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Our commentary "After All, Only Millions?" ([@B1]) highlighted that, in contrast to a recent scaling law-based estimate of global microbial diversity of a trillion species-level operational taxonomic units ([@B2]), the decreasing frequencies of novel 16S rRNA gene sequences recovered by high-throughput sequencing ([@B3], [@B4]) is suggesting that global microbial diversity might well be saturated at a few million. No one knows even the order of magnitude of *Bacteria* and *Archaea* species numbers that ongoing surveys will ultimately yield. Acknowledging that such a census can never be truly completed, it will make a difference for many microbiologists whether we assume for our next experiments that, in addition to each species of higher plants and animals, we also need to consider either just a few hundred or a million unique microbial species in each microbiome.

We are well aware that the short-read 16S rRNA sequence repositories already contain around 1 × 10e7 different sequences, many of those only recorded once or twice. Rather than arguing that a significant part of these short sequences may be erroneous, we would like to reiterate that an accurate census should be based on long (nearly complete), high-quality 16S rRNA sequences ([@B3], [@B4]). Furthermore, the simple identity-based thresholds of operational taxonomic units do not record monophyly, which is a fundamental basis for the species definition of *Bacteria* and *Archaea*.

We certainly agree with our colleagues Jay Lennon and Kenneth Locey ([@B5]) that the census should include more ecosystems and that the number of candidate phyla is high ([@B3]), reflecting the deep divergence and long evolutionary history of *Bacteria* and *Archaea*. Yet, we disagree with their prediction of the extent of the rare biosphere. Our contrasting hypotheses will be brought to a test in a future in which novel sequencing technologies will provide long, error-free, and nonchimeric sequences. And even then, the debate on the global number of species of *Bacteria* and *Archaea* will continue since this relevant question cannot be resolved solely based on comparative 16S rRNA sequence analysis.

This is a response to a letter by Lennon and Locey (http://dx.doi.org/10.1128/mBio.01298-16).

**Citation** Amann R, Rossello-Mora R. 2016. Reply to "The Underestimation of Global Microbial Diversity". mBio 7(5):e01623-16. doi:10.1128/mBio.01623-16.

[^1]: **Editor** Margaret J. McFall-Ngai, University of Hawaii
