How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys

Based on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1) the presence of undetected chimeric sequences; (2) the misplacement of several fast-evolving sequences; and (3) the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups.Environmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel high-level eukaryotic taxa, two of which comprise several phylotypes that were found independently in different studies. To ascertain their taxonomic status, however, the organisms themselves have now to be identified.Over the past few years, cultivation-independent identification of microbial organisms by PCR amplification and sequencing of small-subunit ribosomal RNA (SSU rRNA) genes revealed a huge diversity of eubacterial and archaeal phylotypes in environmental samples, many of which are not represented by cultured organisms [1,2]. Recently, the same techniques have been applied to surveys of eukaryotic diversity in different marine and freshwater biotopes, including planktonic [3,4] and some extreme, anoxic [5], acidic and iron-rich [6] or deep-sea hydrothermal vent [7,8] environments. All these studies revealed an unexpectedly high diversity of new eukaryotic phylotypes at three distinct taxonomic levels. Some of them can be attributed to novel species i