The exponentially increasing number of sequenced genomes necessitates fast, accurate, universally applicable and automated approaches for the delineation of prokaryotic species. We developed specI (species identification tool; http://www.bork.embl.de/software/specI/), a method to group organisms into species clusters based on 40 universal, single-copy phylogenetic marker genes. Applied to 3,496 prokaryotic genomes, specI identified 1,753 species clusters. Of 314 discrepancies with a widely used taxonomic classification, >62% were resolved by literature support.

To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.