Establishment of a 454-amplicon sequenzing method for field applications to study biodiversity with emphasize on Azadinium.

Phytoplankton diversity has been traditionally studied on the basis of descriptive taxonomic and morphological features by using microscopic methods which have their limitations when it comes to very small organisms. The introduction of molecular markers has elucidated many examples of cryptic diversity and demonstrated a high molecular biodiversity within all plankton size-classes. However, insights into the biodiversity of nanoplankton communities are still very limited. The recent discovery of the genus Azadinium with its isolated taxonomic position within the Dinoflagellates has demonstrated the need for further deep examinations of nanoplankton size fractions (2-20 μm). Among the three known Azadinium species, A. spinosum is a toxigenic representative producing azaspiracids. Not much is known of the potential diversity within the new genus, but many different azaspiracid forms have been identified. Hence it is of particular interest to investigate whether A.spinosum is the only toxic species or whether there exist other toxic or nontoxic ones. Within this study we established a 454-amplicon sequencing method to analyze large ribosomal subunit (LSU) D1/D2 rDNA region in order to study biodiversity of the micro- and nanoplankton fraction. In this high throughput approach on the basis of pyrosequencing LSU fragments amplified via PCR are sequenced. We could show for the first time that 454-sequencing works on board a research vessel. Regional differences in the biodiversity and differences between the micro- and nanoplankton communities were observed. We showed that the dinoflagellates and diatoms were the dominant groups within the two size fractions at the time and place of sampling. Azadinium species were only observed in low abundance at the sample sites/time but not at all in the Faroe Islands. In total four potential new Azadinium species, defined as operational taxonomic units (OTUs), were identified on the basis of maximum likelihood phylogenies. The combination of the deep sequencing approach (454 amplicon pyrosequencing) and a powerful molecular marker such as the LSU D1/D2 rDNA region showed a new possibility to investigate biodiversity even online in the field. The existence of the new four Azadinium OTUs has to be further confirmed with living isolates or by visualization of fixed cell within a samples using fluorescence in situ hybridization with OTUs/species specific probes. In future studies, the connection of metatranscriptomic and molecular biodiversity analysis will advance our understanding of the genetic diversity, metabolic function and the ecology and evolution within the plankton community.