Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences

Journal name:
Nature Reviews Microbiology
Year published:
Published online


Publicly available sequence databases of the small subunit ribosomal RNA gene, also known as 16S rRNA in bacteria and archaea, are growing rapidly, and the number of entries currently exceeds 4 million. However, a unified classification and nomenclature framework for all bacteria and archaea does not yet exist. In this Analysis article, we propose rational taxonomic boundaries for high taxa of bacteria and archaea on the basis of 16S rRNA gene sequence identities and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Our analyses show that only nearly complete 16S rRNA sequences give accurate measures of taxonomic diversity. In addition, our analyses suggest that most of the 16S rRNA sequences of the high taxa will be discovered in environmental surveys by the end of the current decade.

At a glance


  1. Variable regions of the 16S ribosomal RNA.
    Figure 1: Variable regions of the 16S ribosomal RNA.

    Secondary structure of the 16S rRNA of Escherichia coli, as generated using the xrna program (see Further information). For our analysis, six R fragments of ~250 nucleotides were designed according to the known V regions (Supplementary information S2 (box)). In red, fragment R1 including regions V1 and V2; in orange, fragment R2 including region V3; in yellow, fragment R3 including region V4; in green, fragment R4 including regions V5 and V6; in blue, fragment R5 including regions V7 and V8; and in purple, fragment R6 including region V9.

  2. Predicting taxa richness using partial 16S ribosomal RNA sequences.
    Figure 2: Predicting taxa richness using partial 16S ribosomal RNA sequences.

    The 16S rDNA data set that was used was obtained from the Living Tree Project (LTP) release 108. Taxa recovery was approached using OTUs (operational taxonomic units) that were calculated using CD-HIT version 4.5 (Ref. 54). In all cases, the general taxonomic thresholds that were used for clustering are: 98.7% (species), 94.5% (genus), 86.5% (family), 82.0% (order), 78.5% (class) and 75.0% (phylum). a | Six R fragments of ~250 nucleotides were designed according to the V regions (Supplementary information S2 (box)); the results that were obtained for the full-length sequence are included for comparison. b | Based on the R fragments, four additional segments were created, all of which start at the 5′ end (that is, all including the V1 and V2 regions) but with different sizes: segment R1 contains the 250 nucleotides at the 5′ end, R1–R2 contains the 5′ 500 nucleotides, R1–R3 contains the 5′ 750 nucleotides, R1–R4 contains the 5′ 1050 nucleotides, R1–R5 contains the 5′ 1300 nucleotides, and 'full' corresponds to the full Escherichia coli 16S rRNA gene (which is 1,542 nucleotides). Analysis of the taxa recovery rate indicates a great underestimation of taxa richness when partial sequences are used. Although the situation tends to ameliorate as longer segments are considered, near full-length 16S rRNA genes sequences are required for accurate richness estimations and accurate classifications of high taxa.

  3. Classification of phylum Spirochaetes into candidate taxonomic units.
    Figure 3: Classification of phylum Spirochaetes into candidate taxonomic units.

    a | The phylum Spirochaetes is currently classified within one class (Spirochaetes), one order (Spirochaetales) and four families (Spirochaetaceae, Brevinemataceae, Brachyspiraceae and Leptospiraceae). A 16S ribosomal RNA phylogenetic reconstruction of the phylum Spirochaetes, based on 82 type strains obtained from the Living Tree Project (LTP) release 111 and using the RAxML algorithm55, shows a clear phylogenetic separation of five lineages within the phylum. Percentage bootstrap values are indicated next to the tree nodes. Scale bar indicates estimated sequence divergence. b | Distribution of the minimum sequence identity that is found in each bacterial and archaeal taxon in LTP release 102 (Supplementary information S1 (table) and Supplementary information S2 (box)). The average sequence identity among the five groups is shown as percentages and is linked to the density plot on the right-hand side to further confirm that the distances are within the values that are commonly displayed between taxa belonging to the rank of class. According to the strong monophyletic support and the low inter-clade sequence identities below the taxonomic threshold of 78.5% (Table 1), our analysis predicts five classes within Spirochaetes rather than one. The ten branches displayed in part a correspond to the following European Nucleotide Archive (ENA) accession numbers: Treponema maltophyla, X87140; Sphaerochaeta globosa, AF357916; Borrelia americana, EU081285; Spirochaeta litoralis, FR733665; Exilispira thermophila, AB364473; Brevinema andersonii, GU993264; Brachyspira aalborgi, Z22781; Leptospira interrogans, Z12817; Leptonema illini, AY714984; Turneriella parva, AY293856.

  4. Phylogenetic reconstruction of the SAR11 group within the Proteobacteria based on 16S ribosomal RNA gene sequences.
    Figure 4: Phylogenetic reconstruction of the SAR11 group within the Proteobacteria based on 16S ribosomal RNA gene sequences.

    The reconstruction was based on Living Tree Project (LTP) release 111. Several phyla and the major alphaproteobacterial families are shown for reference. The CTU (candidate taxonomic unit) analysis that was carried out on the SAR11 group, which was suggested to form a coherent taxon within the class Alphaproteobacteria, revealed the existence of three classes, SAR11.Class1, SAR11.Class1-1 and SAR11.Class2. One class, SAR11.Class2, is composed of two candidate orders (SAR11.Order2 and SAR11.Order3; yellow). The pie charts indicate the percentage of genera that comprise only surface-water sequences (orange), only deep-water sequences (purple), both surface-water and deep-water sequences (green) and other samples (grey). The ecological coherence with respect to the site of sequence retrieval is inversely correlated with the rank of the taxon. In addition to the large phylogenetic depth of Alphaproteobacteria, the positions of classes Epsilonproteobacteria and Deltaproteobacteria in this tree reconstruction strongly suggest that the phylum Proteobacteria is not monophyletic. Scale bar represents estimated sequence divergence.


  1. Godfray, H. C. J. Challenges for taxonomy. Nature 417, 1719 (2002).
  2. Mora, C., Tittensor, D. P., Adl, S., Simpson, S. G. B. & Worm, B. How many species are on Earth and in the ocean. PLoS Biol. 9, e1001127 (2011).
  3. Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590D596 (2013).
    This paper reports the SILVA project, which is a comprehensive web resource (see Further information) for up-to-date, quality-controlled databases of aligned rRNA gene sequences from the Bacteria, the Archaea and the Eukarya.
  4. Mole, B. Microbiome research goes without a home. Nature 500, 1617 (2013).
  5. Amann, R. I., Ludwig, W. & Schleifer, K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143169 (1995).
  6. Rosselló-Móra, R. Towards a taxonomy of Bacteria and Archaea based on interactive and cumulative data repositories. Environ. Microbiol. 14, 318334 (2012).
  7. Dykhuizen, D. E. Santa Rosalia revisited: why are there so many species of bacteria? Antonie Van Leeuwenhoek 73, 2533 (1998).
  8. Richter, M. & Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl Acad. Sci. USA 106, 1912619131 (2009).
  9. Stackebrandt, E. & Ebers, J. Taxonomic parameters revisited: tarnished gold standards. Microbiol. Today 8, 69 (2006).
  10. Tindall, B. J., Rosselló-Móra, R., Busse, H.-J., Ludwig, W. & Kämpfer, P. Notes on the characterization of prokaryote strains for taxonomic purposes. Int. J. Syst. Evol. Microbiol. 60, 249266 (2010).
  11. Philippot, L. et al. The ecological coherence of high bacterial taxonomic ranks. Nature Rev. Microbiol. 8, 523529 (2010).
    This study demonstrates that high bacterial taxa (that is, genus and above) are ecologically meaningful and their coherence is inversely correlated to their taxonomic rank. These observations provide a new perspective for the study of bacterial taxonomy, evolution and ecology.
  12. Gribaldo, S. & Brochier-Armanet, C. Time for order in microbial systematics. Trends Microbiol. 20, 209210 (2012).
  13. Garrity, G. M. & Oren, A. Response to Gribaldo and Brochier-Armanet: time for order in microbial systematics. Trends Microbiol. 20, 353354 (2012).
    In this paper, the International Committee on Systematics of Prokaryotes (ICSP) supports a call for order in microbial systematics to address the lack of criteria to circumscribe high taxa, which represents a major problem in microbiology today.
  14. Ereshefsky, M. Some problems with the Linnaean hierarchy. Phylos. Sci. 61, 186205 (1994).
  15. Yarza, P. et al. The All-Species Living Tree Project: a 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst. Appl. Microbiol. 31, 241250 (2008).
    This paper reports the All-Species Living Tree Project (LTP), which is an initiative of Systematic and Applied Microbiology for the creation and maintenance of highly curated 16S rRNA and 23S rRNA gene sequence databases, alignments and phylogenetic trees for all the type strains of bacteria and archaea.
  16. Cole, J. R., Konstantinidis, K., Farris, R. J. & Tiedje, J. M. in Environmental molecular microbiology (eds Liu, W.-T. & Jansson, J. K.) 119 (Caister Academic Press, 2010).
  17. Ludwig, W., Klenk, H.-P. in Bergey's Manual of Systematic Bacteriology 2nd edn (eds Boone, D. R., Castenholz, R. W. & Garrity, G. M.) 4965 (Springer, 2001).
  18. Ludwig, W. in Molecular Phylogeny of Microorganisms (eds Oren, A. & Papke, R. T.) 6583 (Caister Academic Press, 2010).
    This chapter reports the classification of high ranks of the Bacteria and the Archaea, which is currently based on comparative analyses of rRNA and is supported by other markers and multigene approaches. The high information content and great availability in databases mostly justify the usage of rRNA gene sequences in taxonomy.
  19. Fox, G. E., Pechman, K. R. & Woese, C. R. Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics. Int. J. Syst. Bacteriol. 27, 4457 (1977).
  20. Ludwig, W. & Schleifer, K. H. Bacterial phylogeny based on 16S and 23S rRNA sequence analysis. FEMS Microbiol. Rev. 15, 155173 (1994).
  21. Van de Peer, Y., Chapelle, S. & Wachter, R. D. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Res. 24, 33813391 (1996).
  22. Fuchs, B. M. et al. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64, 49734982 (1998).
  23. Ludwig, W. et al. ARB: a software environment for sequence data. Nucleic Acids Res. 32, 13631371 (2004).
  24. Yarza, P. et al. Update of the all-species living tree project based on 16S and 23S rRNA sequence analyses. Syst. Appl. Microbiol. 33, 291299 (2010).
  25. Shida, O., Takagi, H., Kadowaki, K. & Komagata, K. Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. Int. J. Syst. Bacteriol. 46, 939946 (1996).
  26. Kang, S.-J. et al. Brevundimonas naejangsanensis sp. nov., a proteolytic bacterium isolated from soil, and reclassification of Mycoplana bullata into the genus Brevundimonas as Brevundimonas bullata comb. nov. Int. J. Syst. Evol. Microbiol. 59, 31553160 (2009).
  27. Keswani, J. & Whitman, W. B. Relationship of 16S rRNA sequence similarity to DNA hybridization in prokaryotes. Int. J. Syst. Evol. Microbiol. 51, 667678 (2001).
  28. Chakravorty, S., Helb, D., Burday, M., Connell, N. & Alland, D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J. Microbiol. Methods 69, 330339 (2007).
  29. Mizrahi-Man, O., Davenport, E. R. & Gilad, Y. Taxonomic classification of bacterial 16S rRNA genes using short sequencing reads: evaluation of effective study designs. PLoS ONE 8, e53608 (2013).
  30. Ashelford, K. E., Chuzhanova, N. A., Fry, J. C., Jones, A. J. & Weightman, A. J. At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl. Environ. Microbiol. 71, 77247736 (2005).
  31. Saddler, G. S. International Committee on Systematics of Prokaryotes. Xth International (IUMS) Congress of Bacteriology and Applied Microbiology. Minutes of the meetings, 28 and 30 July 2002, Paris, France. Int. J. Syst. Evol. Microbiol. 55, 533537 (2005).
  32. Ritalahti, K. M. et al. Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochaetes. Int. J. Syst. Evol. Microbiol. 62, 210216 (2012).
  33. Meier-Kolthoff, J. P., Göker, M., Spröer, C. & Klenk, H. P. When should a DDH experiment be mandatory in microbial taxonomy? Arch. Microbiol. 195, 413418 (2013).
  34. Curtis, T. P., Sloan, W. T., & Scannell, J. W. Estimating prokaryotic diversity and its limits. Proc. Natl Acad. Sci. USA 99, 1049410499 (2002).
  35. Salman, V., Amann, R., Shub, D. A. & Schulz-Vogt, H. N. Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria. Proc. Natl Acad. Sci. USA 109, 42034208 (2012).
  36. Klindworth, A. et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41, e1 (2013).
  37. Wang, T. et al. Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers. ISME J. 6, 320329 (2012).
  38. Guazzaroni, M.-E. et al. Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation. ISME J. 7, 122136 (2013).
  39. Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47, 479491 (1997).
  40. Lee, K. C. Y. et al. Phylogenetic delineation of the novel phylum Armatimonadetes (former candidate division OP10) and definition of two novel candidate divisions. Appl. Environ. Microbiol. 79, 24842487 (2013).
  41. Cavalier-Smith, T. The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. Int. J. Syst. Evol. Microbiol. 52, 776 (2002).
  42. Buchanan, R. E. Studies in the nomenclature and classification of the bacteria: II. The primary subdivisions of the Schizomycetes. J. Bacteriol. 2, 155164 (1917).
  43. Hovind-Hougen, K. Leptospiraceae, a new family to include Leptospira Noguchi 1917 and Leptonema gen. nov. Int. J. Syst. Bacteriol. 29, 245251 (1979).
  44. Swellengrebel, N. H. Sur la cytologie comparée des spirochètes et des spirilles. Ann. Inst. Pasteur. 21, 562586 (in French) (1907).
  45. Gupta, R. S., Mahmood, S. & Adeolu, M. A phylogenomic and molecular signature based approach for characterization of the phylum Spirochaetes and its major clades: proposal for a taxonomic revision of the phylum. Front. Microbiol. 4, 217 (2013).
  46. Harris, J. K., Kelley, S. T. & Pace, N. R. New perspective on uncultured bacterial phylogenetic division OP11. Appl. Environ. Microbiol. 70, 845849 (2004).
  47. Giebel, H.-A. et al. Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate. ISME J. 5, 819 (2011).
  48. Wagner, M. & Horn, M. The Planctomycetes, Verrucomicrobia, Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance. Curr. Opin. Biotechnol. 17, 241249 (2006).
  49. Guy, L. & Ettema, T. J. G. The archaeal 'TACK' superphylum and the origin of eukaryotes. Trends Microbiol. 19, 580587 (2011).
  50. Hugenholtz, P., Goebel, B. M. & Pace, N. R. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 47654774 (1998).
  51. Yilmaz, P. et al. The SILVA and 'All-species Living Tree Project (LTP)' taxonomic frameworks. Nucl. Acids Res. 42, D643D648 (2013).
  52. Geissinger, O., Herlemann, D. P. R., Mörschel, E., Maier, U. G. & Brune, A. The ultramicrobacterium 'Elusimicrobium minutum' gen. nov., sp. nov., the first cultivated representative of the Termite Group 1 phylum. Appl. Environ. Microbiol. 75, 28312840 (2009).
  53. R Core Team. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, 2014) [online]
  54. Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 31503152 (2012).
  55. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 26882690 (2006).

Download references

Author information


  1. Marine Microbiology Group, Department of Ecology and Marine Resources, Mediterranean Institute for Advanced Studies (Spanish National Research Council (CSIC)-University of the Balearic Islands (UIB)), E-07190 Esporles, Balearic Islands, Spain.

    • Pablo Yarza &
    • Ramon Rosselló-Móra
  2. Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany.

    • Pablo Yarza,
    • Pelin Yilmaz,
    • Elmar Pruesse,
    • Frank Oliver Glöckner &
    • Rudolf Amann
  3. Ribocon GmbH, Fahrenheitstrasse 1, D-28359 Bremen, Germany.

    • Pablo Yarza
  4. Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany.

    • Frank Oliver Glöckner
  5. Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany.

    • Wolfgang Ludwig &
    • Karl-Heinz Schleifer
  6. Department of Microbiology, University of Georgia, 527 Biological Sciences Building, Athens, Georgia 30605–2605, USA.

    • William B. Whitman
  7. Société de Bactériologie Systématique et Vétérinaire (SBSV) and École Nationale Vétérinaire de Toulouse (ENVT), F-31076 Toulouse cedex 03, France.

    • Jean Euzéby

Competing interests statement

The authors declare no competing interests.

Corresponding authors

Correspondence to:

Author details

  • Pablo Yarza

    Pablo Yarza received his Ph.D. in environmental microbiology and biotechnology from the University of the Balearic Islands, Spain, in 2011. He is currently a postdoctoral researcher at the company Ribocon GmbH in Bremen, Germany. His main interests are the development and analysis of curated sequence databases in the frameworks of microbial systematics and biotechnology.

  • Pelin Yilmaz

    Pelin Yilmaz did her Ph.D. in bioinformatics at the Microbial Genomics and Bioinformatics Group at the Max Planck Institute for Marine Microbiology, Bremen, Germany. She is currently a postdoctoral researcher in this group, focusing on bacterial and archaeal phylogeny and taxonomy, as well as microbial diversity and function in marine ecosystems.

  • Elmar Pruesse

    Elmar Pruesse received his Ph.D. in bioinformatics from the Jacobs University, Bremen, Germany, in 2011. He is currently a postdoctoral researcher in the Microbial Genomics and Bioinformatics group at the Max Planck Institute for Marine Microbiology, Bremen, Germany. His main research interest is the development of scalable methods for the integrative analysis of sequence and contextual data.

  • Frank Oliver Glöckner

    Frank Oliver Glöckner received his Ph.D. in microbiology from the Technical University Munich, Germany, in 1998. He is Head of the Microbial Genomics and Bioinformatics Research Group at the Max Planck Institute for Marine Microbiology in Bremen, Germany, and Professor of Bioinformatics at Jacobs University Bremen, Germany. His group develops enabling technologies and products to transform the wealth of environmental sequence and contextual (meta)data into biological knowledge.

  • Wolfgang Ludwig

    Wolfgang Ludwig received his Ph.D. in microbiology from the Technical University Munich, Germany, in 1981. His research interests focus on the phylogeny and systematics of bacteria and archaea, as well as bioinformatics tools for phylogeny and identification studies.

  • Karl-Heinz Schleifer

    Karl-Heinz Schleifer received his doctoral degree in microbiology from the Technical University Munich (TUM), Germany, in 1967. He was Head of the Department of Microbiology at the TUM from 1974 until 2007. Since 2007, he has been TUM Emeritus of Excellence. He is mainly interested in systematics and molecular ecology of bacteria.

  • William B. Whitman

    William B. Whitman is a professor at the University of Georgia, Athens, USA. He did postdoctoral studies at the University of Illinois at Urbana, USA, and did his doctoral research at the University of Texas at Austin, USA. His laboratory studies the systematics and physiology of free-living bacteria and archaea that catalyse biogeochemically important transformations.

  • Jean Euzéby

    Jean P. Euzéby was a professor at the Ecole Nationale Vétérinaire de Toulouse, France (he retired in June 2013). In 1997, he established the List of Prokaryotic Names with Standing in Nomenclature (LPSN).

  • Rudolf Amann

    Rudolf Amann received his doctoral degree in microbiology from the Technical University Munich, Germany, in 1988. He is currently Head of the Department of Molecular Ecology at the Max Planck Institute for Marine Microbiology, Bremen, Germany, and Professor of Microbial Ecology at the University of Bremen, Germany. His research focus is the analysis of the diversity, composition and function of microbial communities by nucleic acid-based methods.

  • Ramon Rosselló-Móra

    Ramon Rosselló-Móra received his Ph.D. in biology from the University of the Balearic Islands (UIB), Spain, in 1992. He is currently a researcher and Head of the Marine Microbiology Group at the Mediterranean Institute for Advanced Studies (IMEDEA) (Spanish National Research Council-UIB). His research focuses on microbial molecular ecology studies on hypersaline environments, as well as the development of modern tools that can be applied to microbial taxonomy.

Supplementary information

Excel files

  1. Supplementary information S1 (table) (114 KB)

    Intra-taxon sequence identity measures used to calculate the taxonomic thresholds.

  2. Supplementary information S4 (table) (1,080 KB)

    Sequence associated meta-data including CTU classification. Fields: acc,start,stop,tax_CTU,tax_xref_embl

PDF files

  1. Supplementary information S2 (Box) (177 KB)

    Additional tables and figures.

  2. Supplementary information S3 (figure) (11,625 KB)

    Phylogenetic reconstruction of 15 candidate divisions and environmental clades

Additional data