Photosynthesis Research

, Volume 122, Issue 2, pp 171–185 | Cite as

Phylogenetic analysis and molecular signatures defining a monophyletic clade of heterocystous cyanobacteria and identifying its closest relatives

  • Mohammad Howard-Azzeh
  • Larissa Shamseer
  • Herb E. Schellhorn
  • Radhey S. GuptaEmail author
Regular Paper


Detailed phylogenetic and comparative genomic analyses are reported on 140 genome sequenced cyanobacteria with the main focus on the heterocyst-differentiating cyanobacteria. In a phylogenetic tree for cyanobacteria based upon concatenated sequences for 32 conserved proteins, the available cyanobacteria formed 8–9 strongly supported clades at the highest level, which may correspond to the higher taxonomic clades of this phylum. One of these clades contained all heterocystous cyanobacteria; within this clade, the members exhibiting either true (Nostocales) or false (Stigonematales) branching of filaments were intermixed indicating that the division of the heterocysts-forming cyanobacteria into these two groups is not supported by phylogenetic considerations. However, in both the protein tree as well as in the 16S rRNA gene tree, the akinete-forming heterocystous cyanobacteria formed a distinct clade. Within this clade, the members which differentiate into hormogonia or those which lack this ability were also separated into distinct groups. A novel molecular signature identified in this work that is uniquely shared by the akinete-forming heterocystous cyanobacteria provides further evidence that the members of this group are specifically related and they shared a common ancestor exclusive of the other cyanobacteria. Detailed comparative analyses on protein sequences from the genomes of heterocystous cyanobacteria reported here have also identified eight conserved signature indels (CSIs) in proteins involved in a broad range of functions, and three conserved signature proteins, that are either uniquely or mainly found in all heterocysts-forming cyanobacteria, but generally not found in other cyanobacteria. These molecular markers provide novel means for the identification of heterocystous cyanobacteria, and they provide evidence of their monophyletic origin. Additionally, this work has also identified seven CSIs in other proteins which in addition to the heterocystous cyanobacteria are uniquely shared by two smaller clades of cyanobacteria, which form the successive outgroups of the clade comprising of the heterocystous cyanobacteria in the protein trees. Based upon their close relationship to the heterocystous cyanobacteria, the members of these clades are indicated to be the closest relatives of the heterocysts-forming cyanobacteria.


Nostocales Molecular signatures Heterocyst-forming cyanobacteria Akinete-forming cyanobacteria Molecular phylogeny Comparative genomics 



This work was supported by an MRI-ORF Water Round research grant. We thank Mobolaji Adeolu for helpful comments on the manuscript.

Supplementary material

11120_2014_20_MOESM1_ESM.pdf (4.2 mb)
Supplementary material 1 (PDF 4297 kb)


  1. Adeolu M, Gupta RS (2013) Phylogenomics and molecular signatures for the order Neisseriales: proposal for division of the order Neisseriales into the emended family Neisseriaceae and Chromobacteriaceae fam. nov. Antonie Van Leeuwenhoek 104:1–24PubMedCrossRefGoogle Scholar
  2. Baldauf SL, Palmer JD (1993) Animals and fungi are each other’s closest relatives: congruent evidence from multiple proteins. Proc Natl Acad Sci USA 90:11558–11562PubMedCrossRefPubMedCentralGoogle Scholar
  3. Bhandari V, Gupta RS (2014) Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations. Antonie Van Leeuwenhoek 105:143–168Google Scholar
  4. Castenholz RW (2001) Phylum BX cyanobacteria oxygenic photosynthetic bacteria. In Bergey’s manual of systematic bacteriology. Springer, New York, pp 474–487Google Scholar
  5. Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552PubMedCrossRefGoogle Scholar
  6. Cavalier-Smith T (2002) The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. Int J Syst Evol Microbiol 52:7–76PubMedGoogle Scholar
  7. Ciccarelli FD, Doerks T, Von Mering C, Creevey CJ, Snel B, Bork P (2006) Toward automatic reconstruction of a highly resolved tree of life. Science 311:1283–1287PubMedCrossRefGoogle Scholar
  8. De Vos P, Truper HG (2000) Judicial Commission of the International Committee on Systematic Bacteriology; IXth International (IUMS) Congress of Bacteriology and Applied Microbiology. Int J Syst Evol Microbiol 50:2239–2244CrossRefGoogle Scholar
  9. Delwiche CF, Kuhsel M, Palmer JD (1995) Phylogenetic analysis of tufA sequences indicates a cyanobacterial origin of all plastids. Mol Phylogenet Evol 4:110–128PubMedCrossRefGoogle Scholar
  10. Desikachary TV (1959) Cyanophyta, Indian Council of Agricultural Research, monographs on Algae. New Delhi, IndiaGoogle Scholar
  11. Fang G, Rocha E, Danchin A (2005) How essential are nonessential genes? Mol Biol Evol 22:2147–2156PubMedCrossRefGoogle Scholar
  12. Gao B, Mohan R, Gupta RS (2009) Phylogenomics and protein signatures elucidating the evolutionary relationships among the Gammaproteobacteria. Int J Syst Evol Microbiol 59:234–247PubMedCrossRefGoogle Scholar
  13. Geitler L (1932) Cyanophycea. Rabenhorst’s Kryptogamen-Flora von Deutschland, Österreich und der Schweiz, Reprint 1985:14Google Scholar
  14. Giovannoni SJ, Turner S, Olsen GJ, Barns S, Lane DJ, Pace NR (1988) Evolutionary relationships among cyanobacteria and green chloroplasts. J Bacteriol 170:3584–3592PubMedPubMedCentralGoogle Scholar
  15. Gugger MF, Hoffmann L (2004) Polyphyly of true branching cyanobacteria (Stigonematales). Int J Syst Evol Microbiol 54:349–357PubMedCrossRefGoogle Scholar
  16. Gupta RS (1998) Protein phylogenies and signature sequences: a reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol Biol Rev 62:1435–1491PubMedPubMedCentralGoogle Scholar
  17. Gupta RS (2000) The phylogeny of proteobacteria: relationships to other eubacterial phyla and eukaryotes. FEMS Microbiol Rev 24:367–402PubMedCrossRefGoogle Scholar
  18. Gupta RS (2003) Evolutionary relationships among photosynthetic bacteria. Photosynth Res 76:173–183PubMedCrossRefGoogle Scholar
  19. Gupta RS (2009) Protein signatures (molecular synapomorphies) that are distinctive characteristics of the major cyanobacterial clades. Int J Syst Evol Microbiol 59:2510–2526PubMedCrossRefGoogle Scholar
  20. Gupta RS (2010) Molecular signatures for the main phyla of photosynthetic bacteria and their subgroups. Photosynth Res 104:357–372PubMedCrossRefGoogle Scholar
  21. Gupta RS (2013) Molecular markers for photosynthetic bacteria and insights into the origin and spread of photosynthesis. Adv Bot Res 66:37–66CrossRefGoogle Scholar
  22. Gupta RS, Lali R (2013) Molecular signatures for the phylum Aquificae and its different clades: proposal for division of the phylum Aquificae into the emended order Aquificales, containing the families Aquificaceae and Hydrogenothermaceae, and a new order Desulfurobacteriales ord. nov., containing the family Desulfurobacteriaceae. Antonie Van Leeuwenhoek 104:349–368PubMedCrossRefGoogle Scholar
  23. Gupta RS, Mathews DW (2010) Signature proteins for the major clades of Cyanobacteria. BMC Evol Biol 10:24PubMedCrossRefPubMedCentralGoogle Scholar
  24. Gupta RS, Pereira M, Chandrasekera C, Johari V (2003) Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues. Int J Syst Evol Microbiol 53:1833–1842PubMedCrossRefGoogle Scholar
  25. Gupta RS, Chander P, George S (2013) Phylogenetic framework and molecular signatures for the class Chloroflexi and its different clades; proposal for division of the class Chloroflexi class. nov. into the suborder Chloroflexineae subord. nov., consisting of the emended family Oscillochloridaceae and the family Chloroflexaceae fam. nov., and the suborder Roseiflexineae subord. nov., containing the family Roseiflexaceae fam. nov. Antonie Van Leeuwenhoek 103:99–119PubMedCrossRefGoogle Scholar
  26. Harris JK, Kelley ST, Spiegelman GB, Pace NR (2003) The genetic core of the universal ancestor. Genome Res 13:407–412PubMedCrossRefPubMedCentralGoogle Scholar
  27. Henson BJ, Watson LE, Barnum SR (2002) Molecular differentiation of the heterocystous cyanobacteria, Nostoc and Anabaena, based on complete NifD sequences. Curr Microbiol 45:161–164PubMedCrossRefGoogle Scholar
  28. Henson BJ, Hesselbrock SM, Watson LE, Barnum SR (2004) Molecular phylogeny of the heterocystous cyanobacteria (subsections IV and V) based on nifD. Int J Syst Evol Microbiol 54:493–497PubMedCrossRefGoogle Scholar
  29. Hoffmann L (2005) Nomenclature of Cyanophyta/Cyanobacteria: roundtable on the unification of the nomenclature under the Botanical and Bacteriological Codes. Algol Stud 117:13–29CrossRefGoogle Scholar
  30. Hoffmann L, Komarek J, Kastovsky J (2005) System of cyanoprokaryotes (cyanobacteria) state in 2004. Algol Stud 117:95–115CrossRefGoogle Scholar
  31. Honda D, Yokota A, Sugiyama J (1999) Detection of seven major evolutionary lineages in cyanobacteria based on the 16S rRNA gene sequence analysis with new sequences of five marine Synechococcus strains. J Mol Evol 48:723–739PubMedCrossRefGoogle Scholar
  32. Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comp Appl Biosci CABIOS 8:275–282Google Scholar
  33. Komárek J (2002) Problems in cyanobacterial taxonomy: implication for most common toxin producing species. Rapporti Istisan 9:6–43Google Scholar
  34. Labeda DP (2000) International committee on systematic bacteriology; IXth international (IUMS) congress of bacteriology and applied microbiology. Int J Syst Evol Microbiol 50:2245–2247CrossRefGoogle Scholar
  35. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  36. Naushad HS, Lee B, Gupta RS (2014) Conserved signature indels and signature proteins as novel tools for understanding microbial phylogeny and systematics: identification of molecular signatures that are specific for the phytopathogenic genera Dickeya, Pectobacterium and Brenneria. Int J Syst Evol Microbiol 64:366–383PubMedCrossRefGoogle Scholar
  37. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, USAGoogle Scholar
  38. Oren A (2004) A proposal for further integration of the cyanobacteria under the Bacteriological Code. Int J Syst Evol Microbiol 54:1895–1902PubMedCrossRefGoogle Scholar
  39. Oren A, Garrity GM (2014) Proposal to change general consideration 5 and principle 2 of the International Code of Nomenclature of Prokaryotes. Int J Syst Evol Microbiol 64:309–310PubMedCrossRefGoogle Scholar
  40. Oren A, Komarek J, Hoffmann L (2009) Nomenclature of the Cyanophyta/Cyanobacteria/Cyanoprokaryotes: What has happened since IAC Luxembourg? Algol Stud 130:17–26CrossRefGoogle Scholar
  41. Parte AC (2014) LPSN-list of prokaryotic names with standing in nomenclature. Nucleic Acids Res 42:D613–D616PubMedCrossRefPubMedCentralGoogle Scholar
  42. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596PubMedCrossRefPubMedCentralGoogle Scholar
  43. Rajaniemi P, Hrouzek P, Kastovska K, Willame R, Rantala A, Hoffmann L, Komarek J, Sivonen K (2005) Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and Nostoc (Nostocales, Cyanobacteria). Int J Syst Evol Microbiol 55:11–26PubMedCrossRefGoogle Scholar
  44. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61CrossRefGoogle Scholar
  45. Rokas A, Holland PW (2000) Rare genomic changes as a tool for phylogenetics. Trends Ecol Evol 15:454–459PubMedCrossRefGoogle Scholar
  46. Rokas A, Williams BL, King N, Carroll SB (2003) Genome-scale approaches to resolving incongruence in molecular phylogenies. Nature 425:798–804PubMedCrossRefGoogle Scholar
  47. Saatcioglu FAHR, Perry DJ, Pasco DS, Fagan JB (1990) Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene. Mol Cell Biol 10:6408–6416PubMedPubMedCentralGoogle Scholar
  48. Sanchez-Baracaldo P, Hayes PK, Blank CE (2005) Morphological and habitat evolution in the Cyanobacteria using a compartmentalization approach. Geobiology 3:145–165CrossRefGoogle Scholar
  49. Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Federhen S (2010) Database resources of the national center for biotechnology information. Nucleic Acids Res 38:D5–D16PubMedCrossRefPubMedCentralGoogle Scholar
  50. Schoeffler AJ, May AP, Berger JM (2010) A domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function. Nucleic Acids Res 38:7830–7844PubMedCrossRefPubMedCentralGoogle Scholar
  51. Shi T, Falkowski PG (2008) Genome evolution in cyanobacteria: the stable core and the variable shell. Proc Natl Acad Sci 105:2510–2515PubMedCrossRefPubMedCentralGoogle Scholar
  52. Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, Calteau A, Cai F, de Marsac NT, Rippka R (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci 110:1053–1058PubMedCrossRefPubMedCentralGoogle Scholar
  53. Singh B, Gupta RS (2009) Conserved inserts in the Hsp60 (GroEL) and Hsp70 (DnaK) proteins are essential for cellular growth. Mol Genet Genomics 281:361–373PubMedCrossRefGoogle Scholar
  54. Singh P, Singh SS, Elster J, Mishra AK (2013) Molecular phylogeny, population genetics, and evolution of heterocystous cyanobacteria using nifH gene sequences. Protoplasma 250:751–764PubMedCrossRefGoogle Scholar
  55. Stanier RY, Sistrom WR, Hansen TA, Whitton BA, Castenholz RW, Pfennig N, Gorlenko VN, Kondratieva EN, Eimhjellen KE, Whittenbury R (1978) Proposal to place the nomenclature of the cyanobacteria (blue-green algae) under the rules of the International Code of Nomenclature of Bacteria. Int J Syst Bacteriol 28:335–336CrossRefGoogle Scholar
  56. Swingley WD, Blankenship RE, Raymond J (2008) Integrating Markov clustering and molecular phylogenetics to reconstruct the cyanobacterial species tree from conserved protein families. Mol Biol Evol 25:643–654PubMedCrossRefGoogle Scholar
  57. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefPubMedCentralGoogle Scholar
  58. Turner SE, Pryer KM, Miao VPW, Palmer JD (1999) Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis 1. J Eukaryot Microbiol 46:327–338PubMedCrossRefGoogle Scholar
  59. Wilmotte A, Herdman M (2001) Phylogenetic relationships among the cyanobacteria based on 16S rRNA sequences. In: Boone DR, Castenholz RW, Garrity GM (eds) Bergey’s manual of systematic bacteriology. Springer, New York, pp 487–493Google Scholar
  60. Woese CR (1992) Prokaryote systematics: the evolution of a science. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes. Springer, New York, pp 3–18Google Scholar
  61. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ (2009) A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 462:1056–1060PubMedCrossRefPubMedCentralGoogle Scholar
  62. Yarza P, Ludwig W, Euzeby J, Amann R, Schleifer KH, Glockner FO, Rossello-Mora R (2010) Update of the All-Species Living Tree Project based on 16S and 23S rRNA sequence analyses. Syst Appl Microbiol 33:291–299PubMedCrossRefGoogle Scholar
  63. Zehr JP, Mellon MT, Hiorns WD (1997) Phylogeny of cyanobacterial nifH genes: evolutionary implications and potential applications to natural assemblages. Microbiology 143:1443–1450PubMedCrossRefGoogle Scholar
  64. Zhaxybayeva O, Gogarten JP, Charlebois RL, Doolittle WF, Papke RT (2006) Phylogenetic analyses of cyanobacterial genomes: quantification of horizontal gene transfer events. Genome Res 16:1099–1108PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Mohammad Howard-Azzeh
    • 1
    • 2
  • Larissa Shamseer
    • 2
  • Herb E. Schellhorn
    • 1
  • Radhey S. Gupta
    • 2
    Email author
  1. 1.Department of BiologyMcMaster UniversityHamiltonCanada
  2. 2.Department of Biochemistry and Biomedical SciencesMcMaster UniversityHamiltonCanada

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