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We report an in-depth survey of next-generation DNA sequencing of ciliate diversity and community structure in two permanently ice-covered McMurdo Dry Valley lakes during the austral summer and autumn (November 2007 and March 2008). We tested hypotheses on the relationship between species richness and environmental conditions including environmental extremes, nutrient status, and day length. On the basis of the unique environment that exists in these high-latitude lakes, we expected that novel taxa would be present. Alpha diversity analyses showed that extreme conditions-that is, high salinity, low oxygen, and extreme changes in day length-did not impact ciliate richness; however, ciliate richness was 30% higher in samples with higher dissolved organic matter. Beta diversity analyses revealed that ciliate communities clustered by dissolved oxygen, depth, and salinity, but not by season (i.e., day length). The permutational analysis of variance test indicated that depth, dissolved oxygen, and salinity had significant influences on the ciliate community for the abundance matrices of resampled data, while lake and season were not significant. This result suggests that the vertical trends in dissolved oxygen concentration and salinity may play a critical role in structuring ciliate communities. A PCR-based strategy capitalizing on divergent eukaryotic V9 hypervariable region ribosomal RNA gene targets unveiled two new genera in these lakes. A novel taxon belonging to an unknown class most closely related to Cryptocaryon irritans was also inferred from separate gene phylogenies., (© 2014 Marine Biological Laboratory.)