Journal of Molecular Evolution

, Volume 45, Issue 2, pp 168–177 | Cite as

Ribosomal DNA and ITS-2 sequence comparisons as a tool for predicting genetic relatedness

  • Annette W. Coleman
  • Jeffrey C. Mai


The determination of the secondary structure of the internal transcribed spacer (ITS) regions separating nuclear ribosomal RNA genes of Chlorophytes has improved the fidelity of alignment of nuclear ribosomal ITS sequences from related organisms. Application of this information to sequences from green algae and plants suggested that a subset of the ITS-2 positions is relatively conserved. Organisms that can mate are identical at all of these 116 positions, or differ by at most, one nucleotide change. Here we sequenced and compared the ITS-1 and ITS-2 of 40 green flagellates in search of the nearest relative to Chlamydomonas reinhardtii. The analysis clearly revealed one unique candidate, C. incerta. Several ancillary benefits of the analysis included the identification of mislabelled cultures, the resolution of confusion concerning C. smithii, the discovery of mis-identified sequences in GenBank derived from a green algal contaminant, and an overview of evolutionary relationships among the Volvocales, which is congruent with that derived from rDNA gene sequence comparisons but improves upon its resolution. The study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequences, are most useful in systematic and other studies.

Key words

Chlamydomonas Ribosomal RNA-transcribed spacer 


  1. Baldwin B, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS, Donoghue MJ (1995) The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann MO Bot Gard 82:247–277CrossRefGoogle Scholar
  2. Boudreau E, Turmel M (1996) Extensive gene rearrangements in the chloroplast DNAs of Chlamydomonas species featuring multiple dispersed repeats. Mol Biol Evol 13:233–243PubMedGoogle Scholar
  3. Buchheim MA, Chapman RL (1991) Phylogeny of the colonial green flagellates: a study of 18S and 26S rRNA sequence data. BioSystems 25:85–100PubMedCrossRefGoogle Scholar
  4. Buchheim MA, Chapman RL (1992) Phylogeny of Carteria (Chlorophyceae) inferred from molecular and organismal data. J Phycol 28:362–374CrossRefGoogle Scholar
  5. Buchheim MA, Lemieux C, Otis C, Gutell RR, Chapman RL, Turmel M (1996) Phylogeny of the Chlamydomonadales (Chlorophyceae):a comparison of ribosomal RNA gene sequences from the nucleus and the chloroplast. Mol Phylogenet Evol 5:391–402PubMedCrossRefGoogle Scholar
  6. Buchheim MA, Turmel M, Zimmer EA, Chapman RL (1990) Phylo-genetic systematics of Chlamydomonas: an investigation based on cladistic analysis of nuclear 18S rRNA sequence data. J Phycol 26:689–699CrossRefGoogle Scholar
  7. Burrascano CG, VanWinkle-Swift KP (1984) Interspecific matings of five Chlamydomonas species. Genetics 107:sl5Google Scholar
  8. Coleman AW (1996a) The Indian connection, crucial to reconstruction of the historical biogeography of freshwater algae: examples among Volvocaceae (Chlorophyta). Nova Hedwigia 112:477–488Google Scholar
  9. Coleman AW (1996b) Are the impacts of events in the earth’s history discernable in the current distributions of freshwater algae? Hydrobiologia 336:137–142Google Scholar
  10. Coleman AW, Suarez A, Goff LJ (1994) Molecular delineation of species and syngens in volvocacean green algae (Chlorophyta). J Phycol 30:80–90CrossRefGoogle Scholar
  11. Ettl H (1976) Die Gattung Chlamydomonas Ehrenberg. Beih Nova Hedwigia 49:1–1122Google Scholar
  12. Friedl T (1996) Evolution of the polyphyletic genus Pleurastrum (Chlorophyta): inferences from nuclear-encoded ribosomal DNA sequences and motile cell ultrastructure. Phycologia 35:456–469Google Scholar
  13. Gillham NW, Boynton JE, Harris EH (1987) Specific elimination of mitochondrial DNA from Chlamydomonas by intercalating dyes. Curr Genet 12:41–47PubMedCrossRefGoogle Scholar
  14. Goff LJ, Moon DA (1993) PCR amplification of nuclear and plastid genes from algal herbarium specimens and algal spores. J Phycol 29:381–384CrossRefGoogle Scholar
  15. Goodenough U, Ferris P, Woessner J, Armbrust EV (1996) Genes of the mating type locus in Chlamydomonas. J Phycol 32:sl9Google Scholar
  16. Gross CH, Ranum LPW, Lefebvre PA (1988) Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii. Curr. Genet 13:503–508Google Scholar
  17. Harris EH (1989) The Chlamydomonas sourcebook. Academic Press, Inc., New York pp 780.Google Scholar
  18. Harris EH, Boynton JE, Gillham NW, Burkhart BD, Newman SM (1991) Chloroplast genome organization in Chlamydomonas. Archiv Protistenkd 139:183–192Google Scholar
  19. Hershkovitz MA, Zimmer EA (1996) Conservation patterns in angiosperm rDNA ITS2 sequences. Nuc Acids Res 24:2857–2867CrossRefGoogle Scholar
  20. Hershkovitz MA, Lewis LA (1996) Deep-level diagnostic value of the rDNA-ITS region. Mol Bio Evol 13:1276–1295Google Scholar
  21. Hoshaw RW (1965) Mating types of Chlamydomonas from the collection of Gilbert M. Smith. J Phycol 1:194–196CrossRefGoogle Scholar
  22. Hoshaw RW, Ettl H (1966) Chlamydomonas smithii sp. nov.-a chlamydomonad interfertile with Chlamydomonas reinhardtii. J Phycol 2:93–96CrossRefGoogle Scholar
  23. Larson A, Kirk MM, Kirk DL (1992) Molecular phylogeny of the volvocine flagellates. Mol Biol Evol 9:85–105PubMedGoogle Scholar
  24. Liss M, Kirk DL, Beyser K, Fabry S (1997) Intron sequences provide a tool for high resolution phylogenetic analysis of Volvocine algae. Curr Genet 31:214–227PubMedCrossRefGoogle Scholar
  25. Mai JC, Coleman AW (1997) The internal transcribed spacer 2 exhibits a common secondary structure in green algae and flowering plants. J Mol Evol 44:258–271PubMedCrossRefGoogle Scholar
  26. Rausch H, Larsen N, Schmitt R (1989) Phylogenetic relationships of the green alga Volvox carteri deduced from small-subunit ribosomal RNA comparisons. J Mol Evol 29:255–265PubMedCrossRefGoogle Scholar
  27. Ritland CE, Straus NA (1993) High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae). Plant Mol Biol 22:691–696PubMedCrossRefGoogle Scholar
  28. Ritland CE, Ritland K, Straus NA (1993) Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex. Mol Biol Evol 10:1273–1288PubMedGoogle Scholar
  29. Rumpf R, Vernon D, Schreiber D, Birky, Jr C (1996) Evolutionary consequences of the loss of photosynthesis in Chlamydomonadaceae: phylogenetic analysis of Rrn 18 (18S rDNA) in 13 Polytoma strains (Chlorophyta). J Phycol 32:119–126CrossRefGoogle Scholar
  30. Sack L, Zeyl C, Bell G, Sharbel T, Reboud X, Bernhardt T, Koelewyn H (1994) Isolation of four new strains of Chlamydomonas reinhardtii (Chlorophyta) from soil samples. J Phycol 30:770–773CrossRefGoogle Scholar
  31. Schlosser UG (1984) Species-specific sporangium autolysins (cell-wall-dissolving enzymes) in the genus Chlamydomonas. In: Irvine, DEG, John, DM (eds) Systematics of the green algae. Academic Press, New York, pp 409–418Google Scholar
  32. Spanier JG, Graham JE, Jarvik JW (1992) Isolation and preliminary characterization of three Chlamydomonas strains interfertile with Chlamydomonas reinhardtii (Chlorophyta). J Phycol 28:822–828CrossRefGoogle Scholar
  33. Starr RC, Marner FJ, Jaenicke L (1995) Chemoattraction of male gametes by a pheromone produced by female gametes of Chlamydomonas. Proc Nat Acad Sci USA 92:641–645PubMedCrossRefGoogle Scholar
  34. Starr RC, Zeikus JA (1993) UTEX-the culture collection of algae at the University of Texas at Austin. J Phycol 29(Suppl): 1–106CrossRefGoogle Scholar
  35. Swofford DL (1993) PAUP: Phylogenetic analysis using parsimony. Version 3.1.1 Computer program distributed by the Illinois Natural History Survey, Champaign, ILGoogle Scholar
  36. Turmel M, Gutell RR, Mercier J-P, Otis C, Lemieux C (1993) Analysis of the chloroplast large subunit ribosomal RNA gene from 17 Chlamydomonas taxa. J Mol Biol 232:446–467PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag New York Inc 1997

Authors and Affiliations

  • Annette W. Coleman
    • 1
  • Jeffrey C. Mai
    • 1
  1. 1.Department of Molecular and Cellular Biology and BiochemistryBrown UniversityProvidenceUSA

Personalised recommendations