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The diversity and composition of photosynthetic picoeukaryotes (PPEs) in two large shallow lakes in China (Lake Taihu and Lake Chaohu) were investigated from flow cytometry sorted samples using Miseq high-throughput sequencing. We collected 65 samples covering different regions of the two lakes over four seasons to unveil spatial and temporal patterns of PPEs community composition. The use of flow cytometry sorting largely improved the efficiency of detecting PPEs sequences and over 70% of the retrieved reads belonged to PPEs. Chlorophyta and Bacillariophyta dominated PPEs in most of the samples. A distinct but complex seasonality of PPEs composition emerged at the OTUs level. NGS-based Miseq sequencing facilitates an in-depth view of numerous rare OTUs. Nearly 80% of the PPEs OTUs were rare and lots of them were detected only in one season, whereas most of the abundant OTUs were frequently detected in all seasons but only changed in relative abundances. Besides, a close relative of the marine PPEs speciesOstreococcussp. (OTU_1144, 99% identity) was discovered in freshwater systems for the first time and was abundant especially in winter. The diversity and community composition of PPEs were more dependent on season rather than sampling sites. Temperature, phycocyanin and NO3—N concentrations in Lake Taihu explained the PPE composition variations, whereas in Lake Chaohu TN/TP ratios, temperature, pH and nephelometric turbidity units (NTU) seemed to be the most important factors. In addition, a great number of OTUs belong to nonpigmented picoeukaryotes, especially Chytridiomycota, Perkinsozoa, Ciliophora and Cercozoa, which are known to include algae parasites as well as predators. The results of mantel test also showed that the community of photosynthetic and nonpigmented picoeukaryotes were significantly correlated in both lakes.