Plant Systematics and Evolution

, Volume 197, Issue 1–4, pp 87–98 | Cite as

The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement

  • Vincent Savolainen
  • Philippe Cuénoud
  • Rodolphe Spichiger
  • Maria D. P. Martinez
  • Michèle Crèvecoeur
  • Jean-François Manen


During the last few years we have been confronted with the need to use herbarium specimens in the molecular phylogeny studies, since it is generally difficult to obtain living material of some rare species. Ancient DNA has been sequenced, and there are also reports on successful DNA amplification from herbarium specimens. However, it is not easy to obtain amplified DNA from the first herbarium sample tested. In this paper, experiments are described about trials of DNA amplification from two to 151-year-old herbarium specimens of plant species we needed for our projects. Of the 17 herbarium samples tested only two allowed DNA amplification under standard DNA isolation conditions. Different types of PCR inhibiting activities were demonstrated in DNA extracts. In some of the extracts there was extremely low concentration of template with satisfactory quality. In some instances, PCR inhibiting activities were successfully removed by treating them either with insoluble polyvinylpyrrolidone or by adding bovine serum albumin (BSA) to the amplification mixture. However, some PCR-inhibiting activities were resistant to the treatments described above. When the concentration of template was very low, a second PCR amplification with internal primers was necessary to increase the amount of DNA for sequencing. Nevertheless, contamination of either DNA extract or amplification mixture were sometimes observed, and consequently precautions were taken to minimize them. Finally, successful amplification was obtained in eight samples out of the 17 examined.

Key words

Herbarium specimens molecular phylogenetics ancient DNA PCR techniques 


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  1. Adams, R. P., Miller, J. S., Golenberg, E. M., Adams, J. E., 1994: Conservation of plant genes II: utilization of ancient and modern DNA. — St. Louis: Missouri Botanical Garden.Google Scholar
  2. Gano, R. J., Poinar, H. N., 1993: Rapid isolation of DNA from fossil and museum specimens suitable for PCR. — Biotechniques15: 432–435.Google Scholar
  3. ,Poinar Jr., G. O., 1993: Amplification and sequencing of DNA from a 120–135-million-year-old weevil. — Nature363: 536–538.Google Scholar
  4. Carter, M. J., Milton, I. D., 1993: An inexpensive and simple method for DNA purifications on silica particles. — Nucl. Acids Res.21: 1044.Google Scholar
  5. Chase, M. W., Hills, H. H., 1991: Silica gel: an ideal material for field preservation of leaf samples for DNA studies. — Taxon40: 215–220.Google Scholar
  6. , Soltis, D. E., Olmstead, R. G., Morgan, D., Les, D. H., Mishler, B. D., Duvall, M. R., Price, R. A., Hills, H. G., Qiu, Y. L., Kron, K. A., Rettig, J. H., Conti, E., Palmer, J. D., Manhart, J. R., Sytsma, K. J., Michaels, H. J., Kress, W. J., Karol, K. G., Klark, W. D., Hedren, M., Gaut, B. S., Jansen, R. K., Kim, K. J., Wimpee, C. F., Smith, J. F., Furnier, G. R., Strauss, S. H., Xiang, Q. Y., Plunkett, S. E., Soltis, P. S., Swensen, S. M., Williams, S. E., Gadek, P. A., Quinn, C. J., Eguiarte, L. E., Golenberg, E. M., Learn Jr., G. H.,Graham, S. W., Barrett, S. C., Dayanandan, S., Albert, V. A., 1993: Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid generbcL. — Ann. Missouri Bot. Gard.80: 528–580.Google Scholar
  7. Cooper, A., Mourer-Chauvire, C., Chambers, G. K., von Haeseler, A., Wilson, A. C., Pääbo, S., 1992: Independent origin of New Zealand moas and kiwis. — Proc. Natl. Acad. Sci. USA89: 8741–8744.Google Scholar
  8. Coradin, L., Giannasi, D. L., 1980: The effects of chemical preservations on plant collections to be used in chemotaxonomic surveys. — Taxon29: 33–40.Google Scholar
  9. DeSalle, R., Gatesy, J., Wheeler, W., Grimaldi, D., 1992: DNA sequences from a fossil termite in Oligo-Miocene amber and their phylogenetic implications. — Science257: 1933–1936.Google Scholar
  10. Doyle, J. J., Dickson, E. E., 1987: Preservation of plant samples for DNA restriction endonuclease analysis. — Taxon36: 715–722.Google Scholar
  11. , 1990: Isolation of plant DNA from fresh tissue. — Focus12: 13–15.Google Scholar
  12. Eglington, G., Logan, G. A., 1991: Molecular preservation. — Philos. Trans. Roy. Soc. London B333: 315–328.Google Scholar
  13. Golenberg, E. M., 1991: Amplification and analysis of Miocene plant fossil DNA. — Philos. Trans. Roy. Soc. London B333: 419–427.Google Scholar
  14. , 1990: Chloroplast DNA sequence from a MioceneMagnolia species. — Nature344: 656–658.Google Scholar
  15. Gustincich, S., Manfioletti, G., Del Sal, G., Schneider, C., Carninci, P., 1991: A fast method for high-quality genomic DNA extraction from whole human blood. — Biotechniques11: 298–302.Google Scholar
  16. Hagelberg, E., Clegg, J. B., 1993: Genetic polymorphism in prehistoric pacific islanders determined by analysis of ancient bone DNA. — Proc. Roy Soc. London B252: 163–170.Google Scholar
  17. Harris, S. A., 1993: DNA analysis of tropical plant species: an assessment of different drying methods. — Pl. Syst. Evol.188: 57–64.Google Scholar
  18. Höss, M., Pääbo, S., 1993: DNA extraction from pleistocene bones by silica-based purification method. — Nucl. Acids Res.21: 3913–3914.Google Scholar
  19. Janczewski, D. J., Yuhki, N., Gilbert, D. A., Jefferson, G. T., O'Brien, S. J., 1992: Molecular phylogenetic inference from sabre-toothed cat fossils of Rancho La Brea. — Proc. Natl. Acad. Sci. USA89: 9769–9773.Google Scholar
  20. Lewin, R., 1994: Fact, fiction and fossil DNA. — New Scientist29: 38–41.Google Scholar
  21. Lindahl, T., 1993: Instability and decay of the primary structure of DNA. — Nature362: 709–715.Google Scholar
  22. Loockermann, D. J., Jansen, R. K., 1995: The use of herbarium material for DNA studies. — InStuessy, T. F., (Ed.): Sampling the green world. — Columbia Univ. Press. (In press.)Google Scholar
  23. Loomis, W. D., Bataille, J., 1966: Plant phenolic compounds and the isolation of plant enzymes. — Phytochemistry5: 423–438.Google Scholar
  24. McKersie, B. D., Senaratna, T., Walker, M. A., Kendall, E. J., Hetherington, P. R., 1988: Deterioration of the membrane during aging in plants: evidence for free radical mediation. — InNoodeon, L. D., Leopold, A. C., (Eds): Senescence and aging in plants, pp. 441–464. — San Diego: Academic Press.Google Scholar
  25. Pääbo, S., 1989: Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. — Proc. Natl. Acad. Sci. USA86: 1939–1943.Google Scholar
  26. , 1991: Amplifying DNA from archeological remains: a meeting report. — PCR Methods & Protocols1: 107–110.Google Scholar
  27. , 1989: Ancient DNA and the polymerase chain reaction. — J. Biol. Chem.264: 9709–9712.Google Scholar
  28. Pyle, M. M., Adams, R. P., 1989: In situ preservation of DNA in plant specimens. — Taxon38: 576–581.Google Scholar
  29. Reynolds, R. S., 1963: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. — J. Cell Biol.17: 208–212.Google Scholar
  30. Sarkar, G., Sommer, S. S., 1990: Shedding light on PCR contamination. — Nature343: 27.Google Scholar
  31. Savolainen, V., Mannen, J. F., Douzery, E., Spichiger, R., 1994: Molecular phylogeny of families related toCelastrales based on rbcL 5′ flanking sequences. — Mol. Phyl. Evol.3: 27–37.Google Scholar
  32. Scott, N. E., Possingham, J. V., 1983: Changes in chloroplast DNA levels during growth of spinach leaves. — J. Exp. Bot.34: 1756–1767.Google Scholar
  33. Soltis, P. S., Soltis, D. E., Smiley, C. J., 1992: An rbcL sequence from a MioceneTaxodium (bald cypress). — Proc. Natl. Acad. Sci. USA89: 449–451.Google Scholar
  34. Spichiger, R., Savolainen, V., Manen, J. F., 1993: Systematic affinities ofAquifoliaceae andIcacinaceae from molecular data analysis. — Candollea48: 459–464.Google Scholar
  35. Sytsma, K. J., Givnish, T. J., Smith, J. F., Hahn, W. J., 1993: Collection and storage of land plant samples for macromolecular comparisons. — Methods Enzymol.224: 23–37.Google Scholar
  36. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J., 1991: Universal primers for amplification of three non-coding regions of chloroplast DNA. — Pl. Mol. Biol.17: 1105–1109.Google Scholar
  37. Taylor, J. W., Swann, E. C., 1994: DNA from herbarium specimens. — InHerrmann, B., Hummel, S., (Eds): Ancient DNA, pp. 167–181. — Berlin: Springer.Google Scholar
  38. Thomson, D., Henry, R., 1993: Use of DNA from dry leaves fro PCR and RAPD analysis. — Pl. Mol. Biol. Rep.11: 202–206.Google Scholar
  39. Webb, D. M., Knapp, S. J., 1990: DNA extraction from previously recalcitrant plant genus. — Pl. Mol. Biol.8: 180–185.Google Scholar
  40. Weber, A. W., 1982: Mnemonic three-letter acronyms for the families of vascular plants: a device for more effective herbarium curation. — Taxon31: 74–88.Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Vincent Savolainen
    • 1
  • Philippe Cuénoud
    • 1
  • Rodolphe Spichiger
    • 1
  • Maria D. P. Martinez
    • 2
    • 3
  • Michèle Crèvecoeur
    • 4
  • Jean-François Manen
    • 1
  1. 1.Conservatoire et Jardin Botaniques de GenéveChambésySwitzerland
  2. 2.Conservatoire et Jardin Botaniques de GenèveChambésySwitzerland
  3. 3.Centro de Estudios Farmacológicos y Botánicos (CEFyBO), ConicetBuenos AiresArgentina
  4. 4.Laboratoire de Biochimie et Physiologie VégétalesUniversité de GenèveGenevaSwitzerland

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