OBJECTIVES: Diatoms represent one of the most important and diverse groups of primary producers. The number of species that have been described is enormous and estimates range between 50,000 to 200,000 species. The determination and identification of diatoms is very difficult and only few specialists exist. This calls for a phylogenetic approach based on other features, such as ribosomal RNA sequences. However, thus far only a remarkably few sequences, ca. 35, have been determined for diatoms. In this project, which is a NWO-supported Flemish-Dutch cooperation project, we will describe the biodiversity of microphytobenthos and its spatial and temporal variability along the salinity gradient in the Westerschelde using a molecular approach in addition to the classical taxonomy. We will isolate a large number of species and produce a phylogenetic tree of the 18S rDNA of the diatoms. The importance of this knowledge is the fact that we will attempt to relate this to function, i.e. to primary production. APPROACH: Representatives of the major groups of diatoms of the microphytobenthos were isolated and cultivated in pure culture. Phylogenetic affiliation as well as genetic diversity among isolates will be determined by sequence analysis of the almost complete 18S rDNA, and the ITS1 and ITS2. Photosynthesis will be determined in situ by using the pulse amplitude modulated fluorescence (PAM) technique and photosynthesis versus irradiance characteristics of communities as well as freshly isolated species. Photosynthetic activity will also be measured on single species using a microscope PAM. These measurements will be accompanied by the determination of extra cellular polysaccharides and sediment grain size. DNA will be extracted from the sediment and from the phytoplankton to measure the amount of DNA of particular selected species by using real-time PCR, specifically developed for single species. The biodiversity of microphytobenthos and of the pelagic diatoms will be assayed by using TGGE in combination with diatom-specific PCR primers. This project is co-coordinated with the HIMOM project (01MM18) and the photobiology project (98MM04). As benthic diatoms seem much more resistant to photoinhibition, we will specifically investigate the D1-synthesis of the different benthic diatoms. PLANNING FOR 2003: In the begin of this year, monthly field campaigns at 6 locations will be conducted in order to complete annual cycle of monitoring started in 2002. During the field campaigns the photosynthetic properties will be measured and samples are collected for extraction of DNA. The single cell photosynthesis measurements technique will be implemented. We are sequencing 18S rDNA and ITS sequences of isolated and enriched strains in order to develop primers for the PCR-work (TGGE and quantitative PCR).