Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water


  • Research Interests: T.S. and H.-W.B are interested in ecology, diversity and biogeography of marine protests, extremophile protists, anoxic marine systems and eukaryote evolution; D.B. works on microbial ecology and interactions, protist/fungal evolution and phylogenetics, studies on the phylum Cercozoa, uniting phenotypic and genotypics data in environmental microbiology; M.N. works on applied bioinformatics of sequence data and algorithmic analyses of RNA secondary structure predictions; R.C. is interested in computational processing of high-throughout sequencing data; and finally, T.A.R. and M.D.M.J. work on the comparative genomics of eukaryotic microbes and the complexity and branching order of the eukaryotic tree of life.

Thomas A. Richards, Fax: +44 (0)1392 263434; E-mail:, Thorsten Stoeck, Fax: +49 631 205 2496; E-mail: stoeck@rhrk.uni-kl.deThorsten Stoeck and David Bass contributed equally.


Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. 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. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. 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. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. 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.