This approach involves PCR amplification of a marker gene-usually small subunit ribosomal RNA genes (SSU rDNA)-from DNA extracted from environmental samples. These eukaryotic studies were based on earlier pioneering gene cloning techniques for the environmental detection and enumeration of prokaryotes ( Medlin et al. 2001 Richards & Bass 2005 Richards et al. Bass & Cavalier-Smith 2004 López-García et al. Over the past 10 years culture-independent molecular techniques for detecting and elucidating microbial eukaryote communities have been used with increasing regularity across a broad range of terrestrial and aquatic environments, including many ‘extreme’ environments such as anoxic and deep sea habitats (e.g. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. We identified 38 116 V4 and 15 156 V9 unique sequences. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology.
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