Identifying Pseudo-nitzschia species in natural samples using genus-specific PCR primers and clone libraries

The diatom genus Pseudo-nitzschia contains a number of toxic and non-toxic species that are difficult to distinguish using light microscopy (LM) and at times even with electron microscopy (EM). In order to investigate the actual diversity and seasonal occurrence of Pseudo-nitzschia species, we developed genus-specific ribosomal DNA LSU primers to be used in PCR reactions with environmental DNA samples. Using this approach, we constructed clone libraries from samples collected in the Gulf of Naples (Mediterranean Sea) on six dates between April and October 2004 and compared molecular results with those obtained from counts using LM on the same dates. Thirteen distinct genotypes could be distinguished by their LSU sequence, against five species discriminated using the light microscope. Despite the limited number of samples, 10 out of 14 LSU genotypes known in the area were recovered. In addition, three new genotypes were retrieved, two of which were from within the P. galaxiae clade and one possibly corresponding to an undescribed P. delicatissima-like morph. Molecular results matched LM findings in the case of P. multistriata, whereas they provided a much higher resolution for morphs such as P. delicatissima- and P. pseudodelicatissima-like, which include several pseudo-cryptic species. Overall, the direct amplification with the primers developed proved to be an effective and useful tool to assess Pseudo-nitzschia diversity in the natural environment.