With the delivery of millions of sequence reads in a single experiment, next-generation sequencing (NGS) is currently revolutionizing surveys of microorganism diversity. In particular, when applied to Eukaryotes, we are still lacking a rigorous comparison of morphological and NGS-based diversity estimates. In this report, we studied the diversity and the seasonal community turnover of alveolates (Ciliophora and Dinophyceae) in an oligotrophic freshwater lake by SSU amplicon sequencing with NGS as well as by classical morphological analysis. We complemented the morphological analysis by single-cell PCR followed by Sanger sequencing to provide an unambiguous link to the NGS data. We show that NGS and morphological analyses generally capture frequency shifts of abundant taxa over our seasonal samples. The observed incongruencies are probably largely due to rDNA copy number variation among taxa and heterogeneity in the efficiency of cell lysis. Overall, NGS-based amplicon sequencing was superior in detecting rare species. We propose that in the absence of other nuclear markers less susceptible to copy number variation, rDNA-based diversity studies need to be adjusted for confounding effects of copy number variation.
With the advent of molecular methods, it became clear that microbial biodiversity had been vastly underestimated. Since then, species abundance patterns were determined for several environments, but temporal changes in species composition were not studied to the same level of resolution. Using massively parallel sequencing on the 454 GS FLX platform we identified a highly dynamic turnover of the seasonal abundance of protists in the Austrian lake Fuschlsee. We show that seasonal abundance patterns of protists closely match their biogeographic distribution. The stable predominance of few highly abundant taxa, which previously led to the suggestion of a low global protist species richness, is contrasted by a highly dynamic turnover of rare species. We suggest that differential seasonality of rare and abundant protist taxa explains the—so far—conflicting evidence in the ‘everything is everywhere’ dispute. Consequently temporal sampling is basic for adequate diversity and species richness estimates.
The diversity of chrysophytes is often underestimated in morphological surveys because of the limited amount of detectable characters under the light microscope. Addressing biodiversity issues solely based on morphological characters is further problematic since molecular analyses revealed a huge genetic diversity within morphotypes, even showing polyphyly in some morphological defined species. In order to address the seasonal variations of chrysophytes, 10 samples were collected in 2007 from the oligotrophic lake Fuschlsee (Salzkammergut, Austria). By using Dinobryon as a case study, we compared different gene phylogenies of single cells of the 18S, ITS (including ITS1, 5.8S, partial ITS2) and the mitochondrial marker COI. No clear molecular phylogenetic separation between morphological identified cells of D. divergens and D. bavaricum could be accomplished based on the 18S rDNA sequences. ITS and COI sequence data revealed a higher genetic diversity then the 18S, with several independent lineages consisting of D. divergens cells. We further encountered divergent phylogenies by comparing 18S/ITS sequence data with COI data within D. pediforme, D. sociale and D. bavaricum lineages. By combining the results of the single cell approach with seasonal data based on 454 pyrosequencing, we addressed the question in how far one morphological species may dominate in the water column over the course of a single year. The results of the 454 pyrosequencing revealed that the D. divergens/D. bavaricum lineage shows the highest abundance from all Chrysophyceae/Synurophyceae and strong seasonal variations with peaks in May and October.
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