A. Problem, history and goal
Estuaries are often characterized by extensive intertidal mudflats that are important for several reasons.
They form a natural barrier between land and sea, protecting the land from flooding in a natural and
economic way. The water column in estuaries is turbid and any primary production is limited by the
availability of light. This is different for intertidal mudflats that support the growth of dense biofilms of
benthic diatoms, which may provide as much as 50% of the total primary production of estuaries
(Underwood & Kromkamp 1999). While these benthic diatoms migrate into the sediment at high tide, they
move to the surface when the sediment is emersed. During daytime emersion, primary production is not
limited by light and high rates of photosynthesis are usually observed. Growth may be unbalanced as
nutrients may become limiting in this biofilm. As a result, benthic diatoms exude copious amounts of
extracellular polymeric substances (EPS) that form the biofilm matrix in which the diatoms are embedded
(Underwood & Paterson 2003). The diatom biofilm may render stability to the sediment, increasing its
erosion threshold. Acidic EPS have been suggested to serve as a glue, binding the sediment particles
(Decho 2000). During inundation part of the EPS may be lost (‘dissolved’) into the watercolumn or
biofilms may slough off or be grazed by infauna, and as such EPS and the diatoms represent an important
food source for higher trophic levels. Although these benthic communities of diatoms are clearly important,
not much is known about their diversity and how this diversity affects their function. Several studies have
documented relationships between diversity and productivity of ecosystems but very little is known about
the underlying mechanisms (Waide et al. 1999). In the case of microphytobenthic communities hardly any
study has addressed this topic.
The project ‘Diversity-productivity relationships in microphytobenthos’ (DIVPROD) was launched in 2002
with financial support of FWO and NWO in the framework of the Flemish-Dutch collaboration in sea
research (VlaNeZO). This project addresses the relationship between biodiversity of microphytobenthos
and functional aspects of the ecosystem, i.c. primary productivity and stability of tidal sediments along a
salinity gradient in the Westerschelde. DIVPROD selected in total 6 sampling sites at three locations. The 3
locations were Paulina Polder, Biezelingse Ham and Appelzak with salinities of about 20, 17 and 5‰,
respectively. At each location a high and low shore sampling site was chosen, with a difference in emersion
time of 1.5 – 2.5 h. The sites were sampled 9 times between March 2002 and September 2003, to cover a
full seasonal cycle. From these sites we have isolated a large number of diatoms, measured
physicochemical parameters, biomass and primary productivity. The biodiversity was estimated using
classical (morphology-based) taxonomic approaches and molecular techniques such as sequence analyses
of 18S ribosomal RNA gene, 16S ribosomal RNA gene of the chloroplasts and the non-coding internally
transcribed spacer (ITS), and of (18S rRNA-based) DGGE. Analyses of the field data are still in progress
but have already revealed some interesting patterns. Diversity of the microphytobenthic communities is
high (twice as high as in the Scheldt plankton), and our present data may actually underestimate it. There
appears to be no clear relationship between biomass and diversity of the biofilm forming (epipelic)
diatoms. Maximum rate of photosynthesis (Pmax) shows no relationship with diversity or species
composition, but is inversely related to emersion period at low tide. ITS sequence analyses of one of the
dominant epipelic diatoms, Navicula phyllepta, revealed the existence of two distinct clades. Extensive
intraspecific variation also exists in growth rates in relation to salinity for this species. Further analyses of
the nature of this cryptic variation are still in progress, including the development of a real-time PCR
technique that will allow us to determine the quantitative importance of each of the N. phyllepta clades and
analyzing their physiological capacities in response to different and changing environmental parameters.
During the final year of the current project we will also finish the analysis of the DGGE patterns from DNA
extracted during the field campaigns.
The proposed extension of DIVPROD will build on the data and knowledge obtained during the current
successful collaboration. The goal of the extended research will be to assess intra- and interspecific
diversity in diatom biofilms in a salinity gradient and to understand niche differentiation in these
organisms. For the first goal we will extend and mine sequence data obtained from isolated strains and
from communities growing at high and low salinity. The second goal will be achieved by using meso- and
microcosm approaches. Natural communities of microphytobenthos from the Westerschelde will be
incubated under controlled conditions of light, temperature, salinity, nutrients , tidal cycle, as well as of the
composition of micro- and meiobenthic grazers and shifts in community structure, EPS and productivity
will be measured. Simultaneously, we will use high throughput experiments using cultures of diatoms
isolated from the Westerschelde tidal flats to measure the response of individual species and genotypes to
relevant conditions natural communities are exposed to. By investigating the expression of RubisCO genes,
the key enzyme for CO2 fixation, and analyzing pulse amplitude modulated (PAM) variable fluorescence
we will have excellent markers for productivity.
B. Scientific importance and programmatic embedding
Diatoms represent one of the most important and diverse groups of primary producers. The number of
species that have been described is enormous. Kociolek & Spaulding (2000) estimate that about 50,000
species are known, but this is probably only the top of the iceberg. The real number of species of diatoms
that occur on earth may be as large as 200,000 (Mann & Droop 1996). Moreover, it is also not sure whether
all known species are validly described (Mann 1999). This contrasts violently with the relatively small
number of about 100 species described from intertidal biofilms (Underwood & Paterson 2003). The
determination and identification of diatoms is difficult and only a few specialists exist. It is also
cumbersome, because many of the morphological characteristics that are essential for the taxonomic
identification of species, can only be distinguished with the aid of detailed light or even scanning electron
microscopy. This calls for a phylogenetic approach based on other features, such as ribosomal RNA
sequences. However, thus far only a remarkable few sequences, about 100, have been published for
diatoms (Kooistra et al. 2003). In order to be able to describe the biodiversity of microphytobenthos and its
spatial and temporal variability, it will be necessary to apply the molecular approach in addition to the
classical taxonomy.
The importance of this knowledge of diversity is that we will be able to relate it to function, i.e. to primary
production. It is known that microphytobenthos are extremely important primary producers, fuelling the
higher trophic levels and representing the base of the food web in the Westerschelde (Middelburg et al.
2000). In addition, it is known that microphytobenthos produce copious amounts of extracellular polymeric
substances (EPS) and it is supposed that this contributes to stability of the sediment (de Brouwer et al.
2000). It is known that benthic diatoms may contribute significantly to planktonic primary production (de
Jonge 1995) and many species of diatoms that are found in the plankton of the Westerschelde are in fact
benthic species (Muylaert & Sabbe 1996, Sabbe 1997). However, the opposite is also true. Many
planktonic species may occur in the benthos particularly in the freshwater area.
DIVPROD is firmly embedded in the framework of the Flemish-Dutch collaboration of sea research
(VlaNeZO) as is the extension proposed here. It is relevant to the OMES project and other monitoring
activities in the Westerschelde. The project is also relevant to LOICZ and ELOISE. It is furthermore
relevant to a variety of past and current EU projects in which NIOO and TUD participate and of which the
results will be used in this project. This proposal is also relevant for a STW project in which TUD is
involved in which diatom phylogeny is linked to chemical markers.
C. Approach, methods and workplan
The main aim of the current proposal is to assess what mechanisms and processes shape the diversity of
epipelic communities in intertidal estuarine biofilms, and how their taxonomic and functional diversity
affect their function (productivity and EPS production). The project will focus on representatives of the
genus Navicula, which is one of the dominant diatom genera in the Westerschelde biofilms. In order to
study the role of niche differentiation we will characterize inter- and intraspecific differences in
ecophysiological features (a. o. with respect to salinity, temperature) of Navicula strains, and study how
these relate to genotypic variation (ITS, AFLP). We will use real-time PCR techniques based on both
chromosomal ITS and Rubisco (rbcL) mRNA to study the distribution and photosynthetic activity of
Navicula strains in the estuary. Micro- and mesocosm experiments will be used to study how the diversity
of semi-natural and artificial communities affect their productivity and EPS-production. We will also assess
how grazing by micro- and meiobenthos influences diversity and, hence, ecosystem functioning. The
proposed extension of DIVPROD will be firmly based on the results of the investigations of the first part of
the project that is currently ongoing.
DIVPROD is a close collaboration between 3 groups from 3 different institutes (Netherlands: Netherlands
Institute of Ecology (NIOO) and Technical University Delft (TUD); Flanders: Rijksuniversiteit Ghent
(RUG)). The Department of Marine Microbiology (Head: Dr. L.J. Stal) is coordinator of the project and Dr.
V. Créach is appointed as post-doc. Dr. G. Muyzer is the PI of the Laboratory of Microbiology of TUD and
Dr. E. Sahan is the post-doc appointed for the project. Prof. W. Vyverman leads the Section of Aquatic
Ecology of RUG. He has appointed J. van Wichelen as technician and proposes to appoint PhD student
Maureen Fagot for the extension of the project.
Microscopical observations have shown that Navicula species are the most dominant diatoms in biofilms
from the Westerschelde. This result was confirmed by the isolation of different Navicula strains and by
DGGE-sequence analysis of 18S rRNA gene fragments obtained by PCR amplification of genomic DNA
extracted from sediment samples. Navicula phyllepta was identified as a key species in the ecology and
biogeochemistry of the Westerschelde intertidal mudflats. During the genotypic characterization of strains
isolated from the Westerschelde, we found two characteristic internal transcribed spacer sequences (ITS) in
the chromosomal ribosomal operon of different N. phyllepta isolates. A correlation of locality of isolation
(= salinity) and the occurrence of one or the other genotype was observed although the total number of
isolates (10) was too small to draw a definite conclusion. Nevertheless, this observation indicates that there
may be two or more genodemes of N. phyllepta present in the Westerschelde, which cannot be
discriminated morphologically. This intraspecific (micro)-diversity may be the reason of this species’
unusual broad abundance along the salinity gradient. One of the aims of the current proposal is to further
investigate the nature of this cryptic diversity, and its functional significance. To confirm or refute
taxonomic identity of the two N. phyllepta genotypes we will analyze another sequence widely used for
taxonomic assessment, the 16S rRNA gene and the ITS of the plastid ribosomal operon. Chromosomal
AFLP’s will be used to further refine the phylogenetic relationsips between the two genotypes and other
Naviculaceae. On the basis of the combined sequence information, specific primers will be designed that
will be used to analyze the populations. Based on differences in the chromosomal ITS we can develop two
sets of genotype-specific primers for polymerase chain reaction (PCR). With this assay, samples collected
at different seasons and different locations in the Westerschelde will be analyzed qualitatively and
quantitatively for occurrence of one (or both) genotypes. These samples have already been taken and the
DNA extracted and stored for analysis.
The biodiversity of natural communities can be influenced by deterministic or neutral processes. Coexistence of species can be facilitated through niche differentiation (species differ in competitiveness under
fluctuating conditions). On the other hand, more or less neutral sampling effects (sampling from the
regional species pool) can also influence the specific make-up of communities. It is not known to what
extent estuarine biofilm communities are characterized by functional complementarities or redundancy of
the species present. Furthermore, it is not known which role micro- and meiobenthic grazers play in the
maintenance of diversity in these assemblages and how they influence biogeochemical cycling and other
ecosystem processes in intertidal sediments.
The results obtained from field campaigns in the Westerschelde estuary during 2002 – 2003 suggest a
negative relationship between biomass and diversity of microphytobenthos. In addition, the maximum rate
of photosynthesis (Pmax) of the microphytobenthos was related to the abiotic parameters water content,
salinity and irradiance, which, however, all may be linked to emersion time. In order to examine these
links, we propose to run mesocosm experiments using natural communities of microphytobenthos. The
microcosm experiments will be explored for another goal, namely the elucidation of niche differentiation of
co-existing Navicula strains in the Westerschelde. We have conclusively shown that different species (but
also strains belonging to the same species, e.g. Navicula phyllepta) have distinct and stable growth
responses to e.g. salinity. Building on this information, we will design microcosm experiments with
artificial assemblages of different complexity (in terms of taxonomic and functional diversity of the
primary producers) and subject to different environmental regimes. Functional characteristics (growth rate,
colony formation, EPS-production, and steady state biomass) will first be determined for each Navicula
strain grown at different environmental conditions (irradiance, salinity, nutrients and substrate porosity).
We will then construct artificial communities and assess the effects of diversity on functional features of
the communities. These experiments will permit us to manipulate the degree of functional
complementarities and redundancy in the communities, and quantify the effect of functional diversity
independently of taxonomic diversity effects, and vice versa.
The mesocosms consist of uniform areas of washed and autoclaved natural sediment which will be
inoculated by a natural community of benthic diatoms, collected from the different sampling sites and
cleaned from larger infauna. In the mesocosms various environmental parameters (irradiance, light-dark
regime, temperature, nutrients and emersion time) will be controlled and their effects on the development
of benthic biofilms will be studied. Mesocosms will be inoculated with material collected from the test sites
along the estuary. Bulk parameters measured include spectral reflectance and pulse amplitude modulated
(PAM) fluorescence. These techniques give a rapid and accurate indication of the sediment chlorophyll
content (i.e. microphytobenthos biomass) and the functionality (i.e. productivity) of the biofilm (Morris et
al. submitted, Forster & Martin-Jezequel submitted). These measurements will be supplemented by
measuring photosynthetic rates (14C incubation). Biofilm development and changes in species composition
will be followed microscopically and by molecular technique (DGGE) and pigment fingerprints.
Extracellular polymeric substances (EPS) excreted by microphytobenthos may be involved in sediment
stabilization. We have evidence that the benthic diatoms structure the EPS that they excrete and that
sediment stability is the result of this process, rather than the EPS alone (de Brouwer et al. submitted).
Therefore the structure of the biofilm will also be investigated by confocal laser scanning microscopy using
a variety of fluorescent lectins as more or less specific dyes for different EPS. In addition, the composition
of various operational fractions of EPS will be analyzed using HPLC.
While in mesocosm experiments we will inoculate with natural (mixed) communities of
microphytobenthos, we will use clonal cultures of diatoms, ciliates, nematodes and copepods isolated from
intertidal sediments of the Westerschelde estuary to construct artificial assemblages of increasing
complexity (in terms of taxonomic and functional diversity at the level of primary producers and grazers).
Functional characteristics (growth rate, colony formation, photosynthesis, EPS-production, steady state
biomass and RuBisCO gene expression using quantitative RT-PCR) will be first determined for each
diatom strain grown at different salinities and temperatures. Microscopy combined with micro-fiber PAM
will be used to study the photosynthetic activity of individual cells in mixed assemblages. The influence of
grazing will be determined as described in Hamels et al. (2004).
To obtain a better understanding in the productivity of different closely-related co-existing diatom species
the RuBisCO encoding gene (rbcL) is an excellent molecular marker. Detection of rbcL expression in algae
has already been demonstrated (e.g., Xu & Tabita 1996; Pichard et al. 1997; Paul et al. 2000), and rbcL
mRNA abundance nicely correlated to maxima in CO2 fixation (Wyman et al. 1998). To achieve our goal
we will first sequence the RuBisCO gene of the Navicula strains isolated from the Westerschelde.
Thereafter, we will design strain-specific probes (Wyman et al. 2000), which will be used to detect the
expression of the RuBisCO gene in different strains grown at different salinities and different temperatures.
Subsequently, gene expression of a specific group of Navicula will be studied in samples obtained from the
Westerschelde or from mesocosm experiments using real-time PCR (Wawrik et al. 2002). We have already
started to sequence the RuBisCO gene from several phylogenetically different diatom species, including
Navicula sclesviscensis, to create a reference database of sequences for probe design.
In order to carry out the proposed research in the extended DIVPROD project it is important to have the 3
participating research groups working synchronously until the end of 2006. For this we ask for 24 months
salary for a PhD student (Maureen Fagot) at Ghent University, 22 months of post-doc (Dr. V. Créach)
salary for NIOO and 9 months post-doc (Dr. E. Sahan) salary for TUD.
D. Intended publications
1. Diversity – productivity relationships in diatom biofilms maintained under controlled conditions in
mesocosm experiments
2. Quantitative PCR of the ribosomal 18S rRNA gene and ITS of the benthic diatom genus Navicula in
intertidal mudflats of the Westerschelde estuary
3. Microdiversity and niche differentiation in the benthic diatom Navicula phyllepta
4. Expression and diversity of RuBisCO structural gene rbcL in diatom biofilms of the Westerschelde
estuary
5. Role of grazing in maintaining productivity and diversity in a diatom biofilm of the Westerschelde
6. Niche differentiation in benthic diatoms: diversity – productivity relationships
E. Relevance for the Dutch-Flemish co-operation
The project ‘Diversity and productivity relationships of microphytobenthos’ (DIVPROD) is a collaboration
between the Netherlands Institute of Ecology (NIOO), the University of Delft (TUD) in the Netherlands
and the University of Gent (RUG) in Belgium. The three groups have different expertise and role in the
consortium. The Department of Marine Microbiology of NIOO has much expertise in the ecophysiology of
microphytobenthos, the Department of Biotechnology of TUD has much expertise in molecular ecology of
benthic microbial ecosystems and the Section of Protistology and Aquatic Ecology of the RUG has a large
amount of expertise in the taxonomy, biology and culturingof diatoms. The three groups have collaborated
closely and successfully in the DIVPROD project that was launched on January 1, 2002. The proposed
extension can only be carried out with the joined effort of the three groups as it has been shown thus far and
which is one of the prerequisites of the Flemish-Dutch cooperation (VlaNeZO). DIVPROD’s proposed
extension focuses again on diversity –productivity relationships in microphytobenthos on mudflats along
the Westerschelde estuary, the geographic region of the VlaNeZO program. This scientific program
‘Ecology of the Schelde basin and the coastal Zone’ has defined 4 scientific spearheads. DIVPROD is
relevant for spearhead 1: biodiversity. It aims both at the description of habitats at the tidal areas in the
Schelde as well as the diversity of the species that occur in these habitats and their variability. Moreover,
since the project also relates diversity to productivity (‘functional diversity’) it is also very relevant to
spearhead 2: ‘Element cycles and the role of the biota’. DIVPROD assesses primary productivity of
microphytobenthos, a major process in the C-cycle, investigates factors that controls it and relate this to
intra- and interspecific diversity.
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