was
detected in the presence of G. pulex and R. ovata (GR). These abundances were
significantly lower when compared to the treatments A, G, R and AGR.
79
CHAPTER 5
-2
B 400000
300000
total cell numbers * cm
total cell numbers * cm-2
A 400000
200000
100000
0
SD=
SD=
SD=
175975 111796 244419
SD=
160825
300000
200000
100000
0
t0
ctrl
A
G
R
AG
treatment
AR
GR AGR
t0
ctrl
A
G
R
AG
AR
GR AGR
treatment
Figure 8: A, B: Total cell numbers (cells cm-2) at the top 500 µm of the sediment surface in the freshwater
mesocosms after 7 (A) and after 21 days (B) of incubation. The x-axis presents the start values (t0) and the
different treatments: control (ctrl), single-grazer treatments (A, G, R), the combined grazer treatments
(AG, AR, GR) and the all-three grazer-treatment (AGR). Bars present mean values and standard
deviations are given.
Major taxonomic groups
During the short-term freshwater experiment the algal assemblages were dominated by
chain-forming diatoms (28-55%) and Cyanobacteria (32-62%; figure 9A). Chlorophyta were
not present. In contrast to the grazer treatments and the controls, the starting community (t0)
showed higher percentages of prostrate diatoms (34%). However, this pattern changed after
the first week where this taxonomic group made up 10 to 16% of the total algal community.
Although the proportions of prostrate and stalked diatoms remained almost constant in all
grazer units as well as in the controls, the shares of Cyanobacteria and diatom chains
showed high variations. Especially high proportions of Cyanobacteria were detected for the
combined-grazer unit AR (62%) and the single grazer treatment G (55%) whereas diatom
chains contributed approximately 50% to the total algal community in the controls and the
AGR-treatments. Three weeks of incubation produced a shift in the community structure as a
clear dominance of chain-forming diatoms with percentage shares ranging from 72% (GR) to
87% (AGR) occurred (figure 9B). In contrast to the short-term incubation, the relative
importance of stalked and prostrate diatoms declined considerably and Cyanobacteria
contributed between 9% (AGR) and 21% (AR) to the total algal community.
80
CHAPTER 5
A
B
100
80
algal groups (%)
80
algal groups (%)
100
60
40
60
40
20
20
0
0
t0
ctrl
A
G
R
AG
AR
GR AGR
t0
ctrl
A
G
AG
AR
GR
AGR
treatment
treatment
prostrate diatoms
stalked diatoms
diatom chains
Cyanobacteria
Chlorophyta
R
prostrate diatoms
stalked diatoms
diatom chains
Cyanobacteria
Chlorophyta
Figure 9: A, B: Algal group composition at the top 500 µm of the sediment surface in the freshwater
mesocosms after 7 (A) and after 21 days (B) of incubation. Relative proportions of the different taxonomic
groups are calculated as % of the total algal cells. The x-axis presents the start values (t0) and the different
treatments: control (ctrl), single-grazer treatments (A, G, R), the combined grazer treatments (AG, AR,
GR) and the all-three grazer-treatment (AGR).
Detailed taxonomic composition
During the first incubation period, the controls (ctrl) as well as the grazer treatments showed
similar proportions of algal taxa (figure 10A). When comparing the initial community structure
(t0) with the controls, lower proportions of Fragilaria sp. (15%) and Gomphonema sp. (1%)
occurred and higher abundances of Pinnularia sp. (4%), Stauroneis sp. (4%), Nitzschia sp.
(17%) and Merismopedia sp. (50%) were detected at the beginning of the experiment. These
distribution patterns changed in the grazer- and the control-units after one week as they
showed higher proportions of Fragilaria sp. ranging from 28% (AR) to 52% (ctrl) and
relatively higher abundances of Gomphonema sp. (2-5%) were found. The highest amounts
of Merismopedia sp. during the short-term incubation were found for the combination of A.
aquaticus and R. ovata (AR; 62%) and fairly low percentage of 30-40% were detected in the
ctrl-, A-, GR- and AGR-treatments. In the long-term a distinct dominance ranging from 72%
(GR) to 88% (AGR) of Fragilaria sp. was observed in all different treatments (figure 10B).
Merismopedia sp. became less important and made up only 7-21% of the total algal
community. All other taxonomic groups, originally present in the different treatments, showed
proportions of below 2%.
81
CHAPTER 5
A
B
taxonomic composition (%)
taxonomic composition (%)
100
80
60
40
20
0
100
Pinnularia
Caloneis
Stauroneis
Cymbella
Navicula
Synedra
Nitzschia
Aulacoseira
Diploneis
Gomphonema
Amphora
Placoneis
Fragilaria
Pediastrum
Scenedesmus
Merismopedia
80
60
40
20
0
t0
ctrl
A
G
R
AG
AR
GR AGR
t0
ctrl
A
G
treatment
R
AG
AR
GR AGR
treatment
Figure 10: A, B: Taxonomic composition at the top 500 µm of the sediment surface in the freshwater
mesocosms after 7 (A) and after 21 days (B) of incubation. Relative proportions of the different genera are
calculated as % of the total algal cells. The x-axis presents the start values (t0) and the different
treatments: control (ctrl), single-grazer treatments (A, G, R), the combined grazer treatments (AG, AR,
GR) and the all-three grazer-treatment (AGR).
Diversity & Evenness
The highest values for diversity (H’) and evenness (E) were seen at the beginning of the
experiment (t0). However, decreases in H’ and E were observed after one and three weeks
(figure 11 A, B; p<0.5). After seven days evenness was significantly lower in the treatments
with R. ovata as single grazer (R) compared with the t0-values (p<0.5). In terms of diversity
all grazer treatments showed similar H’-values and no significant differences could be
B
2,0
2,0
1,8
1,8
1,6
1,6
diversity indices H' & E
diversity indices H' & E
A
1,4
1,2
1,0
0,8
0,6
0,4
0,2
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0,0
t0
ctrl
A
G
R
AG
AR
GR AGR
t0
ctrl
A
treatment
E (Evenness)
H ' (Diversity)
G
R
AG
AR
GR AGR
treatment
E (Evenness)
H ' (Diversity)
Figure 11: A, B: Diversity indices H’ (Diversity) and E (Evenness) at the top 500 µm of the sediment
surface in the freshwater mesocosms after 7 (A) and after 21 days (B) of incubation. The x-axis presents
the start values (t0) and the different treatments: control (ctrl), single-grazer treatments (A, G, R), the
combined grazer treatments (AG, AR, GR) and the all-three grazer-treatment (AGR). Mean values and
standard deviations are given.
82
CHAPTER 5
detected (p>0.05). During the course of the experiment, the ctrl- as well as the grazer-units
showed a significantly lower diversity and evenness when compared to the initial values (t0;
p<0.5). No differences between the control- and the grazer-treatments were seen (figure
11b). In addition, the correlation between total cell numbers and diversity variables evinces a
decrease in diversity with increasing cell numbers (figure 12).
2,5
diversity indices H' & E
2,0
y = -0,5949x + 3,9542
r² = 0,3994
1,5
1,0
0,5
y = -0,2346x + 1,5548
r² = 0,4215
0,0
2,5
3,0
3,5
4,0
4,5
5,0
5,5
6,0
log total cell numbers * cm-2
Evenness (E)
Diversity (H')
regression line
Figure 12: Correlation between the total algal biomass (log total cell numbers *cm-2) and
the diversity indices H’ (Diversity) and E (Evenness) respectively at the top 500 µm of the
sediment surface in the freshwater mesocosm treatments. Solid lines are linear
regressions.
83
CHAPTER 5
5.4 Discussion
The impact of grazers on the community structure of macrophyte beds and their associated
flora has become an important research field in aquatic benthic ecology over the last
decades. Since nutrient supply is a major variable that regulates primary productivity and
species composition in benthic communities, this topic has also gained in importance. Thus,
the analysis of the combined impacts of consumers and nutrient resources on benthic floral
diversity are of considerable relevance to understand bentho-pelagic systems better.
Marine mesocosms
Total chlorophyll a concentrations and cell numbers
During the marine mesocosm experiment with Z. marina beds the chlorophyll concentrations
at the sediment surface in general showed higher contents after one week compared to the
three incubation week period. Since grazing, in general, is considered to cause decreases in
chlorophyll a contents at the sediment surface these data fit well into previously published
studies (Cattaneo, 1983; Lamberti & Resh, 1983; Feminella & Hawkins, 1995; Steinman,
1996; Hillebrand & Kahlert, 2002). Thus, our assumption, that the presence of invertebrates
affects chlorophyll concentrations negatively over the total duration of the experiment, was
confirmed.
The total chlorophyll concentrations at the sediment surface of our marine mesocosms
ranged from 0.1 µg cm-2 to 1.7 µg cm-2. Since comparable estimates for any other seagrass
beds are rare, comparisons are difficult. Until now, there is only one study known by the
authors that provided chlorophyll a concentration from microphytobenthic assemblages
within a seagrass bed (Daehnick et al.,1992). In contrast to our investigations, this study
showed relatively high chlorophyll a concentrations of 1.4-12.5 µg cm-2 adjacent to and
beneath the seagrass Halodule wrightii in the Mississippi Sound. Additionally, studies from
intertidal or subtidal regions provide chlorophyll a data although the comparability of
sediments within seagrass beds are limited. De Jonge and Colijn (1994) recorded ranges of
2.8-24.7 µg cm-2 and Agatz et al. (1999) of 13.0-23.8 µg cm-2, respectively, for intertidal sand
flats, while Sundbaeck (1983) found a range of 2.3-25.8 µg cm-2 for a shallow subtidal sand
flat. These discrepancies might be explained by the different sediment volumes sampled. All
these mentioned studies determined chlorophyll a concentrations from the first one cm of the
sediment surface. In our case the data was restricted to the uppermost 500 µm of the surface
layer and therefore it can be best compared to an intertidal field study conducted by
Barranguet & Kromkamp (2000) which detected chlorophyll a ranges of 1.0-4.0 µg cm-2 at the
uppermost 1000 µm.
Contrasting with the chlorophyll measurements, the results for total cell numbers were less
easy to interpret. During the first period of the experiment, low cell numbers occurred and no
84
CHAPTER 5
significant differences between the treatments could be detected. However, after three
weeks a sharp increase in cell numbers was found in the controls and also in the majority of
grazer treatments. Since chlorophyll a concentrations and cell numbers are used as biomass
estimates and as they often show good correlation (Karlstroem & Backlund, 1977; Khondker
& Dokulil, 1988; Mitbavkar & Anil, 2002), the discrepancies in our results are not easily
explained. Sediment samples for chlorophyll a and cell count analysis were both obtained
following the same technique, consequently a methodological error seems unlikely. A
possible explanation could be that the increase in algal abundance at the uppermost surface
layer shaded the underlying cell layers and thus overstory algae negatively affected the
pigment composition of understory forms. This might explain the weak correlation between
chlorophyll content and algal cell numbers.
Major taxonomic groups
The initial sediment microflora showed a clear dominance of diatoms, whereby prostrate
forms were the major constituents. Cyanobacteria made up only 8% of the whole sediment
community. This is in good correspondence to earlier microphytobenthos studies in the
intertidal as well as in subtidal macrophyte beds. Edaphic algae dwelling on sediments
consist mainly of diatoms and blue-green or green algae occur only temporarily. In addition,
the diatom populations are usually composed of pennate, prostrate forms, which are either
epipsammic (attached to sand grains) or epipelic (motile forms within the sediment)
(Daehnick et al., 1992; Moncreiff et al. 1992; Agatz et al., 1999; Mitbavkar & Anil, 2002). But
apart from these prostrate forms, chain-forming diatoms occurred in considerable
abundances.
During the course of the marine experiment a shift in taxonomic composition was observed
after one and after three weeks. Apart from grazing activity, there are a variety of abiotic
factors which can regulate species composition in algal assemblages, e.g. temperature,
salinity or nutrient loads. The first fractions were constant in our experiments, nutrients
showed particular dynamics.
Nutrient effects:
Nutrient analyses from the inflow of the mesocosm units showed a clear increase in nitrate
and silicate concentrations during the first week (figure 13; Jaschinski, unpublished data). As
each unit was supplied autonomously with the same source of water, it is highly unlikely that
inter-variations in nutrient supply occurred. Both nutrient components showed a maximum
concentration on day seven of the experiment whereby nitrate reached values of 13 µmol l-1
and silicate of 27 µmol l-1. The nutrient loads in the experimental units were due to the inflow
85
CHAPTER 5
of the nutrient rich seawater from the Kiel Fjord. These values are extremely high when
compared to field data achieved from the Z. marina-site in Falkenstein during the seasons
2001 and 2002 (Jaschinski, pers. comm.). Accordingly the nutrient compositions in our
experiment resembled a spring- or autumn-nutrient situation. It is known, that there is a
nutrient limitation in the water-column from late spring to autumn, especially in terms of
nitrogen and that for example distribution patterns in epilithic communities can be altered by
nutrient supplies from the water-column (Hillebrand & Sommer, 1997). Consequently, the
observed taxonomic composition changes in our mesocosms were highly related to
increased nutrient availabilities. Tendencies towards a shift in community structure after one
week were observed as stalked diatoms and green algae benefited from the changing
nutrient supplies.
-1
nutrient content (µmol * l )
30
day 1-7
day 8-20
25
20
15
10
5
0
18.7. 19.7. 21.7. 22.7. 23.7. 24.7. 25.7. 26.7. 27.7. 28.7. 30.7. 31.7. 1.8. 2.8. 3.8. 5.8. 6.8.
day of incubation
nitrate
ammonium
silicate
phosphate
Figure 13: Nutrient concentrations (µmol * l-1) in the water-column of the experimental units measured
from the inflow during the course of the marine mesocosm experiment (day 1-20).
Grazer effects & competitive interactions:
Compared to the controls, grazer treatments showed distinct composition changes and interspecific variations. Consequently, the community structure of the microphytobenthos was not
only regulated by nutrient supplies but also by feeding preferences and competitive
interactions. The single grazers G. salinus and L. littorea, e.g., influenced the algal
assemblages significantly after seven days by reducing characteristic constituents like
86
CHAPTER 5
chlorophytes, cyanophytes and stalked diatoms, leaving behind a sediment microflora
comprising almost exclusively of prostrate diatoms. In combination with the isopod I. balthica
(IG or IL), however, a clear reduction of prostrate forms was seen. This indicates that while
there was no co-occurring grazer present, the single grazers G. salinus and L. littorea
showed a preference for chlorophytes, cyanophytes and stalked diatoms. But when these
invertebrates had to share food sources with I. balthica they changed from their preferred
diets to prostrate diatoms as food source. Thus, the presence of I. balthica induced a shift in
resource use of G. salinus and L. littorea. The resource use of competitors has been a major
topic in pelagic community ecology for many years. The mechanisms that determine species
coexistence with shared resources are still discussed controversially (Ricklefs & Schluter,
1993; Gaston, 2000) but the central assumption of competition theory is that the strength of
interspecific competition is inversely related to the amount of resource partioning (Pacala &
Roughgarden, 1982). Thus it can be assumed, that some of the distribution patterns obtained
from our mesocosm units were also induced by species competing for resources.
Furthermore, the diverse array of grazer effects might be a result of overlapping trophic
niches.
Nutrient versus consumer effects:
The nitrate and silicate concentrations remained high during the course of the experiment
and persisting high nutrient loads resulted in sharp composition changes of the
microphytobenthic community over the long-term. An overall dominance of chain-forming
diatoms appeared and no differences between treatments occurred. Thus, under these
circumstances, diatom chains benefited most from continuously high nutrient supplies
resulting in tremendous growth rates and biomasses. Due to their high abundances, diatom
chains remained almost unaffected by consumers and they out-competed other taxonomic
groups that were originally residents of the sediment community. At such high algal
biomasses, grazing activity was no longer successful in affecting microphytobenthic
community structures.
Detailed taxonomic composition
The consideration of different microalgal taxa provided a more detailed knowledge of how
special genera interact and in which way single taxonomic groups are affected by changing
nutrient availability and grazer presence.
When comparing the initial community structure with the controls after one week, lower
proportions of Fragilaria sp., Amphora sp. and Navicula sp. were detected and increasing
proportions of Diploneis sp., Synedra sp. and chlorophytes were seen. These changes in
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CHAPTER 5
community composition most likely resulted from a combination of increased nutrient
supplies in the water column and from grazers’ selectivity.
Nutrient effects:
In contrast to the short-term incubation, the duration of the experiment showed a
considerable increase in cell numbers which was directly related to higher biomasses of the
chain-forming diatoms Fragilaria sp. and M. nummuloides. Due to this mass occurrence,
other taxonomic forms were almost overgrown and their relative importance decreased,
however it should be stressed that in terms of absolute cell numbers small, prostrate genera
remained almost unaffected. The viability of prostate forms despite the high productivity of
chain-forming diatoms is noteworthy, as the light conditions at the uppermost surface layer
most probably had declined due to shading from overstory algae. Competition for nutrients
between prostrate and erect forms was obviously not given, since microalgae, living closely
attached to or within the sediment, mainly depend on the sediment nutrient pool (Admiraal,
1984). From pore water analysis at the end of the experiment it was shown that nutrient
concentrations were high in the sediment (5 µmol l–1nitrate, 158 µmol l
µmol l
–1
silicate, 5 µmol l
–1
–1
ammonium, 130
phosphate; Jaschinski, unpublished data) and thus a nutrient
limitation of the sediment microflora was not given. The chain-forming diatoms Fragilaria sp.
and M. nummuloides clearly dominated the sediment microflora after three weeks of
incubation and therefore these forms played a tremendous role in forming the
microphytobenthic community. Both genera can be characterized as bentho-pelagic diatoms
(Round et al, 1990). They temporarily reside at the sediment surface where they often
contribute to large amounts to the population dynamics of the sediment microflora by forming
an overstory within the microalgal mat (MacIntire & Overton, 1971; Nicotri, 1977). This
habitat is in contrast to traditional microphytobenthic communities, that are usually
characterized by prostrate diatoms, forming distinctly flat, two-dimensional communities
(Miller et al., 1987). But beside their benthic stage, Fragilaria sp. and M. nummuloides have
neritic phases either as they get frequently resuspended by hydrodynamic processes and
they then dwell in the water column and contribute to the planktonic community (Drebes,
1974). In our experiment one pattern that showed out was the fluctuation of Fragilaria sp. in
the marine mesocosm units. At the beginning of the experiment this genera was quite
abundant but after one week of incubation these chains almost disappeared. The Fragilariachains emerged again in interaction with the centric filaments of M. nummuloides and both
taxa clearly dominated the sediment microflora after three weeks. In this context the
occurrence of M. nummuloides is of considerable interest since the vegetative cells of that
species were not observed in the sediments initially and they first occurred at low numbers in
some of the grazer treatments after one week. Possibility M. nummuloides germinated from
88
CHAPTER 5
resting stages and appeared in culture due to the high nutrient and light availabilities. Factors
that assure success in microalgal growth are a high resilience to external parameters (e.g.
predation, hydrodynamics, nutrient stress) that favours a high competitiveness. In contrast to
prostrate diatoms, which reside permanently in the sediments and which are more
dependent on nutrient conditions at the sediment-water interface (Admiraal, 1984; Hillebrand
& Kahlert, 2002), the chain-forming, temporarily edaphic, microalgae are known to be highly
influenced by nutrient supplies from the water column (Mitbavkar & Anil, 2002). A study
published by Sommer (1997), e.g. clearly showed that M. nummuloides was favoured by
high silicate concentrations and a similar effect was detected by Hillebrand & Sommer (1997)
in case of low nitrogen enrichments. The three-dimensional habit of Fragilaria sp. and M.
nummuloides enables a nutrient uptake from the water column which is impossible for small
pennate forms living closely attached to or within the sediment (see Chapter 2). In case of
high nitrate and silicate concentration in the water column, the productivity of long-chained
diatoms seems to be stimulated and therefore high nutrient loads in the water column favour
their competitiveness.
Grazer effects & competitive interactions:
As pointed out in the section on “major taxonomic groups”, the grazers G. salinus and L.
littorea demonstrate the importance of feeding preferences and inter-specific competition.
Both grazers preyed predominantly upon Cyanobacteria, chlorophytes and the stalked
diatoms Synedra sp. and thus, an active selection for large, filamentous and erect genera
was detected. As a result of selectivity, the proportions of the small prostrate forms Nitzschia
sp. and Diploneis sp. increased in the G- and L-treatments. Thus, their feeding preferences
differed considerably from the feeding habits of I. balthica which was shown to predominantly
reduce these small, prostrate forms.
There are many studies dealing with invertebrate grazers such as amphipods, isopods and
gastropods preying on benthic microalgae (van Montfrans et al., 1984; Brendelberger, 1995;
Jernakoff & Nielsen, 1997; Sommer, 1997; Duffy & Hay, 2000; Duffy et al., 2001). The
majority of studies have focused on these types of grazers because these are the most
abundant and productive ones. In general, gastropods, amphipods and isopods are
considered as obligatory or optionally herbivore with a broad dietary overlap and gastropods
being more efficient but slightly less selective as opposed to crustaceans (Klumpp et al.,
1992; Jernakoff & Nielsen, 1997; Sommer 2000). These differences in food selectivity are
most likely related to the grazers’ feeding mode and different morphologies of the
invertebrates mouthparts. Gastropods appear to be a more generalised browser (see also
Chapter 2) and L. littorea in particular is known to scrape surfaces with its radula thus
sequestering rather unselectively as much food as possible with a large spatial efficiency
89
CHAPTER 5
(Sommer, 2000). However, food items not only consist of algae and plant material but also of
detritus, although a certain preference for large-sized, overstory microalgae was observed for
gastropods in general and therefore the removal of algal cells is also assumed to be
facilitated by morphological and size-dependent features (Nicotri, 1977; Hunter, 1980;
McCormick & Stevenson, 1991). Despite the fact that the mandibles of crustaceans are
specialized mouthparts, the isopod I. balthica was found to have a rather broad food range
grazing on microalgae, macroalgae and seagrass. It does however show a preference for
microalgae instead of macroalgae (Shacklock & Doyle, 1983; Sommer; 2000; Worm et al.,
2000; Duffy et al. 2001). Furthermore, amphipods are also known to exploit a wide variety of
food types, shapes and sizes, however they appear to be selective feeders with a preference
for softer food material (Jernakoff & Nielsen, 1997). The genera Gammarus in particular is
known to graze extensively on microalgae, detritus and associated microbes (Zimmermann
et al., 1979; Smith et al. 1982; Duffy et al. 2001).
Unlike the studies conducted by Sommer (1997) where green filamentous algae and the
diatom Synedra (= Fragilaria) tabulata suffered high grazing losses by the isopod I. balthica,
our short-term incubation showed that this species mainly influenced the abundances of
unicellular, prostrate diatoms. Stauroneis sp., Nitzschia sp., Navicula sp. and Diploneis sp.
were preferred by I. balthica as opposed to green filamentous ones and Synedra sp. which
were selected by the gastropods and amphipods. The reduction of filamentous
Cyanobacteria and chlorophytes by amphipod grazers confirm the findings of Jernakoff &
Nielsen (1997) that showed a high grazing loss for these algal groups in the presence of
amphipods. In addition, L. littorea-only treatments showed similar distributions, and as the
feeding habit of gastropods is characterized by a “bulldozer”-like biofilm grazing, it seems
likely that this feeding mode reduces primarily large, overstory algal species as opposed to
small, prostrate forms. This was also supported by the fact, that the stalked diatom Synedra
sp. was affected considerably by grazing L. littorea.
Nutrient versus consumer effects:
But even though Fragilaria sp. and M. nummuloides showed extremely high production rates,
it is still surprising that the presence of invertebrate grazers did not regulate the mass
occurrence of these diatom chains. In this respect, only the occurrence of L. littorea seemed
to reduce the occurrence of M. nummuloides to any degree. In general, consumers are
known to minimize the algal crop and, furthermore, past research detected a selective
removal of large, overstory species (Nicotri, 1977; Van Montfrans et al., 1982; Steinman et
al. 1987; McCormick & Stevenson, 1989). In addition, some studies even showed that
grazers stimulate the growth of understory species by removing overlying algae and
senescent cells (Lamberti et al., 1987; McCormick & Stevenson, 1989). However, in our case
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CHAPTER 5
the high abundances of Fragilaria sp. and M. nummuloides showed, that the population grew
faster than grazed. Therefore, intermediate grazer abundances were not successful in
controlling mass occurrences of particular algal genera.
Diversity & evenness
At the beginning of the experiment and after one week relatively high and constant values
were determined, both for diversity and evenness. When compared to data from Hillebrand &
Sommer (1997) for epiphytic communities in the western Baltic, our mean results of 1.7 for H’
and 0.8 for E in control- and grazer-treatments after one week of incubation are in a similar
range albeit at the upper end. Throughout the experiment both indices showed higher
variances and, in general, diversity and evenness declined considerably. This is in close
correlation with total cell numbers, as with an increase in algal abundance a decreasing trend
in diversity could be detected (figure 6). However, the decrease in evenness resulted from
the dominance of the two chain-forming diatoms Fragilaria sp. and M. nummuloides rather
than from a loss in taxonomic groups, as the number of species and genera remained almost
constant within the whole mesocosm experiment. In general, high nutrient supplies are
known to function as triggers for decreasing diversity in microalgal communities (Sullivan,
1976; Carrick et al.1988; Hillebrand & Sommer, 1997) and therefore the herein detected
decline in diversity after three weeks seems most likely to be linked to nutrient conditions. As
opposed to grazer presence, the consistently high silicate and nitrate contents in the water
column affected the microphytobenthic community structure to a higher degree especially in
cases where populations increase faster than they are grazed.
The results from this experiment support the ‘consumer vs. resource control’-model
developed by Worm et al. (2002). These authors stated that shifts in diversity are induced by
a combined change in grazer activity and nutrient supply. In addition, their model assumes
that in case of low or intermediate consumer pressure and high productivity, diversity
increases. After one week diversity and evenness in all treatments showed relatively high
values which resembled intermediate grazer activity and productivity. During this period
obviously both consumers and nutrients regulated the microphytobenthic community. On the
long run, however, the productivity increased significantly resulting in mass occurrences of
only few genera while consumer pressure remained constant. Nutrients then clearly took on
a governing role resulting in a decrease in diversity.
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CHAPTER 5
Conclusions
There were three main results of the marine mesocosm experiment: nutrient effects, grazer
effects and competitive interaction. Nutrients significantly affected the sediment microflora
from the beginning on and their impact steadily increased with the duration of the
experiment. In this respect the decrease in diversity as a result from constantly high nutrient
availabilities were especially remarkable. Effects from grazer activity and competition clearly
influenced the sediment community on the short-term of the experiment since large
differences between grazer treatments occurred. Consumer effects were best detected from
taxonomic composition patterns of the microphytobenthos.
General trends observed on microphytobenthic assemblages in the marine mesocosms are
summarized in table 2. The marine mesocosms showed contradictory trends for chlorophyll
contents and cell numbers during the course of the experiment. Although pigment
concentrations decreased after three weeks, possibly due to shading effects from overstory
algae, an increase in cell numbers was observed at the same period. Due to these
contradictory results future quantification of grazer and nutrient effects from chlorophyll or
cell count analyses alone seems inappropriate. Community shifts in the microphytobenthos
as a consequence of grazer presence and nutrient enrichment were best detected both from
morphological characteristics of major taxonomic groups and from detailed community
structure analysis. Hence a shift from a community that was dominated by Cyanobacteria,
Chlorophyta and prostrate diatoms after one week, to an assemblage mainly comprising of
chain-forming diatoms after three weeks was observed. In this respect, the governing role of
M. nummuloides- and Fragilaria-chains was remarkable. Grazer presence did not affect the
microphytobenthic community homogeneously since a shift in food sources was observed in
the cases of species coexistence.
Table 2: General trends in marine mesocosms during the course of the experiment
parameter
incubation time: 7 days
incubation time: 21 days
chlorophyll a concentration
+
-
cell number
-
+
diversity & evenness
constant
major taxonomic groups
prostrate diatoms, Cyanobacteria,
Chlorophyta
chained & prostrate diatoms
main genera
Synedra, Diploneis, Navicula,
Nitzschia, filamentous Cyanobacteria
& Chlorophyta
Fragilaria, Melosira nummuloides
-
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CHAPTER 5
Freshwater mesocosms
Total chlorophyll a concentrations and cell numbers
The chlorophyll contents at the sediment surface of the freshwater mesocosm ranging from
1.0 to 3.0 µg cm-2 showed similar concentration ranges when compared to studies on benthic
microalgae in freshwater lakes. For example Khondker & Dokulil (1988) showed average
chlorophyll a concentrations of 2.0 µg cm-2 for microphytobenthic communities in a shallow
Austrian lake (Neusiedlersee) and Nozaki et al. (2003) detected concentration ranges from 06.0 µg cm-2 for sand microflora from lake “Biwa”. Although direct comparisons to similar sand
communities adjacent to or beneath macrophyte beds with P. perfoliatus are missing, our
data are within the expected range. During the short-term period the grazers A. aquaticus
and G. pulex together reduced algal biomass significantly, whereas a fertilizing effect could
be detected for single A. aquaticus. This pattern changed after three weeks of incubation
since this time algal biomass benefited from the presence of A. aquaticus and G. pulex and
decreases were detected for G. pulex-only units. Thus, the macrograzers in the freshwater
units affected chlorophyll concentrations more strongly than the hydrobiid snail P.
antipodarum in a previously described study (see Chapter 3). For the freshwater mesocosms
the assumption, that the presence of invertebrates affects chlorophyll a concentrations and
that the degree of grazing may differ when species coexist, was confirmed. In addition, this
data fits well with past grazing studies that detected decreasing chlorophyll contents at
sediment surfaces caused by grazing activity (Cattaneo, 1983; Lamberti & Resh, 1983;
Feminella & Hawkins, 1995; Steinman, 1996; Hillebrand & Kahlert, 2002). Thus, detecting
feeding patterns on the basis of such rough estimates as chlorophyll concentrations (Wolff,
1979; De Jonge & Colijn, 1994) also seems possible for our system.
In contrast to the relatively constant chlorophyll concentrations, the cell numbers showed
lower abundances after seven days than after three weeks of incubation and grazer effects
could only be detected over the long-term. Disproportionably high cell numbers occurred in
the presence of all three single grazers, whereas the combination of grazers resulted in lower
productivities. In this context it can be speculated that the co-occurrence of G. pulex and R.
ovata stimulated grazing activity, as considerably lower algal biomass occurred in the
presence of these consumers. Numerous ecological studies have focused on the effect of
inter-specific competition on the resource use of competitors and the effects of coexistence
are still controversially discussed. The central assumption of competition theory, however, is
that the strength of interspecific competition is inversely related to the amount of resource
partioning (Pacala & Roughgarden, 1982). Two grazer species with similar feeding
preferences could therefore be regarded as highly competitive and a stimulation of feeding
activity seems plausible.
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CHAPTER 5
Major taxonomic groups
Diatoms and Cyanobacteria dominated the sediment microflora from the start of the
experiment whereas chlorophytes were found in only negligible abundances. This is in good
correspondence with other lake sediments, where diatoms usually dominate the sediment
communities and where Cyanobacteria are known to be highly abundant in some seasons
for example during the summer period (Kann, 1940; Kann, 1993; Khodker & Dokuli, 1988;
Hillebrand & Kahlert, 2002). In contrast to grazer treatments and the controls, the initial
community showed higher percentages of prostrate diatoms, but this form was almost
completely replaced by chain-forming diatoms after one week. In general, freshwater diatom
assemblages are considered to predominantly comprise of pennate, prostrate forms living
closely attached to or within the sediment (Steinman et al. 1987; Miller et al. 1987;
McCormick & Stevenson, 1989). However, apart from these prostrate forms, chain-forming
diatoms, e.g. Fragilaria sp., Diatoma sp., Bacillaria sp., Melosira sp., are also known to occur
in considerable abundances in some benthic microflora assemblages (Miller et al. 1987;
Kann, 1993; Hillebrand & Kahlert, 2002, Hillebrand et al., 2002). The occurrence of such
chained forms changes the community structure considerably since this results in a shift from
a two- to a three-dimensional community.
Nutrient effects:
The detected large proportions of chained diatoms and cyanophytes in the control- and
grazer units need to be interpreted in combination with water column nutrient data provided
by Jaschinski (unpublished data). Nutrient measurements from the freshwater inflow to the
mesocosm units showed extremely high nitrate and silicate contents with mean nitrate values
of 33 µmol l-1 and 410 µmol l-1 for silicate (figure 14). When compared to field data from the
Schluensee in August and September 2001/2002 (Jaschinski, unpublished data), the
concentrations detected in this study were disproportionably high. This was due to the
nutrient-rich tap water used for the constant water exchange in the experimental units. The
same was true for nutrient contents in the pore water of the sediments, which showed values
of 15 µmol l-1 for nitrate, 10 µmol l-1 ammonium, 247 µmol l-1 silicate, 3 µmol l-1 phosphate;
Jaschinski, unpublished data). Thus, the high nitrogen and silicate concentrations in the
water column resembled more an autumn or winter situation, whereas even then the silicate
concentrations were ten times higher in our experiment as opposed to the field. The impact
of water column nutrient enrichment on benthic algal biomasses are discussed
controversially in the literature. Although Admiraal (1984) suggested that the nutrient pool in
the sediment pore water should prevent nutrient limitation for the sediment microflora, other
studies showed that an increased nutrient supply had a positive effect on algal growth
94
CHAPTER 5
(Nilsson et al., 1991; Sundbaeck & Snoeijs, 1991, Rosemond et al., 1993). On the other
hand, Hillebrand & Kahlert (2002) demonstrated that a high nutrient supply in the water
column did not affect benthic algal biomass and, furthermore, they speculated that water
column nutrients remained almost unavailable to benthic microalgae at least on short time
scales. Nevertheless, especially benthic Cyanobacteria and diatom chains are known to be
somehow related to water column nutrients as several studies detected biomass increases of
those forms at high nutrient levels (Kann, 1940; Kann 1993; Yallop et al., 1994; Taylor &
Paterson, 1998; Hillebrand et al., 2002; Nozaki et al., 2003).
600
550
day 0-7
day 9-19
-1
nutrient content (µmol * l )
500
450
400
350
300
250
200
50
0
22.8. 23.8. 24.8. 25.8. 26.8. 27.8. 28.8. 30.8. 31.8. 1.9. 2.9. 3.9. 4.9. 5.9. 6.9. 7.9. 8.9. 9.9. 10.9.
day of incubation
nitrate
ammonium
silicate
phosphate
Figure 14: Nutrient concentrations (µmol * l-1) in the water-column of the experimental units measured
from the inflow during the course of the freshwater mesocosm experiment (day 1-19).
Grazer effects & competitive interactions:
It was remarkable, that the proportions of prostrate and stalked diatoms remained almost
constant in the grazer units and in the controls in the first week, whereas the shares of
Cyanobacteria and diatom chains showed high variability. In some cases grazer presence
and inter-specific competition favoured the dominance of cyanophytes (AR + G), whereas
others showed a clear dominance of diatom cells (control, A, GR, AGR). This is in good
correspondence to a grazing study conducted by Rosemond et al. (1993) which detected
high cyanophyte biomass in the presence of grazers. In addition, Hillebrand & Kahlert (2001)
have supported these findings in that they showed composition changes in algal
95
CHAPTER 5
assemblages under grazing pressure, whereby the relative importance of Cyanobacteria was
often enhanced.
Nutrient versus consumer effects:
Our data shows that shifts in the community compositions within the freshwater mesocosms
most likely resulted from a combination of effects, both nutrient enrichment and grazerspecific selectivity. Throughout the experiment, the nitrate and silicate concentrations
remained constant and an overall dominance of chain-forming diatoms occurred. Since the
contributions of Cyanobacteria to the benthic food-web are still poorly understood, it remains
unclear whether the relative proportion of cyanophytes declined due to grazing losses or as a
result from nutrient competition. Since neither diatom chains nor cyanophytes showed
grazer-dependent variations on the long-term, and as Cyanobacteria are in general
considered to be a low quality or even toxic food source (Lampert, 1987; Blomqvist, 1996;
DeMott, 1998), it can only be speculated that the dominance of chain-forming diatoms was
triggered by the high nutrient supplies rather than by the invertebrates’ grazing activity.
Detailed taxonomic composition
During the first incubation period, the controls as well as the grazer treatments showed
similar proportions of algal taxa. These units differed slightly from the initial composition as a
decrease of prostrate forms like Pinnularia sp., Stauroneis sp., and Nitzschia sp. was
detected, whereas Fragilaria sp. and Gomphonema sp. showed higher proportions. In
general, the taxonomic composition of the benthic microflora adjacent to or beneath the P.
perfoliatus–beds in our experiment mainly comprised of taxonomic groups that are
considered either as epiphytic or epipsammic. This is especially true for Fragilaria sp. which
is known to occur on sediment surfaces (Miller et al., 1987; Winterboum & Fegley, 1989;
Hillebrand & Kahlert, 2002) and also as an epiphyte on macrophyte leaves in general (Ho,
1979) and on Potamogeton-blades specifically (Carlton & Wetzel, 1987). In addition, the
stalked diatom Gomphonema sp. which was present at a lower proportion in the grazer units
and the controls, has been documented as epipelic (Kann, 1940; Hill et al., 1992), as an
epiphyte on P. perfoliatus-leaves (Kann, 1940) or on Potamogeton sp. (Carlton & Wetzel,
1987), as well as epipsammic (Wasmund; 1984). This genus is also characterised by its
ability to adhere to solid surfaces as well as to sediment particles by forming mucilaginous
pads.
The coccoid cyanobacterium Merismopedia sp. is characterized by a bentho-pelagic life
cycle as this form typically comprises to the plankton and settles periodically from the water
column in considerable amounts where it lives on (Potter et al., 1975; Blomqvist, 1996).
Merismopedia sp. can be frequently found as “Aufwuchs” and on sediment surfaces (Kann,
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CHAPTER 5
1940; Agatz et al. 1999; Riethmueller et al., 2000). The only mentionable prostrate diatoms
that made up the microphytobenthos to some extent were Nitzschia sp. and Navicula sp..
Those forms played a minor role within the total mesocosm community. This pattern is
contradictory to studies conducted by Miller et al. (1987) and Wasmund (1984) as well as to
our data from the Schöhsee, where epipelic and epipsammic assemblages were found to
form distinct flat, two-dimensional communities, mainly comprising of prostrate, attached
diatoms like Navicula sp., Nitzschia sp., Pinnularia sp. and Stauroneis sp.. In contrast,
stalked or erect forms are usually considered to be minor components of the
microphytobenthos. Thus, the three-dimensional habit of the sediment microflora in our
experiment seems to have been atypical.
Nutrient effects:
A possible explanation for the replacement of prostrate forms with overstory algae could be
an increase in water-column nutrients in the experimental units. As already mentioned in the
section on “Major taxonomic groups”, the persistently high nutrient loads obviously favoured
the overall dominance of Merismopedia sp. and Fragilaria sp.. Nutrient enrichment
experiments conducted by Hillebrand et al. (2002) showed that the chain-forming diatom
Fragilaria sp. increased in abundance when nutrients were added to the water column.
Similar effects have been shown for cyanophytes as they are known to be triggered by water
column nutrients and several studies detected biomass increases at higher nutrient loadings
(Kann, 1940; Kann 1993; Hillebrand et al., 2002). But a potential fertilizing effect of
Merismopedia sp. must be treated with caution, since Blomqvist (1996) documented that this
cyanobacterium did not respond to nutrient additions and hence concluded that
Merismopedia sp. was unable to use nitrate as nitrogen source. This is supported by a study
of Agatz et al. (1999), who showed that Merismopedia sp. was mainly found at nutrient poor
sites on a tidal flat. This could indicate, that the variations in relative proportions of
Cyanobacteria in the grazer treatments we observed were probably related to the grazing
activity of invertebrate grazers rather than to nutrient supply.
Grazer effects & competitive interactions:
However, the assumption that Cyanobacteria-consumption might have occurred is in contrast
to studies which showed that grazing affects predominantly large erect or chain-forming
species (Nicotri, 1977; Van Montfrans et al., 1982; Steinman et al. 1987; McCormick &
Stevenson, 1989). Thus, a higher mechanical vulnerability of Fragilaria-cells rather than of
the coccoid cells of Merismopedia sp. could be assumed. Furthermore, Cyanobacteria are in
general considered as an inadequate, toxic food source (Lampert, 1987; DeMott, 1998) and,
as pointed out by Blomqvist (1996), Merismopedia sp. in particular is not grazed in the
97
CHAPTER 5
plankton. As a consequence of this he concluded, that this cyanobacterium may act as a
dead-end in energy flow towards higher trophic levels. Unlike Merismopedia-cells, Fragilaria
sp. is known to suffer high grazing losses. Other research has shown a high grazing
efficiency by a variety of freshwater and marine consumers preying on Fragilaria-cells
(Nicotri, 1977; Winterboum & Fegley, 1989; Sommer, 1997). Chains of Fragilaria sp. seem to
be a very edible and, additionally, a fast growing food source. Taking this into account it is
most likely that varying Cyanobacteria proportions resulted from different degrees of
Fragilaria-consumption rather than from Cyanobacteria grazing.
However, the proportions of Fragilaria-cells were not only generally related to grazer
presence, but they also showed variation between grazer treatments. For example in the
presence of G. pulex (G) and also when A. aquaticus and R. ovata co-occurred (AR), lowest
proportions of Fragilaria sp. were detected. Thus, the degree of Fragilaria-consumption
differed also between species and when grazers coexisted. Several previous studies have
shown that co-occurring mesograzer species can have quite different feeding preferences
(Moore, 1975; Shacklock & Doyle, 1983; Brendelberger, 1995; Constantini & Rossi, 1998;
Duffy et al., 2001). The relative decline of Fragilaria sp. in the single-grazer treatments with
G. pulex is interesting, since the impact of grazing amphipods is discussed controversially in
the literature. Gammaridae are known to feed on a diverse array of food items, e.g.
microalgae, detritus, and associated microbes (Moore, 1975; Zimmermann et al. 1979; Smith
et al., 1982; Friberg & Jacobsen, 1994) and hence, this genus is considered more a
generalist. Past research considered amphipods to be less efficient and less selective
grazers compared with isopods and gastropods (Jernakoff & Nielsen, 1997; Duffy et al,
2001). However, their strong impact on benthic communities has been stressed (Duffy and
Hay, 2000) and their selective potential shown (Shacklock & Doyle, 1983; Friberg &
Jacobsen, 1994). A study conducted by Moore (1975) showed that diatoms were the most
common algae ingested by G. pulex and that cyanophytes were consumed in relatively small
amounts. Therefore, an active consumption of Fragilaria-cells by G. pulex at the sediment
surface can be hypothesized. Similar patterns occurred for combinations of A. aquaticus and
R. ovata, whereas the single-grazer units of both species did not show a reduction of
Fragilaria sp.. A. aquaticus is generally considered as herbivore or detritivore (Moore, 1975;
Marcus et al., 1978; Constantini & Rossi, 1998). Gastropods are known to have overlapping
food spectra (Lodge, 1986; McCormick & Stevenson, 1989; Norton et al., 1990; Rosemond
et al., 1993; Jernakoff & Nielsen, 1998). Interestingly, both grazers did not show grazing
pressure on Fragilaria-cells as long as they were separated, but in combination these
diatoms declined. Potentially, one of these grazers changed its feeding mode slightly in the
presence of a co-occurring species and this might indicate a shift in resource-partioning.
However, as this pattern occurred only in one of the combined grazer treatments this effect
98
CHAPTER 5
was the exception rather than the rule. This is especially true as high food availabilities
occurred in all of the treatments and resources were far from limited.
Nutrient versus consumer effects:
Even though slight grazing impacts could be detected after one week of incubation, this
pattern disappeared after three weeks. In the long-term of the experiment a distinct
dominance of Fragilaria sp. appeared in all different treatments and Merismopedia sp.
became less important. This truly indicates that Fragilaria sp. was favoured by the constantly
high nitrogen and silicate concentrations from the water column, and thus these diatom
chains could out-compete other microphytobenthic forms. Finally, a monoculture of Fragilaria
sp. developed and, as the population increased faster than it was grazed, the presence of
consumers at natural abundances was unable to regulate the algal community. This is in
contrast with studies conducted by Hillebrand & Kahlert (2001) as well as Rosemond et al.
(1993) which showed that grazing had stronger effects on microalgal assemblages than
nutrient supplies. However our results show the opposite.
In comparison with other enrichment studies, it must be pointed out that the nutrient level in
the freshwater mesocosms were extremely high, especially in terms of silicate. To date
studies on nutrient effects on benthic microalgal assemblages mainly focused on the aspect
of eutrophication as a result from elevated nitrate, nitrite, ammonium or phosphorus
concentrations whereas silicate levels were usually neglected. Thus, the comparability to
other studies is limited. Nevertheless, it has been shown that water column nutrient
enrichments affect only those species that are directly dependent on nutrient contents from
the overlying water body (e.g. planktonic, bentho-pelagic or periphytic forms) but not
sediment-dwelling algae (Blumenshine et al., 1997; Vadeboncoeur & Lodge, 2000;
Hillebrand & Kahlert, 2002). The chain-forming Fragilaria sp. were previously shown to
largely increase their biomass when nutrients were added to the water column (Hillebrand et
al., 2002). Thus, the colonization- and growth-success of this form in the benthos seems to
be highly related to water-column nutrient supplies and as long as sufficient resources are
available this genus seems to be able to out-compete and to overgrow prostrate taxa. The
described composition shift from prostate diatoms to a dominance of the chained Fragilaria
sp. resulted in a complete restructuring of the whole microphytobenthos community in the
freshwater mesocosms.
This experiment provides evidence that extreme nutrient loadings in the long-term can affect
microphytobenthic assemblages to a considerable degree and that consumers, at
intermediate abundances, are unable to regulate algal biomasses. The high food quantities,
however, seemed to boost the grazers’ growth rates since all three grazers showed extreme
biomass increases in the experimental units and, in addition, high reproduction rates were
99
CHAPTER 5
observed during the course of the experiment for the gastropod R. ovata (Gohse-Reimann,
pers. comm.). It can be speculated that at a longer duration of the experiment (> 3 weeks)
the grazer effects could have potentially increased in the experimental units as increasing
biomass and abundance of adult consumers could have then shown higher efficiencies.
These patterns confirm the general assumption that the factors controlling the ups and
downs in microphytobenthic communities are complex and that it is not useful to consider
both, grazer consumption and nutrient, separately.
Diversity & evenness:
In general, high nutrient supplies are known to decrease the diversity of benthic microalgal
assemblages (Sullivan, 1976; Carrick et al.1988; Hillebrand & Sommer, 1997). Since the
diversity and evenness in our mesocosm experiment declined after three weeks of incubation
and no differences between grazer-treatments and controls occurred, this decline in diversity
seems most likely to be linked to the high nutrient levels from the water column. This is
closely correlated with total cell numbers, as, for example, with an increase in algal
abundance a decrease in diversity was detected (figure 12). However, the observed
decrease in evenness resulted from the dominance of the chain-forming diatom Fragilaria sp.
rather than from a general loss in taxonomic groups, since the number of species and genera
remained constant within the whole experiment.
When compared to diversity data obtained by Khondker & Dokulil (1988) for lake
“Neusiedlersee” or to Schöhsee values (see Chapter 2), the diversity variables in the controls
and the grazer treatments of our freshwater mesocosms can be considered relatively low.
Therefore, our data could be best described by a univariate model predicting that diversity
variables peak at intermediate resource supply or productivity and that H’ and E decline as
soon as nutrient concentrations increase as it was stated by several authors (Sullivan, 1976;
Carrick et al.1988; Hillebrand & Sommer, 1997; Kassen et al., 2000). Thus, the continuous
decreases in diversity and evenness in our mesocosm were the consequence of persisting
nutrient enrichment. The result was a shift from a relatively diverse and even distribution of
the benthic microflora at the beginning of the experiment to an almost monocultural
community after three weeks.
Conclusions
The microphytobenthic community in the freshwater mesocosms was severely affected by
nutrient supplies since constantly high water-column nutrients resulted in a restructuring of
the microphytobenthos over the course of the experiment. Significant decreases in diversity
were the consequence. Grazer presence and competitive interaction influenced the sediment
100
CHAPTER 5
microflora only on the short-term and consumers’ impact decreased with increasing
productivity.
General trends resulting from the freshwater mesocosm experiment are listed in table 3.
Although constant chlorophyll concentrations were observed in the freshwater mesocosm,
the number of algal cells steadily increase during the course of the experiment. Both
parameters are usually considered to give good correlations, but possibly due to self-shading
from overstory algae, this was not confirmed in our study. Taxonomic analysis showed that
the sediment microflora was dominated by diatoms and Cyanobacteria after one week and
that the proportion of cyanophytes decreased considerably after three weeks. However, due
to the fact that Cyanobacteria are in general considered an inadequate food source, it can be
speculated that the reduction of cyanophytes was more a result of them being out-competed
by the very productive chained diatoms rather than from grazing loss. The persistently high
nutrient loads in the freshwater mesocosms favoured the overall dominance of the
cyanobacterium Merismopedia sp. and the diatom Fragilaria sp. during the first experimental
period whereas on the long run chains of Fragilaria sp. persisted. Despite the high food
availabilities in the experimental units, however, the consumption of microalgae in the shortterm showed inter-specific variations and, in some cases, also effects of coexistence.
Furthermore, diversity and evenness in the mesocosms declined during the course of the
experiment and thus, these results support one of the major predictions of community
ecology claiming that high nutrient supplies lead to decreased diversities in an ecosystem.
Table 3: General trends in freshwater mesocosms during the course of the experiment
parameter
chlorophyll a concentration
incubation time: 7 days
constant
incubation time: 21 days
constant
cell number
+
++
diversity & evenness
-
--
major taxonomic groups
chained diatoms, Cyanobacteria
chained diatoms
main genera
Fragilaria, Merismopedia
Fragilaria
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CHAPTER 6
Chapter 6
Selectivity and competitive interactions between two
benthic invertebrate grazers (Asellus aquaticus and
Potamopyrgus antipodarum)- an experimental study
using 13C- and 15N-labelled diatoms.
The relevance of feeding selectivity and interspecific competition between herbivore grazers
were the primarily focus of the study presented in this chapter. The aim was to investigate
the aspect of assimilation and to distinguish between active and passive selectivity patterns.
A stable isotope enrichment approach was successfully used elucidating major aspects that
regulate microphytobenthic communities such as selectivity, competition, assimilation,
nutrients as well as coexistence.
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CHAPTER 6
6.1 Introduction
Benthic microalgae contribute significantly to the primary production of shallow aquatic
systems and serve as an ideal diet for small sized, grazing biota. They therefore play an
important role in benthic food webs and are valuable food sources for a large variety of
organisms (protists, meio- and macrofauna).
The literature on benthic grazing patterns consists of a diverse array of studies focussing on
grazer-microalgae interactions to elucidate the impact of herbivory in benthic food-webs.
Until now most studies concentrated on the possible effects of grazing on algal cell numbers,
biomass and chlorophyll a (Steinman 1996, Feminella and Hawkins 1995, Hillebrand 2002).
Most studies indicate a strong, direct impact of benthic grazers on benthic microalgal
biomass, which often correlates with grazer density, specific grazer types, and feeding
morphology (Lodge, 1986; Underwood & Thomas, 1990; Sommer, 1997; Chase, Wilson &
Richards, 2001). Furthermore, the degree of digestion and the survival of gut passage by
some microalgal species are factors that can also regulate grazer-microalgae interactions
(Porter, 1973; Moore, 1975; Underwood & Thomas, 1990; Brendelberger, 1997 a,b) but this
aspect of assimilation has mostly been neglected.
In this context, aspects gaining in importance are selectivity and feeding preferences, as
these may be regulatory mechanisms for grazer–prey interactions in freshwater and marine
systems (Steinmann, 1996; Feminella & Hawkins, 1995; Chase et al., 2001; Hillebrand,
2002). We distinguish between active selectivity (active choice of food component) and
passive feeding preferences (by mechanism of food intake or digestibility). Most evidence
points to a predominance of passive feeding preferences in grazer–microalgae interactions
(Steinman 1996, Brendelberger, 1997a; Hillebrand et al. 2000). If grazers have differing
assimilation efficiencies for different diatom species, microalgal community structure could
affect benthic grazer communities because different food sources have different qualities.
Selective or differential feeding may also be the primary cause for coexistence among grazer
species that share resources. Differentiation in size ratios between coexisting snail species is
a classical example of reduced niche overlap between competing species (Fenchel, 1975).
In recent years the use of stable isotope techniques have been used increasingly to
investigate trophic interactions in freshwater and marine food-webs. Natural isotope
compositions can be used for the analysis of food sources and trophic interactions (Peterson
& Fry, 1987; Fry, 1988) as the stable isotope signatures of a consumer generally reflect the
isotope composition of their diets in a relatively dependable manner (De Niro & Epstein,
1981, Post 2002). Moreover, labelling with stable isotopes can serve in a tracer concept
allowing the investigation of flux processes or feeding habits. For example the stable isotope
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CHAPTER 6
13
C has successfully been used as a tracer for enrichment studies quantifying the uptake and
incorporation of tracer carbon into body tissues (Levin et al., 1999; Middelburg et al. 2000,
Aberle & Witte, 2003). Herman et al. (2000) also used stable isotopes as tracers in a dual
labelling experiment where pelagic algae were enriched with
15
N and benthic algae with 13C.
We used this new tool to investigate the grazing of two co-occurring herbivores on prey of
different size. The isopod Asellus aquaticus and the gastropod Potamopyrgus antipodarum
differ in their feeding mechanism. Both invertebrates are abundant herbivores in the littoral
zones of European freshwaters and are known to feed on, among other things, a variety of
microalgal species (Marcus, Sutcliffe & Willoughby, 1978; James et al., 2000). Due to their
high abundances they play an important role in freshwater food-webs and therefore serve as
model organisms for the detection of trophic relations.
We used stable isotopic enrichment of food sources to investigate interspecific trophic
interactions between two co-occurring herbivores, and specifically the differentiation of
feeding selection, during competition as compared to isolated species treatments.
6.2 Material & Methods
Experimental design
Grazer preference experiments with P. antipodarum (size range 3 mm) and A. aquaticus
(size range 5-6 mm) were conducted using four different treatments: a control treatment
without grazers (C), two single-grazer treatments with A. aquaticus or P. antipodarum (A or
P) and a combined-grazer treatment where both grazers were present (PA). Each treatment
was replicated four times and the complete experimental set-up was duplicated to retrieve
independent samples for two different incubation times (day 1; day 2). Erlenmeyer flasks
(300 ml) served as experimental units and were filled with 100 ml filtered (0.2 µm) and
autoclaved water from lake Schöhsee, Germany. Each culture flask from each treatment was
inoculated with a mixed, labelled algal solution of 10ml of Nitzschia palea (26000 cells ml-1)
and 2 ml of Fragilaria crotonensis (61000 cells ml-1). Different initial volumes of algal solution
represented a comparable biovolume of each algal species. The grazer addition followed a
supplementary design where grazer biomass in each treatment was kept constant. The
number of individuals added to each experimental unit were calculated from the individual
dry weights: eight A. aquaticus (5.6 mg total dry weight) in treatment A; ten P. antipodarum
(6.0 mg total dry weight) in treatment P; and the mixed-grazer units (PA) containing four A.
aquaticus and five P. antipodarum (5.8 mg total dry weight). At the end of the experiment, the
animals were picked live from the flasks and oven dried at 60°C for 24h. Snail body tissues
were removed from the shells after treating with 1M HCl-solution to dissolve inorganic
carbon. For the determination of cell numbers and biovolume, 10 ml of the algal suspension
were transferred into brown-glass bottles and fixed with Lugol’s solution. To collect faecal
104
CHAPTER 6
pellets for measurements of excretion of
13
C and
15
N by the animals, the remaining
suspension was sieved through a 100 µm-gauze and the sieve-residues collected on a precombusted GF/F-filter. The residues were checked for purity under a binocular microscope to
ensure that only faecal pellets, and no algal colonies were retained on the filters. Faecal
pellet material from all four replicates of each treatment were pooled to obtain sufficient
material for stable–isotope analysis.
For the determination of algal cell numbers and biovolumes, the Lugol’s-fixed samples were
mixed gently and 10ml of the sample immediately transferred to Utermöhl counting chambers
(total volume 10ml). After leaving to settle for 24h, algal cells were counted under an inverted
microscope and converted to biovolume following the methods of Hillebrand et al. (1999).
The grazing rates per hour were calculated from the difference between the gross growth
rate µ = (ln Vc – ln V0) x h-1) and the net growth rate r = (ln Vgr – V0) x h-1 (Vc= biovolume
control; V0= biovolume start; Vgr= biovolume grazer treatment).
Stable isotope labelling
Prior to the experiment, the diatoms Fragilaria crotonensis (large-celled, sessile colonies)
and Nitzschia palea (single-celled, mobile) were cultured at 17°C in artificial freshwater
amended with WC medium (Guillard & Lorenzen, 1972). The axenic F. crotonensis cultures
contained 30% NaH13CO2 (99 atom%; Chemotrade Leipzig) whereas 30% Na15NO3 (95
atom%; Chemotrade Leipzig) was added to the cultures of N. palea. The algae were
cultivated in 500ml Erlenmeyer flasks under a 16h light:8h dark regime for four weeks. At the
beginning of the in-situ labelling experiment the isotope signatures of the labelled cultures of
F. crotonensis (δ13C 30.4‰; δ15N -15.4‰) and N. palea (δ13C -19.6‰; δ15N 488.8‰) as well
as the background isotope signatures of unlabelled algae (F. crotonensis: δ13C -22.5‰; δ15N
-15.9‰; N. palea: δ13C -19.4‰; δ15N -7.7‰) were determined.
Stable isotope analysis
Individual A. aquaticus were weighed into tin cups, whereas two or three individual P.
antipodarum were pooled to obtain sufficient weight of nitrogen for a representative analysis.
Tin cups were oxidised in a Carlo Erba NA 1500 elemental analyser coupled to a Micromass
IsoPrime continuous flow isotope ratio mass spectrometer. Isotope ratios are expressed
using the U-notation (δ13C, δ15N) in units of per mil (‰). Reference materials used were
atmospheric nitrogen, and a secondary standard of known relation to the international
standard of Vienna Pee Dee belemnite for carbon. Repeat analyses of an internal standard
resulted in typical precision and accuracy of <0.2‰ for δ13C and <0.4‰ for δ15N. The uptake
of
13
C (and similarly for
15
N) by the herbivores was calculated as excess (above background)
and is expressed as the specific uptake Uδ13C (Uδ13C = δ13Csample-δ13Cbackground). Thus, prior
105
CHAPTER 6
to the labelling experiment, background (natural) isotope signatures of each grazer species
obtained directly from Lake Schöhsee, were measured to substitute into the calculation of
specific uptake. A selectivity index (Q) was defined as the quotient Uδ13C/Uδ15N, which
expresses the relative uptake of 13C compared to the uptake of 15N.
Statistical analysis
To test for a significant impact of herbivores on algal biomass, and on
13
C and
15
N-uptake, a
full-factorial ANOVA was used. Independent factors in both cases comprised time (F1) and
treatment (F2). Algal biomass was log-transformed to reduce the observed heterogeneity in
variance. No transformation was necessary for the
13
C- and
15
N-uptake as the variances
showed no significant deviation from homogeneity. To test for significant relationships
between the biomass-specific grazing rate and the
13
C- and
15
N-uptake, linear regression
analysis were implemented. In addition, an ANOVA on selectivity was performed using the
log-transformed dependent variable Q (13C/15N) and the independent factors time (F1) and
treatment (F2).
A
B
SD= 148247
SD= 232387
day 1
day 2
400000
200000
*
day 1
day 2
600000
biovolume (µm3 * cm-2)
biovolume (µm3 * cm-2)
600000
400000
200000
*
*
*
*
*
0
*
*
*
*
*
*
0
C
P
A
treatment
PA
C
P
A
PA
treatment
Figure 1: A) Biovolume (mean ± SE) of F. crotonensis in control (C), single-grazer (P and A), and mixed
grazer treatments (PA) on day 1 and day 2 of incubation. B) Biovolume (mean ± SE) of N. palea in control
(C), single-grazer (P and A), and mixed grazer treatments (PA) on day 1 and day 2 of incubation.
6.3 Results
Algal biovolume
The two grazers significantly reduced the biovolume of both algal species during both days of
incubation (table 1). The biovolume of F. crotonensis increased in both control treatments,
but showed a significant decline in all grazer treatments (figure 1A; p= 0.0000, table 1).
106
CHAPTER 6
Grazer presence reduced the biovolume of F. crotonensis by 74-95 %. Although there
appeared to be a marked decline in biovolume in the treatments containing only A. aquaticus
(A) relative to treatments with the single grazer P. antipodarum (P) and the mixed grazers A.
aquaticus and P. antipodarum (PA), a significant difference between grazer species was not
detected. The grazing rates on F. crotonensis ranged from 0.04 (P. antipodarum as singlegrazer, day 2) to 0.09 (A. aquaticus as single-grazer, day 1) µm³ biovolume h-1. A significant
grazer effect was also detected for the reduction in biovolume of N. palea (figure 1B; p=
0.0000, table 1), both in the single, and the mixed-grazer treatments (78-95 %). Again, there
was no significant difference between grazer species. The biovolume of N. palea in the
control treatments increased from day 1 to day 2. The hourly grazing rates for N. palea
ranged from 0.04 (A. aquaticus as single-grazer, day 2) to 0.10 (P. antipodarum as singlegrazer, day 1) µm³ biovolume h-1.
Table 1: Grazing on N. palea + F. crotonensis . Results of a full factorial ANOVA for total algal
biovolume, with time and treatment as independent factors and total biovolume as dependent variable.
Log-transformed data gave homogeneity of variance.
(df)
MS
F-ratio
p-level
Time
1
0.0172
0.23
0.6346
Treatment
3
1.8085
24.38
0.0000
Time x treatment
3
0.1323
1.78
0.1771
Error
24
0.0742
Time
1
0.1924
1.245
0.2755
Treatment
3
2.0684
13.382
0.0000
Time x treatment
3
0.0976
0.631
0.6020
Error
24
0.1546
Grazer effect on N. palea
Grazer effect on F. crotonensis
Background isotope signatures
The two species showed clear differences in their natural (Lake Schöhsee) isotope
composition, especially in δ13C . The mean δ13C of P. antipodarum was –21.5 ± 1.3‰ while
A. aquaticus was –23.6 ± 0.1‰. Mean δ15N values were 4.4 ± 0.4‰ for P. antipodarum and
4.3 ± 0.1‰ for A. aquaticus.
107
CHAPTER 6
Uptake 13C and 15N
Potamopyrgus antipodarum typically showed a higher Uδ13C in comparison to A. aquaticus
(figure 2A). After two days the specific uptake of P. antipodarum declined slightly whereas
the uptake of A. aquaticus remained constant over time. Differences between single- and
mixed-grazer treatments of both grazers were negligible. Despite these slight differences in
specific
13
C-uptake, a univariate test of significance showed that the differences between
grazer treatments were not significant (table 2). However, a Tukey HSD posthoc test
revealed slightly significant differences between the single-grazer treatments with P.
antipodarum and A. aquaticus. Both grazers can be considered isotopically identical to the
13
C-enriched cultures of F. crotonensis.
A
B
40
3000
P. antipodarum
A. aquaticus
2500
P. antipodarum
A. aquaticus
15N(‰)
Uδ
20
Uδ
13C(‰)
30
2000
1500
1000
10
500
0
0
single (d1) mixed (d1) single (d2) mixed (d2)
single (d1) mixed (d1) single (d2) mixed (d2)
treatment
treatment
Figure 2: A) Uδ13C (mean ± SE) of P. antipodarum and A. aquaticus in the single- and mixed-grazer
treatments after one day (day 1) and after the second day (day 2) of incubation. B) Uδ15N (mean ± SE) of
P. antipodarum and A. aquaticus in the single- and mixed-grazer treatments after one day (day 1) and after
the second day (day 2) of incubation.
Mean specific uptake of N. palea led to
15
N-enrichment of both grazers (figure 2B). Uδ15N
values were generally higher for P. antipodarum than for A. aquaticus, and were highest
within the mixed-grazer treatments. The Uδ15N of P. antipodarum appeared to increase on
day 2, whereas the Uδ15N of A. aquaticus remained relatively constant within the different
treatments and with time. However, a univariate test of significance showed that these
differences between treatments were not significant (table 2). The grazers exhibited δ15N
values two to four times higher relative to the 15N-labelled N. palea-cultures.
There was no clear correlation between biomass-specific grazing rates and stable isotopeuptake, either for different treatments or for incubation time. The only positive correlation was
found on day 1 between the 13C-uptake and the biomass-specific grazing rate (p= 0.049), but
this relation disappeared on day 2.
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CHAPTER 6
Table 2: Grazer 13C /15N-uptake. Results of a full factorial ANOVA for tracer uptake, with time and
treatment as independent factors and total 13C- and 15N-uptake as dependent variable. Untransformed
data gave homogeneity of variance.
(df)
MS
F-ratio
p-level
Time
1
82.480
1.5123
0.2346
Treatment
2
183.747
3.3690
0.0572
Time x treatment
2
42.392
0.7772
0.4745
Error
18
54.541
Time
1
483903
3.4028
0.0816
Treatment
2
461023
3.2419
0.0627
Time x treatment
2
222760
1.5665
0.2359
Error
18
142206
Grazer 13C-uptake
Grazer 15N-uptake
Faecal pellets
Stable isotope analyses of faecal material revealed distinctive signatures for P. antipodarumpellets; the degree of
15
N-enrichment on days 1 and 2 was greater than
(figure 3). Faecal pellets of A. aquaticus were also
15
N- and
13
C-enrichment
13
C-enriched, but to a lesser
extent compared to P. antipodarum. Pellets measured from the mixed-grazer treatments
showed intermediate values.
Selectivity Q
The selectivity index Q (Uδ13C/Uδ15N) showed significant variation over time and between
single and mixed species treatments (figure 4, table 3). Both grazers showed a significant
decline in Q between day 1 and day 2 (A. aquaticus p= 0.0013; P. antipodarum p= 0.034).
Thus, a significant change in feeding preferences from day 1 to day 2 could be detected for
both species, with a higher uptake of F. crotonensis (13C) at the beginning of the experiment.
Differences in selectivity between the single and mixed-grazer treatments of A. aquaticus
could not be detected (table 3, figure 4). In contrast, the Q-values for P. antipodarum showed
significant differences between the single- and mixed-grazer treatments (p= 0.0039,table 3;
figure 4). During the first day of incubation, P. antipodarum alone showed Q-values two times
higher compared to individuals from the mixed-grazer treatments.
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CHAPTER 6
18000
P
P
A
A
PA
PA
16000
14000
δ 15N(‰)
12000
day 1
day 2
day 1
day 2
day 1
day 2
10000
8000
6000
4000
2000
-20
-10
0
10
20
30
40
δ 13C(‰)
Figure 3: δ13C and δ15N of faecal pellets (single measurements) in single-grazer (P and A)
and mixed grazer treatments (PA) on the first (day 1) and the second day (day 2) of
incubation.
50
day 1
day 2
P. antipodarum
A. aquaticus
Selectivity (Q*1000)
40
30
*
20
10
0
single
mixed
single
mixed
Figure 4: Selectivity index Q (Uδ13C/Uδ15N *1000) of P. antipodarum and A. aquaticus in
single-grazer (P and A) and mixed grazer treatments (PA) on the first (day 1) and the second
day (day 2) of incubation (mean ± SE).
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CHAPTER 6
Table 3: Selectivity Q. Results of an ANOVA for selectivity, with time and treatment as independent
factors and Q (δ13C/δ15N) as dependent variable. Log-transformed data gave homogeneity of variance.
(df)
MS
F-ratio
p-level
Time
1
0.3951
10.95
0.0013
Treatment
1
0.0179
0.495
0.4834
Time x treatment
1
0.0092
0.256
0.6144
Error
90
0.0361
Time
1
0.464
4.71
0.034
Treatment
1
0.890
9.08
0.0039
Time x treatment
1
0.0041
0.042
0.837
Error
53
0.0985
Q for A. aquaticus
Q for P. antipodarum
6.4 Discussion
We were able to detect differences between grazing and assimilation, as well as significant
changes in food preference, of two co-occurring species as a result of interspecific
competition, using differential labelling of algal food with stable isotopes. Such an outcome
would have been difficult to observe with traditional methods.
Algal biovolume
A decrease in biovolume within microphytobenthic or epiphytic communities in the presence
of invertebrate grazers is an expected pattern which has been detected in numerous studies
(Steinman 1996, Feminella and Hawkins 1995, Hillebrand 2002). In general, it is assumed
that the diet of A. aquaticus and P. antipodarum largely consists of diatoms, whereas both
grazers are known to show selectivity patterns for different algal taxa (Moore, 1975; Marcus
et al., 1978; James et al., 2000). The two grazer species consumed both microalgal species
within our experimental set-up, indicating that the chosen diatoms were a suitable food
source. No biovolume differences in consumption of algae were to be found between the
grazer species, or over time. Therefore, using this particular methodological approach, no
implications for active selectivity could be found. This is in direct contrast to our data derived
from incorporation of stable isotopes. Evidently then, uptake and assimilation are not
necessarily correlated, supported by the weak or absent correlations between grazing rates
111
CHAPTER 6
per biomass and uptake of
15
N or
13
C. Stable isotope analyses appear to provide insights
unobtainable from more classical grazing studies.
Background isotope signatures
The background isotope signatures of both grazers in our study were similar to values
reported in the literature. James et al. (2000), observed δ13C values of –19.5 to –22.5 ‰ for
P. antipodarum from New Zealand lakes. Animals from the Schluensee, a neighbouring lake
to the Schöhsee, were isotopically enriched in comparison to individuals from Schöhsee
(δ13C of –19.1 ‰ and δ15N of 10.1 ‰ (Brendelberger, pers. comm.). A. aquaticus from
Schluensee showed comparable δ13C and δ15N of –23.4 and 9.5 ‰ respectively
(Brendelberger, pers. comm.), and similar to individuals from Windermere (–26.5 and 8.8 ‰)
and Esthwaite (–26.5 and 6.0 ‰) (Grey, unpublished data). The natural isotope signatures of
both these species reflects a dependence on microalgae as a food source, since microalgae
typically show values of –22.6 ‰ to –19.4 ‰ (Hecky & Hesslein, 1995, James et al., 2000).
Indeed, A. aquaticus and P. antipodarum both use macrophyte/epiphyte-communities in the
littoral zones of lakes as habitats, and so an overlap of trophic niches between both grazer
species seems likely.
Uptake of 13C and 15N
The Uδ13C values for both invertebrates indicated a rapid uptake of
13
C from the labelled F.
crotonensis but uptake by P. antipodarum was higher than by A. aquaticus (sketch 1).
Although these results were not significant they still infer differences in
13
C-uptake from the
large-celled, colony forming diatom F. crotonensis. Similarly, the uptake of
consumption of the single celled diatom N. palea resulted in considerable
15
N via
15
N-enrichment of
15
the animals on day 1. The Uδ N values were slightly higher for P. antipodarum relative to A.
aquaticus but the differences were not as marked as for F. crotonensis.
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CHAPTER 6
Sketch 1: Simplified diagram on the uptake of 13C (top sketch) and 15N (bottom sketch) by both
grazers. Arrow thickness presents the relative amounts of uptake and excretion.
A possible explanation for the contrast in results from tracer uptake and algal biovolume
might be due to different degrees of assimilation and digestion. Traditional methods such as
the analyses of cell numbers, biovolumes or chlorophyll a content provide an overall view of
the total amount of algae ingested within a particular time and therefore they can be used to
detect active selectivity patterns. However, such methods do not provide detailed information
on the actual degree of assimilation and digestibility. Digestion efficiency is known to be a
function of cell wall structure, morphotype and defensive strategy that can influence digestive
pathways (Moore 1975; Underwood & Thomas, 1990; Brendelberger, 1997b). Moore (1975)
reported a very low digestive efficiency for A. aquaticus but could not find any evidence for
cell size-dependent explanations. The digestive enzymes of A. aquaticus appear to show low
penetration of diatom cells despite a gut evacuation time of 25 hours (Moore, 1975). Thus we
might expect a less efficient uptake of
13
C and
15
N from the labelled algal material in our
experiment. Not so much is known regarding the assimilation efficiency of P. antipodarum,
but the much higher gut evacuation time of P. antipodarum (4.5 hrs.; James et al. 2000)
suggests more efficient assimilation. We infer that P. antipodarum was able to digest F.
crotonensis and N. palea more efficiently than A. aquaticus from the Uδ15N and Uδ13C
113
CHAPTER 6
values generated, and therefore that passive selectivity patterns occurred between both
grazer species. The difference in digestion efficiency is even more striking for F. crotonensis,
but we can only speculate whether this might be due to colony type, larger cell size or thicker
cell walls.
Faecal pellets
When the Uδ13C and Uδ15N values derived for the animals are compared to the isotope
composition of their faecal pellets it becomes obvious that an accumulation of
15
N and
13
C in
the faecal pellets had occurred (sketch 1). The extent to which the accumulation took place
differed, 15N more so than 13C, and particularly in the pellets of P. antipodarum (single grazer)
after both experimental days. In comparison the pellets from the mixed-grazer treatments
and the treatments with A. aquaticus as a single grazer showed much lower U15N
suggesting an active selectivity towards the lighter isotopes. Variability in isotope
fractionation is becoming more widely recognised, but the causative factors are difficult to
define in many circumstances and there are few experimental studies addressing such
variation. Needoba et al. (2003) described significant differences in isotope discrimination
between different algal groups and species. Studies on natural stable isotope signatures
have shown that fractionation by metazoans can be rather variable or even species-specific
(Macko, Lee & Parker, 1982; DeNiro & Epstein, 1981; Vander Zanden & Rasmussen, 2001;
Post, 2002). Possible explanations for species-specific discrimination of heavier isotopes
include differences in metabolic processes (e.g. protein synthesis), gastrointestinal
assimilation, and excretion (Vanderklift & Ponsard, 2003). In addition, there seems to be a
correlation between the level of isotope enrichment, and the C:N ratios of diets and
consumers (Gorokhowa & Hansson, 1999; Adams & Sterner, 2000; Vanderklift & Ponsard,
2003). Our results suggest that the gastropod P. antipodarum discriminates more strongly
between
15
N and
14
N compared to the isopod A. aquaticus. Indeed, both invertebrates
exhibited discrimination in both carbon and nitrogen stable isotopes. Similar patterns have
already been shown from natural stable isotope studies (Vander Zanden & Rasmussen,
2001; Post, 2002) and our data provide further support that an active fractionation had taken
place and that these patterns were species-dependent.
Selectivity Q
Interpretation of Uδ13C and Uδ15N values requires care since the degree of isotope
enrichment of both algal species was initially very different. Drawing comparisons between
the 13C- and 15N-uptake is difficult. We do not treat the values derived from isotope uptake as
absolute values. Direct comparisons should be drawn only between treatments rather than
between the δ13C and δ15N values.
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CHAPTER 6
In contrast to a discrete consideration of δ13C and δ15N values, the Q value represents a ratio
between both signatures and therefore it can be used to evaluate the relative importance of
each diatom species to the diet of each grazer. This index enables detection of relative shifts
in preference despite the difficulties in comparing the δ13C values directly with the δ15N
values. Many studies have used two-end-member-mixing- models to determine the relative
importance of two food sources to the diet of consumers (e.g. Vander Zanden & Rasmussen,
2001; Post, 2002). The advantage of this method is, that when using this model, the isotope
data is corrected for fractionation effect first and afterwards the actual importance of each
food source can be estimated. As
15
N fractionates more strongly than
13
C, that is generally
considered to be constant, this correction is especially useful as far as nitrogen isotope
signatures are concerned. However, an implicit assumption of the model is that the
consumer is in isotopic equilibrium with its diet. Our experiment was of two days duration, a
too short period for complete turnover of the experimental animal tissues, and thus precludes
us from using such a model. We used the selectivity index Q even though it does not
incorporate a fractionation factor. Excluding trophic fractionation may lead to an
underestimation of the relative importance of 13C within this study.
Each grazer treatment showed a significant effect of time indicating both grazers consumed
a higher percentage of F. crotonensis during the first day of incubation, and subsequently
switched to a N. palea-based diet on the second day (sketch 2a,b).
The shift from one food source to the other can easily be explained by changing food source,
and the increased effort in gathering the more firmly attached N. palea compared to F.
crotonensis. Since the biovolume of each algal species already had declined significantly
after day 1 it seems likely that consuming the single-celled diatom N. palea (which presented
a more uniform distribution within the experimental units) was a better feeding strategy than
having to scavenge actively to find the few remaining colonies of F. crotonensis. The shift in
preference from day 1 to day 2 indicates that as long as large amounts of different algae are
available, an active selectivity takes place. However, as soon as food sources become
limited, a rather unselective but more efficient feeding strategy is chosen. The correlation
between food concentration and selectivity is a well known phenomenon in planktonic
systems (Cowles et al., 1979; DeMott, 1995; Boenigk et al., 2002). Thus our assumption of
concentration-dependent shifts in preference seems more likely.
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CHAPTER 6
Sketch 2: Simplified diagram on feeding preferences of the two invertebrate grazers based on Q-ratios.
(A) Showing single-grazer treatments and (B) mixed-grazer treatments.
In addition to the effect of time, a significant difference between single- and mixed-grazer
treatments of the gastropod P. antipodarum was detected. This species only showed a
preference for F. crotonensis while there was no co-occurring grazer present. When both
invertebrates had to share food sources, P. antipodarum changed from a F. crotonensisbased to a N. palea-based diet (sketch 2b). The presence of A. aquaticus induced a shift in
resource use of P. antipodarum. Differing mobility of the two species might be an explanation
for the change in food preference. It is noteworthy that the more mobile grazer species (the
isopod, A. aquaticus) was able to maintain the relative preference of F. crotonensis, whereas
the less mobile snail switched to less preferred food. Many studies in community ecology
have investigated the effect of inter-specific competition on the resource use of competitors.
The central assumption of competition theory is that the strength of interspecific competition
is inversely related to the amount of resource partioning (Pacala & Roughgarden, 1982) but
much discussion and debate regarding mechanisms that determine species coexistence with
shared resources still remains (Ricklefs & Schluter, 1993; Gaston, 2000). Studies on the
coexistence of species have often produced contradictory results (Costantini & Rossi,1998;
Rossi et al.,1983), whereas it appears clear from our study that there is an induced shift in
116
CHAPTER 6
feeding preferences in the case of interspecific competition. However, the differentiation was
based on digestion rather than ingestion and this gives evidence that passive selection may
occur even if active selection does not. Since the biovolumes of F. crotonensis declined in
the single- as well as in the mixed-grazer treatments, it is assumed that differential digestion
results from different gut evacuation times, digestion efficiencies, or digestive enzymes. Our
data confirm that the actual abundance of grazed algal cells does not automatically reflect
the real amount of digested material. Insights into grazer feeding preferences in
microphytobenthic systems achieved from a new combination of stable isotope labelling,
have provided us with a basis for further experiments on feeding preferences and resource
partitioning.
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CHAPTER 7
Chapter 7
General Discussion
The present study outlines system-specific characteristics of microphytobenthic communities
and it provides further insights into their ecological role in shallow aquatic environments.
Microphytobenthic assemblages in various habitats were investigated emphasising their
overall significance in benthic food-webs and the complexity of trophic linkages. This work
was divided up into an assessment of communities in situ and this provides the basic
information for investigations of grazer-microalgae interactions.
Microphytobenthic community structures
Life cycles & dynamics
Microphytobenthic assemblages in the intertidal of the Wadden Sea site (Dorum) and on
sediments in the Schöhsee were characterized by distinct seasonality and succession
patterns. These changes in microalgal abundance were directly linked to community shifts
and thus each season was characterized by a particular taxonomic composition. The
observed seasonality and succession patterns are similar to pelagial communities and they
were also confirmed for microphytobenthic assemblages. However, such studies dealing with
seasonal variability in the microphytobenthos mainly focussed on intertidal marine or
estuarine habitats (Admiraal & Peletier, 1980; Blanchard & Cariou-Le Gall, 1994; De Jonge &
Colijn, 1994) whereas similar investigations on the freshwater microphytobenthos are rare.
However, the few seasonality and succession studies addressing freshwater sediment
communities (Khodker & Dokuli, 1988; Nozaki et al., 2003) are in good correspondence to
data achieved for Schöhsee sediments. Thus, the fact that microphytobenthic communities in
the Wadden Sea and in the Schöhsee showed similar seasonality patterns confirms the
assumption that such successions are a general feature of temperate sediment communities
rather than distinct characteristics of particular aquatic habitats.
CHAPTER 9
Growth structure & habits
The taxonomic composition of the microphytobenthos at the marine and the freshwater site
in this study were characterized by similar two-dimensional communities. Both habitats were
dominated by pennate, prostrate diatom taxa with either epipsammic or epipelic life styles.
These forms are typical for variable environments (sand, mud) where disturbance plays an
important role in structuring the algal community. Stalked or chained forms occurred only
sporadically and thus they were a minor component of the microphytobenthos at the Wadden
Sea site and on Schöhsee sediments. Miller et al. (1987) originally described the
morphological habit of distinctly flat, two-dimensional communities for freshwater sediment
communities and opposed them to the three-dimensional community structure of epiphytic
and periphytic community structures. The field assessments at both sites confirm these
growth patterns and thus, despite the differences in habitat characteristics, such habits most
likely can be generalized for microphytobenthos assemblages.
These findings are in direct contrast to community patterns observed in the freshwater and
marine mesocosms where a transition from an initially prostrate to an erect community
structures had developed with course of the experiments. In the long run, the sediment
microflora in the mesocosms was dominated by large, chain-forming diatoms which
considerably restructured the microphytobenthic community. As already pointed out before
(Chapter 2.1), such three-dimensional growth forms are typical for epiphytic or periphytic
algal assemblages and thus, the question was posed whether sheltered microphytobenthic
communities adjacent to or beneath macrophyte beds would probably contradict the typical
microphytobenthos habit. It can be speculated that the mesocosm sediments in close vicinity
to macrophytes were potentially colonized by epiphytic diatom species which have settled
from the macrophytes’ leaves. However, the literature to date neglects the coupling between
macrophyte-epiphyte communities and the sediment microflora and until now only few
studies considered this habitat as a whole (Sullivan & Moncreiff, 1990; Moncreiff & Sullivan,
2001). The present study indicates that the microphytobenthos in association with vascular
plants or macroalgae shows pronounced differences to microphytobenthic communities on
macroscopically un-vegetated, open sand or mud surfaces. Thus, a three-dimensional habit
might not only be a characteristic feature of algal assemblages on hard substrate but also of
the sediment microflora sheltered by macrophyte beds. It must be pointed out that there is an
ecological need for investigations on such complex macrophyte-epiphyte-microphytobenthos
ecosystems and that further developments in this research field are required if we wish to fill
the gaps in this field.
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CHAPTER 9
Food web interactions and trophic linkages
Studies on grazer-microalgae interactions have a long tradition in benthic aquatic ecology
and despite intense research in this field for decades a lot of debate still remains
(Underwood & Thomas, 1990; Miller et al., 1996). Past research has provided extensive
information on the significance of herbivory in regulating such complex ecosystems like salt
marshes, macrophyte beds and sediment communities. Grazer effects can be considered as
highly complex and variable since they depend on grazer types, abundances as well as on
external parameters e.g. nutrient contents or food availabilities. Most of the former
experimental studies have considered the grazer community to be a relatively homogeneous
functional group grazing unselectively on microalgae and detritus (Edgar 1990; Jernakoff et
al, 1996). However, investigations on feeding preferences and selectivity by consumers have
emphasized the overall importance of particular grazer species in regulating benthic
ecosystems (Duffy & Hay, 1994; Brendelberger, 1995; Jernakoff & Nielsen, 1997; Sommer,
1997; Duffy & Hay, 2000).
Grazing efficiency and active selectivity of P. antipodarum
The hydrobiid snail Potamopyrgus antipodarum was subject of two different grazing studies;
the first one emphasising grazing patterns and feeding preferences in general, and the
second one focussing on competitive interactions as well as on the aspect of active versus
passive selectivity. Gastropods are assumed to be generalised browsers grazing rather
unselectively but with a high spatial efficiency on a diverse array of food items (Underwood &
Thomas, 1990; McCormick & Stevenson, 1991; Sommer, 1997). However, several studies
from periphytic or epiphytic communities also demonstrated particular preference of
gastropods for large-sized, overstory microalgae (Nicotri, 1977; Hunter, 1980; McCormick &
Stevenson, 1991).
In this study the emphasis lay on the grazer impact of P. antipodarum as this species is
known to be an effective grazer on benthic microalgal assemblages in freshwater lakes
(Fenchel, 1975a; Dorgelo & Leonards, 2001; Broekhuizen et al. 2002) and it was a dominant
species in our system. In the first experiment on the grazing activity of P. antipodarum clear
trends for grazer efficiency were not found. It was shown that these snails can have both
positive (fertilizing) and negative effects on microphytobenthic algal biomass. In the literature
it has been assumed that such interplays between positive and negative effects are mainly
caused by selective or differential grazing. However, the present study showed that in some
cases specific genera benefited from grazer presence and in other cases the same taxa
were strongly preyed upon. Accordingly, the diversity of the algal assemblage remained
unaffected by these gastropods eluding to their rather unselective feeding mode. Thus, the
morphology and size-dependent features of benthic microalgae that are known to facilitate
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CHAPTER 9
snail grazing in periphyton or epiphytic communities seem to be neglectable in case of P.
antipodarum grazing on sediment surfaces. However, the selective potential of this species
when grazing on epiphytes on macrophyte leaves was stressed by James et al. (2000a).
This may originate from the fact that the structure of the microphytobenthos contrasts
considerably the three-dimensional habit of biofilms on solid substrates, e.g. epiphytic and
periphytic communities. As already pointed out before, large, erect or chain-forming
microalgae are usually missing on sediment surfaces. Consequently, morphology-triggered
feeding preferences might be irrelevant for grazers preying on microphytobenthic
assemblages. Distinct differences in grazing patterns between sediment communities and
solid substrates can be assumed.
Despite the typical characteristics of a microphytobenthic community, it has increasingly
been seen that benthic algae may not be strictly edaphic and that planktonic forms can
temporarily dwell on sediments (Drebes, 1974, Gätje, 1992; De Jong & De Jonge, 1995).
Such bentho-pelagic microalgae are characterised by distinct morphotypes e.g. long chains,
and thus, such forms can occasionally play an important role in restructuring
microphytobenthic assemblages. In order to intensify investigations on the selective potential
of P. antipodarum and to expand these studies on different growth forms of algae, an
additional grazing experiment was conducted. The emphasis was to show whether P.
antipodarum can really be considered as the unselective grazer as proposed above, or
whether it shows feeding preferences in case of distinct morphological differences between
diatom taxa. If so, this would indicate changing feeding habits depending on the substrate
type grazed by the snails. Since P. antipodarum is known to prey upon microphytobenthos
as well as on epiphytes this aspect was of considerable relevance in order to clarify its
selective potential. For this purpose two different diatom species were chosen as food
sources; the large-celled, chain-forming Fragilaria crotonensis and the single-celled, prostate
form Nitzschia palea. The genus Fragilaria sp. is characterized by a bentho-pelagic life cycle
(Round et al., 1990) and it is known to temporarily reside at sediment surfaces. This genus
frequently contributes to large amounts to the population dynamics of the sediment
microflora by forming an overstory within the microalgal mat (MacIntire & Overton, 1971;
Nicotri, 1977). In order to investigate potentially morphology-triggered feeding preferences of
P. antipodarum, tracer experiments with these diatoms labelled with stable isotopes were
conducted. Data from the labelling experiment showed clear feeding preferences of P.
antipodarum for chains of F. crotonensis and thus, this supports the assumption that these
snails are potentially able to select for particular morphotypes. On exclusively twodimensional sediment communities dominated by small prostrate forms, however, there
seems to be no necessity for the development of feeding preferences by P. antipodarum.
However, it can be speculated that food choices may be of higher relevance when these
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CHAPTER 9
snails are grazing on three-dimensional communities were a large variety of overstory algae
are known to occur. These results indicate that P. antipodarum has the potential to select
food items by its growth forms or morphology and consequently, these gastropods’ can be
regarded as facultative selective depending on the community structure of the grazed
surfaces.
Passive selection and competitive interactions
A labelling experiment was, however, not only used to provide further insights on the role of
active selectivity by P. antipodarum. In addition, it was carried out to understand competitive
interactions between the gastropods and the herbivore isopod Asellus aquaticus and to
detect pronounced differences in tracer uptake in case of species coexistence. From stable
isotope ratios it was shown that P. antipodarum only showed a preference for F. crotonensis
while there was no co-occurring grazer present. When both invertebrates had to share food
sources, P. antipodarum changed from a F. crotonensis-based to a N. palea-based diet.
Thus, the presence of A. aquaticus induced a shift in resource use of P. antipodarum.
Differing mobility of the two species were assumed to cause the change in food preference.
Many studies in community ecology have investigated the effect of inter-specific competition
on the resource use of competitors. The central assumption of competition theory is that the
strength of interspecific competition is inversely related to the amount of resource partioning
(Pacala & Roughgarden, 1982) but much discussion and debate regarding mechanisms that
determine species coexistence with shared resources still remains (Ricklefs & Schluter,
1993; Gaston, 2000). It appears clear from this study that there is an induced shift in feeding
preferences in the case of interspecific competition and thus, the present study support the
theory of divergences of trophic niches through competitive interactions. It was distinguished
between the qualitative uptake of carbon and nitrogen sources and on the quantitative
selection of different food items. Thus, these techniques successfully enabled the direct
quantification of the grazers’ uptake and assimilation and therefore served as an ideal tool for
the detection of passive selectivity. The differentiation, however, was based on digestion
rather than ingestion and this provides evidence that passive selection may occur even if
active selection does not.
Induced diversity shifts- consumer versus nutrient effects
These simplified, straight-forward grazing experiments were expanded into more complex
investigations on grazer-microalgae interactions including higher trophic levels. For this
purpose mesocosm experiments were conducted to study the impact of various
combinations of macrograzers on the sediment microflora both by simulating a freshwater
and a marine habitat in the laboratory. Since the coupling between plant-epiphyte
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CHAPTER 9
communities and the sediment microflora have mostly been neglected in benthic research to
date, these mesocosm experiments focussed on microphytobenthic communities beneath
and adjacent to macrophyte beds. These studies were aimed at evaluating the trophic
significance of the sediment microflora in macroscopical vegetated, submersed habitats.
The emphasis of this investigation was to outline the functional role of several herbivore
species in influencing ecosystem processes whereby the aspects of feeding preferences,
competitive interactions and induced diversity shifts were of special interest. Among other
grazers, the impact of snail grazers on microphytobenthic communities was also of particular
interest in the present study, although gastropods with larger body sizes were considered
this time (Littorina littorea and Radix ovata).
The diversity of a system can be used as an indicator for the detection of environmental
conditions since diversity variables are known to reflect external parameters like physical
disturbance, grazing pressure or resource supply (Agatz et al., 1999; Worm et al., 2002;
Mitbavkar & Anil, 2002). However, the amplitude to which each of these factors contributes to
a systems’ diversity is still a key question in aquatic ecology leading to controversial
discussions. Feeding preferences and selectivity by grazing organisms are known to form
distinct community structures and thus their governing role in benthic food webs has been
pointed out by several authors (Duffy & Hay, 1994; Jernakoff & Nielsen, 1997; Sommer,
1997; Duffy & Hay, 2000). In addition, Pacala & Roughgarden (1982) have stressed the
significance of inter-specific competition on resource partioning and the overall impact of
coexistence.
Apart from grazing activity, productivity and resource supply are factors that can influence
diversity (Sullivan, 1976; Carrick et al.1988; Hillebrand & Sommer, 1997; Kassen et al., 2000;
Worm et al, 2002). Thus, nutrient concentrations in the sediment and in the overlying water
body can regulate community structure of benthic algal assemblages (Admiraal, 1984;
Hillebrand & Sommer; 1997; Hillebrand & Kahlert, 2002; Mitbavkar & Anil, 2002).
To date there is a consensus that all the previously mentioned variables show a relationship
with diversity, when manipulated in isolation (Connell, 1978; Tilman, 1994, Kassen et al.,
2000). Recent studies, however, emphasized that multivariate models should be addressed
since the diversity of a system is triggered by an interaction of factors rather than from
unimodal relationships (Worm et al., 2002). Thus, these authors developed a ‘consumer
versus resource control’-model stating that shifts in diversity are induced by a combined
change in grazer activity and nutrient supply. Moreover, they showed that in case of low or
intermediate grazing pressure and high productivity the diversity of a systems decreases
significantly.
In this study community shifts in the freshwater and the marine microphytobenthic
assemblages were found to be induced by grazer presence and nutrient enrichment
123
CHAPTER 9
respectively. Despite the high food availabilities in the experimental units resulting from
constantly high nutrient concentrations, the consumption of microalgae in the short-term
indicated inter-specific variations and competitive interactions. On the long run, however,
persisting nutrient enrichment induced decreases in diversity and evenness in the
mesocosms leading to enormous microphytobenthos biomass and almost monoculture
conditions. Accordingly, consumers at intermediate abundances were able to control algal
biomass but, as soon as productivity exceeded grazing losses, the equilibrium collapsed.
Over the course of the experiments clear shifts from a relatively diverse and even distribution
of the sediment microflora to mass occurrences of only few genera were observed and
nutrients took on a governing role. Thus, data from the mesocosm experiments support the
‘consumer versus resource control’-model predicting that diversity variables peak at
intermediate grazer abundance and intermediate productivity and that diversity variables
decline as soon as productivity exceeds grazing pressure (Worm et al., 2002).
Future prospects
The present study outlines several trends which contribute considerably to further
understanding on microphytobenthic assemblages and on trophic linkages between the
sediment microflora and invertebrate consumers. Furthermore, some fundamental ideas
have been raised and applying these aspects in future approaches could consolidate a
profound knowledge on grazer-microalgae interactions. Three aspects can be summarized
as major interest:
Improved methods for large scale assessments
The development of a new algal sensor in this study now allows monitoring algal
assemblages on larger scales enabling non-retrospect assessments. Apart from monitoring
the total chlorophyll concentrations at the sediment surface, the benthic fluoroprobe now
enables a rapid evaluation of the community structure and distribution in situ. The necessity
of developing such a device arose from the fact that sampling sediment surfaces for the
determination of microphytobenthic assemblages with traditional methods rapidly revealed
several drawbacks: a time-consuming enumeration and large financial effort of adequately
sampling sediment communities on larger scales. In order to improve quantitative and
qualitative assessments with high spatial and temporal resolution, this benthic sensor was
devised. Apart from monitoring the total chlorophyll concentrations at the sediment surface,
the benthic fluoroprobe now enabled a rapid evaluation of the community structure and
distribution in situ. Applying the benthic fluorometer will facilitate field assessments allowing
higher resolution of both seasonality and community patterns with higher accuracy in situ.
124
CHAPTER 9
Comparisons between numerous habitats and even inter-annual variability might be easily
assessed and thus, this technique represents a challenge for future application.
Uptake versus assimilation
The technique of labelling various food items with different stable isotopes was developed in
the present study in order to investigate the aspect of assimilation and to distinguish between
active selectivity (active choice of food component) and passive feeding preferences (by
mechanism of food intake or digestibility). These factors are hardly detectable with traditional
methods and thus, the stable isotope enrichments successfully enabled the direct
quantification of the grazers’ uptake and assimilation. Passive selectivity patterns were
detected indicating that passive selection may occur even if active selection does not. This
first attempt to use the labelling-technique to differentiate between active and passive
selection seems very promising. Therefore, applying this method to further investigations on
food-web interactions and on resource partioning of coexisting species would be a challenge.
Consumer versus nutrient effects
The mesocosm experiments simulating macrophyte habitats pointed to the overall relevance
of consumer presence and nutrient effects in affecting the microphytobenthos population. In
this study community shifts from a diverse microphytobenthic assemblage to an assemblage
mainly comprising of only few genera were induced predominantly by high water column
nutrient concentrations. An initially two-dimensional community was restructured by
developing a third dimension. It can be hypothesised that the sediment microflora and
adjacent macrophyte beds are in strong conjunction with one another and that in the case of
high water column nutrients the microphytobenthos is invaded by epiphytes from macrophyte
leaves and by bentho-pelagic forms. The strong competitive potential of these forms
observed in the present study indicate that three-dimensional community structures on
sediment surfaces might be the rule in case of high water column nutrients. Thus, the
transition from a two-dimensional to a three dimensional community and an outline of the
turning points are of special interest for further investigations.
Outlook
The present study addressed manifold aspects of interactions between microphytobenthos
populations and herbivore consumers. Several characteristics of the complex sediment
community structure were elucidated leading to new ideas for future approaches. To fill the
gaps in microphytobenthos research some of the posed questions could stimulate intense
125
CHAPTER 9
investigations especially in the field of freshwater sediment microflora. Applying these future
approaches could establish a consolidated knowledge on microphytobenthic ecosystems.
126
CHAPTER 8
Chapter 8
Summary
Microphytobenthos represents an important component in freshwater and marine habitats as
it contributes significantly to the primary production in shallow-water ecosystems and benthic
microalgae biomass supports higher trophic levels. Microphytobenthic assemblages are
found in all sorts of sediments ranging from salt marshes, intertidal sand and mud flats,
submerged macrophyte beds as well as subtidal sediments. The microphytobenthos modifies
substrate characteristics by forming special matrices in which sediment particles and algal
cells are embedded and it is a reliable and highly nutritious food source for micro-, meio- and
macrofaunal grazers in shallow aquatic ecosystems.
In marine systems it has become clear that microphytobenthic communities are very
significant both in terms of their wide-spread spatial distribution and ecological relevance. In
contrast, their potential importance in freshwater littoral zones and especially in lakes has
received little attention. Where benthic microalgae in limnic habitats have been addressed,
investigations focussed predominantly on epiphytic or periphytic communities and studies on
the sediment microflora are rare.
This study had the main aim of elucidating which microphytobenthic community structures
and key organisms were important in situ and then, based on this, to differentiate key grazermicroalgae interactions. In order to achieve this I measured diversity and evenness over
short seasonal ranges in freshwater and in intertidal marine sediments. The assessments of
microphytobenthic communities were carried out to evaluate succession patterns at two
ecologically relevant and contrasting sites. These investigations were to provide enhanced
knowledge on taxonomic composition, seasonal and temporal variations, structures and
morphological habits of the microphytobenthos.
Pronounced seasonality and succession patterns were a typical feature of the marine,
intertidal sediments both in the Wadden Sea and of freshwater subtidal regions in the
Schöhsee. Chlorophyll concentration and cell numbers at these sites were concentrated
predominantly on the spring period and decreasing values were found in summer. The
chlorophyll concentrations in surface sediments showed similar values for Schöhsee and
Dorum although cell numbers were much higher at the Wadden Sea site. The freshwater
microphytobenthic assemblage was characterized by higher diversities than the marine
CHAPTER 9
microphytobenthos. Clear shifts in community composition occurred at both sites showing
changing taxonomic compositions at different times of the year. The algal assemblages on
Dorum and on Schöhsee sediments were characterized by a distinctly flat, two-dimensional
community which is considered a typical feature of the microphytobenthos. However, the
dominant taxa found on Schöhsee sediments showed distinct differences to those observed
at the Wadden Sea site. In the Schöhsee mainly the genera Synedra, Fragilaria, Nitzschia,
Stauroneis and Navicula were common. On Wadden Sea sediments Navicula, Cylindrotheca
and Merismopedia dominated the community.
Because of the man-hour problems associated with differentiating the variability of
microphytobenthos populations spatially in situ a new multi-algal fluorometer was devised
and tested. This probe enabled a rapid evaluation of microphytobenthic communities,
instantaneous monitoring of total chlorophyll concentrations and differentiation of major
taxonomic groups on an non-retrospect approach. The prototype of the benthic fluorometer
was tested under laboratory and under field conditions and the applicability of the probe in
determining algal populations on sediments in situ has successfully been realised.
The information on community structures gleaned from the in situ investigations was then
used as a fundament to carry out three types of grazer-microphytobenthos experiments: (1)
Investigations on the grazer efficiency and active selectivity of the freshwater snail
Potamopyrgus antipodarum, (2) Experiments on the functional role of consumer presence
and nutrient supply on microphytobenthic assemblages in macrophyte ecosystems and (3)
Labelling experiments on selectivity patterns and competitive interactions between coexisting
benthic grazers.
Grazer efficiency and active selectivity of P. antipodarum
To elucidate the role of grazer-microalgae interactions on natural Schöhsee sediments
laboratory experiments with varying densities of the hydrobiid snail P. antipodarum were
conducted. It was shown that the gastropods had both positive and negative effects on
microphytobenthic algal biomass and that taxonomic composition and diversity remained
unaffected by these snails. Moreover, predominately size- and density-dependent grazing
patterns were observed and no morphology-triggered feeding preferences were found. Thus,
it was assumed that P. antipodarum has a rather unselective feeding mode.
Functional role of consumers versus nutrient effects
The functional role of several herbivore grazers in influencing the microphytobenthos
beneath macrophyte beds was subject to complex mesocosm studies simulating freshwater
and marine vegetated habitats. Grazer presence did not affect the microphytobenthic
community homogeneously and tendencies for a shift in food sources were observed in case
128
CHAPTER 9
of coexistence. Apart from grazer activity, an impact of constantly high nutrient availabilities
in the water column in changing taxonomic compositions was found. Both mesocosm
experiments showed a community shift from a diverse microphytobenthic assemblage to an
assemblage mainly comprising of large, chain-forming overstory diatom species. These data
strongly
suggest
that
both
grazer
presence
and
nutrient
enrichment
induced
microphytobenthic community shifts and on the long run nutrients took over a governing role
in restructuring the microphytobenthos.
Selectivity patterns and competitive interactions
Feeding selectivity and interspecific competition between two herbivore grazers were
investigated by conducting stable isotope enrichment experiments. The emphasis of this
study was to investigate the aspect of assimilation and to distinguish between active
selectivity and passive feeding preferences. Measurements on isotope signatures enabled a
quantification of the tracer uptake and the actual degree of assimilation. Strong differences
between both factors were observed. The results indicated a shift in feeding preferences
related to intra-specific competition and a potential divergence of trophic niches through
competitive interactions. Thus, this techniques served as an ideal tool for distinguishing
between active and passive selectivity patterns.
This work represents the first step to differentiate grazer-microalgae interactions particularly
in freshwater sediments and it elucidates major aspects regulating microphytobenthic
communities such as selectivity, competition, assimilation, nutrients as well as coexistence.
129
CHAPTER 9
Chapter 9
Zusammenfassung
Das Mikrophytobenthos repräsentiert einen wichtigen Bestandteil von limnischen und
marinen
Lebensräumen.
Es
trägt
in
hohem
Maße
zur
Primärproduktion
in
Flachwasserökosystemen bei und dient als Nahrungsgrundlage für höhere trophische
Ebenen.
Diese
Sedimentlebensgemeinschaften
findet
man
in
nahezu
allen
Küstenlebensräumen, wie z.B. Salzmarschen, Watten und Makrophytenbestände.
Durch das Formen charakteristischer Algenmatten und die Ausscheidung von Schleim
tragen sie wesentlich zur Stabilisierung und Umformung von Sedimenten bei. Des weiteren
stellen benthische Mikroalgen eine zuverlässige und hochwertige Nahrungsquelle für MikroMeio- und Makrofaunaorganismen dar.
Bisher wurde die Bedeutung des Mikrophytobenthos bezüglich ihrer räumlichen Ausdehnung
und ihrer ökologischen Relevanz besonders in marinen Systemen betont. Im Gegensatz
hierzu konnte seine potentielle Bedeutung im Littoral limnischer Systemen noch nicht
hinreichend geklärt werden. Wenn überhaupt, dann beschränkten sich Untersuchungen in
Süsswasserlebensräumen lediglich auf epiphytische oder periphytische Gemeinschaften;
Sedimentmikroalgen wurden jedoch bis dato vernachlässigt.
Das Hauptziel dieser Studie lag darin, die Struktur von Mikrophytobenthosgemeinschaften
näher zu beleuchten und Schlüsselorganismen herauszuarbeiten. Basierend auf diesen
Untersuchungen wurden Interaktionen zwischen Mikroalgen und deren Fraßfeinden genauer
untersucht. Die Freilanduntersuchungen wurden durchgeführt um Sukkzessionsmuster an
zwei unterschiedlichen Standorten zu erfassen. Sie sollten Aufschlüsse darüber geben
welche taxonomischen Zusammensetzungen, Saisonalitäten und Fluktuationen in den
Sedimentgemeinschaften vorherrschen.
Ausgeprägte Saisonalitäts- und Sukkzessionsmuster waren ein charakteristisches Merkmal
sowohl des marinen Standortes im Wattenmeer als auch des Schöhsees. Die
Chlorophyllkonzentrationen und Zellzahlen erreichten jeweils im Frühjahr ihr Maximum, eine
Biomasseabnahme
wurde
im
Verlauf
des
Sommers
beobachtet.
Die
Chlorophyllkonzentrationen an der Sedimentoberfläche zeigten vergleichbare Werte für den
Schöhsee
und
dem
Wattenmeerstandort
Wattstandort
deutlich
höher.
Dorum.
Die
Die
Zellzahlen
waren
Süsswassergemeinschaften
jedoch
waren
am
hierbei
wesentlich diverser als die marinen Gemeinschaften. Deutliche Veränderungen in der
CHAPTER 9
Gemeinschaftsstruktur zu unterschiedlichen Jahreszeiten waren für beide Standorte
charakteristisch.
Die
Struktur
des
Mikrophytobenthos
zeigte
eine
ausgeprägte
zweidimensionale Ausdehnung. Aufrechte Wuchsformen stellten die Ausnahme dar. Diese
Wuchsform gilt als typisches Merkmal für das Mikrophytobenthos. Die taxonomische
Zusammensetzung zeigte jedoch deutliche Unterschiede zwischen den Schöhsee und den
Wattsedimenten. Im Schöhsee dominierten hauptsächlich die Gattungen Synedra, Fragilaria,
Nitzschia, Stauroneis und Navicula, auf den Wattoberflächen waren hauptsächlich Navicula,
Cylindrotheca und Merismopedia.
Aufgrund des hohen Zeit- und Arbeitsaufwandes, der beim erfassen von Sedimentalgen mit
herkömmlichen Untersuchungsmethoden anfällt, wurde ein Multialgenfluorometer in dieser
Arbeit
entwickelt.
Dieses
Instrument
ermöglicht
eine
schnelle
Erfassung
des
Mikrophytobenthos und eine sofortige Ermittlung der Chlorophyllkonzentrationen. Außerdem
konnten
die
Hauptalgengruppen
in
situ
ermittelt
werden.
Der
Prototyp
des
Benthosfluorometers wurde unter Labor- und Freilandbedingungen auf seine Tauglichkeit
überprüft. Diese Untersuchungen zeigten, das die neuentwickelte Sonde ein ideales
Hilfsmittel zur großflächigen Erfassung von Mikroalgenpopulationen darstellt.
Die aus den Freilanduntersuchungen gewonnenen Erkenntnisse wurden im Folgenden als
Grundlage für drei verschiedene Versuchsansätze verwendet: (1) Untersuchungen zur
Grazingeffektivität
und
aktiver
Selektivität
der
Süßwasserschnecke
Potamopyrgus
antipodarum, (2) Experimente zum Einfluss von Konsumenten und Nährstoffbedingungen
auf
Mikrophytobenthosgemeinschaften
in
Makrophytenbeständen,
(3)
Markierungsexperimente zur Selektivität und Interaktionen zwischen konkurrierenden Arten.
Fraßeffizienz und aktive Selektivität von P. antipodarum
Um die Interaktion zwischen dem Mikrophytobenthos und dessen Fraßfeinden genauer zu
untersuchen, wurden Laborexperimente mit unterschiedlichen Abundanzen von P.
antipodarum durchgeführt. Es konnte gezeigt werden, das diese Gastropoden das
Mikrophytobenthos
sowohl
positive
als
auch
negative
beeinflussen
können.
Die
taxonomische Zusammensetzung und Diversität blieb jedoch von der die Fraßaktivität
unberührt. Es zeigten sich keine Fraßpräferenzen im Bezug auf morphologische
Charakteristika der Mikroalgen. Eine unselektive Ernährungsweise von P. antipodarum wird
somit vermutet.
Einfluss von Konsumenten und Nährstoffbedingungen.
Die
Bedeutung
von
verschiedenen
herbivoren
Grazern
für
die
Mikroalgensedimentgemeinschaft mariner und limnischer Makrophytzenhabitate wurde in
unterschiedlichen Mesokosmenszenarien simuliert. Das Mikrophytobenthos zeigte keine
131
CHAPTER 9
einheitliche Reaktion auf die Anwesenheit von Grazern. Im Falle von koexistierenden Arten
wurden jedoch keine Anzeichen für eine Verschiebung von Nahrungspräferenzen gefunden.
Zusätzlich
zu
dem
Einfluss
durch
Grazing
beeinflussten
auch
die
hohen
Nährstoffkonzentrationen in der Wassersäule die taxonomische Zusammensetzung der
Sedimentmikroalgen. Sowohl die marinen als auch die limnischen Mesokosmenexperimente
zeigten eine Verschiebung der Mikrophytobenthosgemeinschaft von divers zu artenarm. Die
artenarmen
Gemeinschaften
waren
geprägt
durch
kettenbildende
Diatomeen.
Die
Ergebnisse aus diesen Experimenten zeigten, daß sowohl die Präsenz von Fraßfeinden als
auch hohe Nährstoffkonzentrationen die Gemeinschaftsstruktur in höchstem Maße
beeinflussen.
Selektivität und Interaktionen zwischen konkurrierenden Arten
Zur Untersuchung von Konkurrenz- und Fraßverhalten von zwei koexistierenden
Invertebraten wurden Anreicherungsversuche mit stabilen Isotopen durchgeführt. Aspekte
wie passive und aktive Selektivität sowie Assimilationseffizienz waren hierbei von
besonderem Interesse. Mit Hilfe der Isotopensignaturen konnte die Traceraufnahme
quantifiziert und der tatsächliche Assimilationsgrad bestimmt werden. Das Experiment
zeigte,
dass
eine
Verschiebung
der
Fraßpräferenzen
in
Abhängigkeit
der
Konkurrenzsituation auftrat. Außerdem wurde eine Tendenz zur Nischenverschiebung unter
Konkurrenzdruck deutlich. Die Methode stellte hierbei ein ideales Mittel dar um aktive und
passive Selektivitätsmuster aufzudecken.
Diese Arbeit repräsentiert eine der wenigen Studien, die sich mit Grazer-MikroalgenInteraktionen, besonders in limnischen Sedimenten beschäftigt. Viele Hauptmechanismen,
die zur Regulierung des Mikrophytobenthos beitragen, wie z.B. Selektivität, Konkurrenz,
Assimilation, Nährstoffbedingungen sowie Koexistenz wurden im Besonderen betrachtet und
neue Aspekte aufgezeigt.
132
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146
Danksagung
•
Mein besonderer Dank gilt meiner Betreuerin Karen Wiltshire, die mich während der gesamten
Zeit uneingeschränkt gefördert, unterstützt und motiviert hat. Ihrer wissenschaftliche
Herangehensweise und Ihr Engagement waren bei der Durchführung dieser Arbeit
unersetzlich.
•
Prof. Dr. W. Lampert möchte ich für seine Anregungen und seine zuverlässige Unterstützung
auch nach dem „Übersiedeln“ nach Helgoland ganz herzlich danken.
•
Helmut Hillebrand danke ich für die anregende Zusammenarbeit, die hilfreichen Diskussionen
und seine Gastfreundschaft.
•
Für die gute Zusammenarbeit und den freundschaftlichen Zusammenhalt möchte ich meinen
„Mitstreiterinnen“ Sybill Jaschinski und Sandra Gohse-Reimann ganz herzlich danken.
•
Karen Stumm danke ich für die Kooperation bei der gemeinsamen Versuchsdurchführung und
Bestandsbeprobung.
•
Vielen Dank an Martin Beutler für die nette Zusammenarbeit, die anregenden Diskussionen
und seinen unersetzlichen Einsatz als Sonden „Trouble Shooter“.
•
Jon Grey danke ich für die Kooperation bei der Messung der stabilen Isotopenverhältnisse.
•
Den Mitarbeitern des MPI für Limnologie (Plön) möchte ich an dieser Stelle meinen herzlichen
Dank aussprechen; allen voran Tina, Kirsten, Conny, Claes, Eckart, Heinke und Anita.
•
Den Kollegen an der Biologischen Anstalt Helgoland danke ich für das nette Arbeitsklima, das
mir ein schnelles Einleben auf der Insel ermöglichte. Ein besonderes Dankeschön geht hierbei
an die „Food Webber“, sowie an Silvia, Peter und Kristine.
•
Der Deutschen Forschungsgemeinschaft danke ich für die Finanzierung dieses Projektes.
•
Den Kollegen aus HIMOM, besonders Wolfgang, Mireille und Maria, möchte ich für den netten
Austausch und die gute Zusammenarbeit danken.
•
Ein „Grazie mille“ geht an Petra Ringeltaube und Thomas Pillen die mich auf diesen Weg
gebracht haben und mit denen ich die Freude am aquatischen Milieu stets teilen konnte.
•
Meinen lieben Eltern möchte ich an dieser Stelle von Herzen danken. Sie haben mir diesen
Weg ermöglicht, mich stets mit vereinten Kräften unterstützt und es ist immer ein Genuss
gemeinsam mit Ihnen die Seele auf der Mühle baumeln zu lassen.
•
Abschließend möchte ich Arne, meinem allerliebsten Gefährten, dem besten Mann aller
Zeiten und meinem Lieblingsbiologen, für seinen grenzenlosen Einsatz, die unendliche
Geduld und die vielen, schönen gemeinsamen Stunden danken.
Lebenslauf
Name
Aberle-Malzahn
Vorname
Nicole
Persönliche Angaben
•
Geburtsdatum: 25.03.1974
•
Geburtsort: Rheinfelden
•
Staatsangehörigkeit: deutsch
Ausbildung
1980 - 1984
Grundschule Wehr
1984 - 1991
Theodor-Heuss Gymnasium Schopfheim
1991 - 1993
Scheffelgymnasium Bad Säckingen
Mai 1993
Abitur
1994 - 1997
Grundstudium Biologie an der Christian- Albrechts
Universität zu Kiel
November 1997
Vordiplom Biologie
1997 - 2001
Hauptstudium an der CAU zu Kiel (Diplom: Februar 2001)
November 1999
Diplomprüfungen in den Fächern: Biologische
Meereskunde (Hauptfach), Zoologie (Nebenfach),
Physikalische Ozeanographie (Nebenfach)
Januar 2000 – Januar 2001
Diplomarbeit am Max-Planck Institut für Marine Mikrobiologie (Bremen) mit dem Thema: „Reaktionen der
Tiefseemakrofauna auf einen Nahrungspuls: in-situ
Experimente mit 13C/15N-markiertem Algenmaterial“
unter der Betreuung von Dr. U. Witte
Januar 2001
Beginn der Doktorarbeit am Max-Planck Institut für
Limnologie (Plön) in dem DFG-finanzierten Projekt :
„Diversität und Funktion von benthischen Mikroalgen“
unter der Betreuung von PD Dr. K. H. Wiltshire
August 2003
Anstellung an der Biologischen Anstalt Helgoland
(Stiftung Alfred-Wegener Institut) und dortige Weiterführung
der Arbeit innerhalb des DFG-Projektes
Tätigkeiten neben dem
Juni 1996 – Dezember 1998
Studium
Wissenschaftliche Hilfskraft am Forschungszentrum
GEOMAR (Kiel) bei Dr. U. Witte
Januar 1999 – Oktober 1999
Aushilfstätigkeit bei der Firma MariLim (Kiel)
März 2000 – Mai 2000
Wissenschaftliche Hilfskraft am Max- Planck Institut für
Marine Mikrobiologie (Bremen) bei Prof. Dr. B.B. Jörgensen
Auslandsaufenthalte
August 1993 – November 1993
Research Assistant an der Heron Island Research Station
(Great Barrier Reef, Australien) bei Dr. P. Ringeltaube
August 1994 – September 1994
Research Assistant am Institut für Marine Biologie
(Isola del Giglio, Italine) bei Dr. T. Pillen
Forschungsfahrten
28.03.1997 – 12.05.1997
Forschungsreise mit FS Sonne ins Arabische Meer
(Forschungsprojekt BIGSET I, GEOMAR)
29.06.1999 – 30.07.1999
Forschungsreise mit FS Sonne Honolulu-Oregon
(Forschungsprojekt TECFLUX, GEOMAR)
14.02.2000 – 26.02.2000
Forschungsreise mit FS Heincke in den Sognefjord
(Norwegen) (Forschungsprojekt BIGSET II, GEOMAR)
03.05.2000 –28.05.2000
Forschungsreise mit FS Poseidon in den Nordost- Atlantik
(Forschungsprojekt BIGSET II, GEOMAR)
Spezielle Qualifikationen
Juli 1998
Ausbildung zur staatliche geprüften Forschungstaucherin
der Berufsgenossenschaft Tiefbau
Kiel, den 15. März 2004
149
Erklärung
Hiermit erkläre ich, dass die vorliegende Dissertation selbstständig von mir angefertigt wurde
und dass sie nach Form und Inhalt meine eigenen Arbeit ist. Sie wurde keiner anderen Stelle
im Rahmen eines Prüfungsverfahrens vorgelegt. Dies ist mein einziges und bisher erstes
Promotionsverfahren. Die Promotion soll im Fach Limnologie erfolgen. Des weiteren erkläre
ich, dass ich Zuhörer bei der Disputation zulasse.
Kiel, den 15.3.2004 ......................................................
Nicole Aberle-Malzahn
150
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