The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes. To date, there are more than 30,000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ. This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology. A group of sequences appearing monophyletic in one dataset may not be so in another. This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level. In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data. The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.


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