Skip to content
1887

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY logo International Journal of Systematic and Evolutionary Microbiology

Volume 76, Issue 2

Genome-based phylogenomic reassessment of with proposals for species synonymization and new subspecies No Access

Abstract

The genus encompasses phylogenetically diverse, obligately anaerobic, Gram-positive, spore-forming bacteria, yet taxonomic resolution for several members remains limited by the conservatism of the 16S rRNA gene. We reassessed four closely related species pairs: and ; and ; and ; and subsp. and subsp. . A polyphasic framework was applied, integrating 16S rRNA and core-genome phylogenies, whole-genome relatedness metrics (digital DNA–DNA hybridization, average nucleotide identity and average amino acid identity), phenotypic characterization and carbohydrate-active enzyme profiling. Concordant genomic and phenotypic evidence supported the synonymization of (Prévot 1940) Bernard . 2018 with (Prévot 1938) Holdeman and Moore 1970, Greetham . 2003 with Soh . 1991 and subsp. (Kalchayanand . 1993) Spring . 2003 with subsp. (Collins . 1993) Spring . 2003. By contrast, despite high genomic relatedness, (van Ermengem 1896) Bergey . 1923 and (Prévot and Laplanche 1947) Bernard . 2018 displayed reproducible phenotypic and functional distinctions consistent with subspecies status. We, therefore, propose subsp. comb. nov., alongside subsp. comb. nov., thereby refining genome-based classification within the genus.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): average amino acid identity (AAI) , average nucleotide identity (ANI) , Clostridium , digital DNA–DNA hybridization (dDDH) , genomic analysis and taxonomic reclassification
© 2026 The Authors
Loading

Article metrics loading...

/content/journal/ijsem/10.1099/ijsem.0.007048
2026-02-02
2026-02-15

Metrics

Loading full text...

Full text loading...

References

  1. Prażmowski A. Untersuchung Über Die Entwickelungsgeschichte Und Fermentwirking Einiger Bacterien-Arten. Inaugural Dissertation. Leipzig (Germany): Hugo Voigt; 1880
  2. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J et al. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 1994; 44:812–826 [View Article] [PubMed]
     [Google Scholar]
  3. Lawson PA, Rainey FA. Proposal to restrict the genus Clostridium prazmowski to Clostridium butyricum and related species. Int J Syst Evol Microbiol 2016; 66:1009–1016 [View Article]
     [Google Scholar]
  4. Lúquez C, Halpin JL, Dykes J. Unintended consequences: renaming botulinum neurotoxin-producing species of clostridium and related species. Toxicon 2023; 224:107036 [View Article] [PubMed]
     [Google Scholar]
  5. Yutin N, Galperin MY. A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ Microbiol 2013; 15:2631–2641 [View Article] [PubMed]
     [Google Scholar]
  6. Stackebrandt E. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006; 33:152
     [Google Scholar]
  7. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 2014; 12:635–645 [View Article] [PubMed]
     [Google Scholar]
  8. Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351 [View Article] [PubMed]
     [Google Scholar]
  9. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 2009; 106:19126–19131 [View Article] [PubMed]
     [Google Scholar]
  10. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 2013; 14:60 [View Article] [PubMed]
     [Google Scholar]
  11. Riesco R, Trujillo ME. Update on the proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2024; 74:006300 [View Article] [PubMed]
     [Google Scholar]
  12. Lawson PA, Saavedra Perez L, Sankaranarayanan K. Reclassification of Clostridium cocleatum, Clostridium ramosum, Clostridium spiroforme and Clostridium saccharogumia as Thomasclavelia cocleata gen. nov., comb. nov., Thomasclavelia ramosa comb. nov., gen. nov., Thomasclavelia spiroformis comb. nov. and Thomasclavelia saccharogumia comb. nov. Int J Syst Evol Microbiol 2023; 73: [View Article]
     [Google Scholar]
  13. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 2014; 42:D490–5 [View Article] [PubMed]
     [Google Scholar]
  14. Holdeman LV, Moore WEC et al. Clostridia. In Cato EP, Cummins CS, Holdeman LV, Johnson JL, Moore WEC et al. eds Outline of Clinical Methods in Anaerobic Bacteriology, 2nd revision. Blacksburg, VA: Virginia Polytechnic Institute and State University - Anaerobe Laboratory; 1970 pp 57–66
     [Google Scholar]
  15. Bernard K, Burdz T, Wiebe D, Alfa M, Bernier A-M. Clostridium neonatale sp. nov. linked to necrotizing enterocolitis in neonates and a clarification of species assignable to the genus Clostridium (Prazmowski 1880) emend. Lawson and Rainey 2016. Int J Syst Evol Microbiol 2018; 68:2416–2423 [View Article] [PubMed]
     [Google Scholar]
  16. Lawson Anani Soh A, Ralambotiana H, Ollivier B, Prensier G, Tine E et al. Clostridium thermopalmarium sp. nov., a moderately thermophilic butyrate-producing bacterium isolated from palm wine in Senegal. Syst Appl Microbiol 1991; 14:135–139 [View Article]
     [Google Scholar]
  17. Greetham HL, Gibson GR, Giffard C, Hippe H, Merkhoffer B et al. Clostridium colicanis sp. nov., from canine faeces. Int J Syst Evol Microbiol 2003; 53:259–262 [View Article] [PubMed]
     [Google Scholar]
  18. Bergey DH, Harrison FC, Breed RS, Hammer BW, Huntoon FM. Bergey’s Manual of Determinative Bacteriology, 1st ed. Baltimore: The Williams & Wilkins Co; 1923
     [Google Scholar]
  19. Spring S, Merkhoffer B, Weiss N, Kroppenstedt RM, Hippe H et al. Characterization of novel psychrophilic clostridia from an Antarctic microbial mat: description of Clostridium frigoris sp. nov., Clostridium lacusfryxellense sp. nov., Clostridium bowmanii sp. nov. and Clostridium psychrophilum sp. nov. and reclassification of Clostridium laramiense as Clostridium estertheticum subsp. laramiense subsp. nov. Int J Syst Evol Microbiol 2003; 53:1019–1029 [View Article] [PubMed]
     [Google Scholar]
  20. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 2017; 67:1613–1617 [View Article] [PubMed]
     [Google Scholar]
  21. Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 2021; 38:3022–3027 [View Article] [PubMed]
     [Google Scholar]
  22. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16:111–120 [View Article] [PubMed]
     [Google Scholar]
  23. Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 1981; 17:368–376 [View Article] [PubMed]
     [Google Scholar]
  24. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4:406–425 [View Article] [PubMed]
     [Google Scholar]
  25. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 2004; 101:11030–11035 [View Article] [PubMed]
     [Google Scholar]
  26. Felsenstein J. Phylogenies and the comparative method. Am Nat 1985; 125:1–15 [View Article]
     [Google Scholar]
  27. Chaudhari NM, Gupta VK, Dutta C. BPGA- an ultra-fast pan-genome analysis pipeline. Sci Rep 2016; 6:24373 [View Article] [PubMed]
     [Google Scholar]
  28. Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One 2010; 5:e9490 [View Article] [PubMed]
     [Google Scholar]
  29. Goecks J, Nekrutenko A, Taylor J. Galaxy Team Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 2010; 11:R86 [View Article] [PubMed]
     [Google Scholar]
  30. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res 2019; 47:W256–W259 [View Article] [PubMed]
     [Google Scholar]
  31. Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 2019; 10:2182 [View Article]
     [Google Scholar]
  32. Henz SR, Huson DH, Auch AF, Nieselt-Struwe K, Schuster SC. Whole-genome prokaryotic phylogeny. Bioinformatics 2005; 21:2329–2335 [View Article]
     [Google Scholar]
  33. Lefort V, Desper R, Gascuel O. FastME 2.0: a comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol Evol 2015; 32:2798–2800 [View Article] [PubMed]
     [Google Scholar]
  34. Kreft L, Botzki A, Coppens F, Vandepoele K, Van Bel M. PhyD3: a phylogenetic tree viewer with extended phyloXML support for functional genomics data visualization. Bioinformatics 2017; 33:2946–2947 [View Article] [PubMed]
     [Google Scholar]
  35. Meier-Kolthoff JP, Hahnke RL, Petersen J, Scheuner C, Michael V et al. Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy. Stand Genomic Sci 2014; 9:2 [View Article] [PubMed]
     [Google Scholar]
  36. Lee I, Ouk Kim Y, Park SC, Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 2016; 66:1100–1103 [View Article]
     [Google Scholar]
  37. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 2018; 9:5114 [View Article] [PubMed]
     [Google Scholar]
  38. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. BLAST+: architecture and applications. BMC Bioinformatics 2009; 10:1–9 [View Article]
     [Google Scholar]
  39. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M et al. Versatile and open software for comparing large genomes. Genome Biol 2004; 5:1–9 [View Article] [PubMed]
     [Google Scholar]
  40. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 2016; 32:929–931 [View Article] [PubMed]
     [Google Scholar]
  41. Rodriguez-R LM, Konstantinidis KT. Estimating coverage in metagenomic data sets and why it matters. ISME J 2014; 8:2349–2351 [View Article] [PubMed]
     [Google Scholar]
  42. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 2015; 25:1043–1055 [View Article] [PubMed]
     [Google Scholar]
  43. Tanizawa Y, Fujisawa T, Nakamura Y. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics 2018; 34:1037–1039 [View Article] [PubMed]
     [Google Scholar]
  44. Yin Y, Mao X, Yang J, Chen X, Mao F et al. dbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic Acids Res 2012; 40:W445–W451 [View Article]
     [Google Scholar]
  45. Wiegel J, Tanner R, Rainey FA. An introduction to the family Clostridiaceae. In Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E. eds The Prokaryotes New York: Springer; 2006 pp 654–678
     [Google Scholar]
  46. Skarin H, Segerman B. Horizontal gene transfer of toxin genes in Clostridium botulinum: Involvement of mobile elements and plasmids. Mob Genet Elements 2011; 1:213–215 [View Article] [PubMed]
     [Google Scholar]
  47. Auch AF, von Jan M, Klenk H-P, Göker M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2010; 2:117–134 [View Article] [PubMed]
     [Google Scholar]
  48. Wambui J, Cernela N, Stevens MJA, Stephan R. Whole genome sequence-based identification of Clostridium estertheticum complex strains supports the need for taxonomic reclassification within the species Clostridium estertheticum. Front Microbiol 2021; 12:727022 [View Article] [PubMed]
     [Google Scholar]
  49. Oren A, Garrity GM. Valid publication of names of prokaryotes according to the international code of nomenclature of prokaryotes. Int J Syst Evol Microbiol 2021; 71:005062
     [Google Scholar]
  50. Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev 1990; 3:66–98 [View Article] [PubMed]
     [Google Scholar]
  51. Peck MW, Stringer SC, Carter AT. Clostridium botulinum in the post-genomic era. Food Microbiol 2011; 28:183–191 [View Article] [PubMed]
     [Google Scholar]
  52. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 2009; 37:D233–8 [View Article] [PubMed]
     [Google Scholar]
/content/journal/ijsem/10.1099/ijsem.0.007048
Loading
Genome-based phylogenomic reassessment of Clostridium with proposals for species synonymization and new subspecies
Int J Syst Evol Microbiol 76, 007048 (2026); https://doi.org/10.1099/ijsem.0.007048
/content/journal/ijsem/10.1099/ijsem.0.007048
/content/journal/ijsem/10.1099/ijsem.0.007048
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error