An improved methodology for the identification of protists and microalgae from plankton samples preserved in Lugol's iodine solution: Combining microscopic analysis with single cell PCR

Institute for Limnology, Austrian Academy of Sciences, Mondseestr. 9, 5310 Mondsee, Austria

^* To whom correspondence should be addressed. Email: jens.boenigk{at}oeaw.ac.at.

	Abstract

Here we introduce a method for quantitative analysis of planktonic protists and microalgae from preserved field samples combining morphological and SSU rRNA gene sequence analysis. We linked a microscopic screening with PCR of single cells using field samples preserved with Lugol's iodine solution. Cells possessing a rigid cell wall were incubated with Viscozyme and subsequently with proteinase K for cell disruption; this was unnecessary for fragile cells. The addition of sodium thiosulfate to the PCR reaction tube considerably decreased the inhibiting effect of the fixative (iodine) on the PCR reaction and thus allowed for successful single cell PCR even of long DNA fragments (up to as many as 3000 base pairs). We further applied the protocol to investigate the dominant SSU genotypes in distinct flagellate morphospecies originating from different samples. We hypothesised that despite the morphological similarity, protist morphospecies in different habitats or sampled during different seasons are represented by different genotypes. Our results indicate species-specific differences: the two species Ochromonas sp. and Dinobryon divergens were represented by several different genotypes each and for the latter species the dominating genotype differed with habitat. In contrast, Dinobryon pediforme, D. bavaricum and Synura sphagnicola were exclusively represented by a single genotype each and the respective genotype was the same in different samples. In summary, our results highlight the significance of molecular variation within protist morphospecies.