Organisms Diversity & Evolution

, Volume 13, Issue 1, pp 1–13 | Cite as

Divergence time estimation in Cichorieae (Asteraceae) using a fossil-calibrated relaxed molecular clock

  • Karin Tremetsberger
  • Birgit Gemeinholzer
  • Holger Zetzsche
  • Stephen Blackmore
  • Norbert Kilian
  • Salvador Talavera
Original Article


Knowing the age of lineages is key to understanding their biogeographic history. We aimed to provide the best estimate of the age of Cichorieae and its subtribes based on available fossil evidence and DNA sequences and to interpret their biogeography in the light of Earth history. With more than 1,550 species, the chicory tribe (Cichorieae, Asteraceae) is distributed predominantly in the northern Hemisphere, with centres of distribution in the Mediterranean region, central Asia, and SW North America. Recently, a new phylogenetic hypothesis of Cichorieae based on ITS sequences has been established, shedding new light on phylogenetic relationships within the tribe, which had not been detected so far. Cichorieae possess echinolophate pollen grains, on the surface of which cavities (lacunae) are separated by ridges. These lacunae and ridges show patterns characteristic of certain groups within Cichorieae. Among the fossil record of echinolophate pollen, the Cichorium intybus-type is the most frequent and also the oldest type (22 to 28.4 million years old). By using an uncorrelated relaxed molecular clock approach, the Cichorieae phylogenetic tree was calibrated with this fossil find. According to the analysis, the tribe originated no later than Oligocene. The species-rich core group originated no later than Late Oligocene or Early Miocene and its subtribes diversified no later than Middle/Late Miocene or Early Pliocene—an eventful period of changing geological setting and climate in the Mediterranean region and Eurasia. The first dispersal from Eurasia to North America, which resulted in the radiation of genera and species in North America (subtribe Microseridinae), also occurred no later than Middle or Late Miocene, suggesting the Bering land bridge as the route of dispersal.


Bering land bridge Lactuceae Miocene Oligocene Pollen evolution Uncorrelated relaxed molecular clock 



We thank P. Hochuli (Zurich) for sharing information on Cichoraearumpollenites, S. Ho (Sydney) and G. Schneeweiss (Vienna) for help with the program BEAST, and A. Wortley (Edinburgh) for helpful discussion on Cichorieae pollen. The comments of two anonymous reviewers greatly improved the manuscript. We also acknowledge a Juan de la Cierva fellowship of the Ministerio de Educación y Ciencia (Spain) and the financial support of the European Commission’s Research Infrastructure Action via the SYNTHESYS Project (both to K.T.).


  1. Álvarez, I., & Wendel, J. F. (2003). Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution, 29, 417–434.PubMedCrossRefGoogle Scholar
  2. Babcock, E. B. (1947). The genus Crepis: part one. The taxonomy, phylogeny, distribution, and evolution of Crepis. Berkeley: University of California Press.Google Scholar
  3. Baldwin, B. G., Sanderson, M. J., Porter, J. M., Wojciechowski, M. F., Campbell, C. S., & Donoghue, M. J. (1995). The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden, 82, 247–277.CrossRefGoogle Scholar
  4. Blackmore, S. (1981). Palynology and intergeneric relationships in subtribe Hyoseridinae (Compositae: Lactuceae). Botanical Journal of the Linnean Society, 82, 1–13.CrossRefGoogle Scholar
  5. Blackmore, S. (1982a). The apertures of Lactuceae (Compositae) pollen. Pollen et Spores, 24, 453–462.Google Scholar
  6. Blackmore, S. (1982b). A functional interpretation of Lactuceae (Compositae) pollen. Plant Systematics and Evolution, 141, 153–168.CrossRefGoogle Scholar
  7. Blackmore, S. (1982c). Palynology of subtribe Scorzonerinae (Compositae: Lactuceae) and its taxonomic significance. Grana, 21, 149–160.CrossRefGoogle Scholar
  8. Blackmore, S. (1984). The Northwest European Pollen Flora, 32: Compositae—Lactuceae. Review of Palaeobotany and Palynology, 42, 45–85.CrossRefGoogle Scholar
  9. Blackmore, S. (1986). The identification and taxonomic significance of lophate pollen in the Compositae. Canadian Journal of Botany, 64, 3101–3112.CrossRefGoogle Scholar
  10. Blackmore, S., Van Campo, E., & Crane, P. R. (1986). Lophate Compositae pollen from the Miocene and Pliocene of the Mediterranean region. Pollen et Spores, 28, 391–401.Google Scholar
  11. Blattner, F. R. (1999). Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. BioTechniques, 27, 1180–1186.PubMedGoogle Scholar
  12. Bremer, K. (1994). Asteraceae: cladistics and classification. Portland: Timber.Google Scholar
  13. Bremer, K., Friis, E. M., & Bremer, B. (2004). Molecular phylogenetic dating of asterid flowering plants shows early Cretaceous diversification. Systematic Biology, 53, 496–505.PubMedCrossRefGoogle Scholar
  14. Demarcq, G., Méon-Vilain, H., Miquet, R., & Kujawski, H. (1976). Un bassin paralique Néogène: celui de Skanes-Monastir (Tunisie orientale). Notes du Service Géologique de Tunisie, 42, 97–147.Google Scholar
  15. Donoghue, M. J., Bell, C. D., & Li, J. (2001). Phylogenetic patterns in northern Hemisphere plant geography. International Journal of Plant Sciences, 162(6 Suppl.), S41–S52.CrossRefGoogle Scholar
  16. Drummond, A. J., Ho, S. Y. W., Phillips, M. J., & Rambaut, A. (2006). Relaxed phylogenetics and dating with confidence. PLoS Biology, 4, e88.PubMedCrossRefGoogle Scholar
  17. Drummond, A. J., & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7, 214.PubMedCrossRefGoogle Scholar
  18. Drummond, A. J., Rambaut, A., & Suchard, M. A. (2002–2010). BEAST, Version 1.6.1, available from
  19. Drummond, A. J., Rambaut, A., & Xie, W. (2002–2010). BEAUti, Version 1.6.1, available from
  20. Eldenäs, P., Anderberg, A. A., & Källersjö, M. (1998). Molecular phylogenetics of the tribe Inuleae s. str. (Asteraceae), based on ITS sequences of nuclear ribosomal DNA. Plant Systematics and Evolution, 210, 159–173.Google Scholar
  21. Enke, N., & Gemeinholzer, B. (2008). Babcock revisited: new insights into generic delimitation and character evolution in Crepis L. (Compositae: Cichorieae) from ITS and matK sequence data. Taxon, 57, 756–768.Google Scholar
  22. Fehrer, J., Gemeinholzer, B., Chrtek, J., Jr., & Bräutigam, S. (2007). Incongruent plastid and nuclear DNA phylogenies reveal ancient intergenic hybridization in Pilosella hawkweeds (Hieracium, Cichorieae, Asteraceae). Molecular Phylogenetics and Evolution, 42, 347–361.PubMedCrossRefGoogle Scholar
  23. Funk, V. A., Bayer, R. J., Keeley, S., Chan, R., Watson, L., Gemeinholzer, B., et al. (2005). Everywhere but Antarctica: using a supertree to understand the diversity and distribution of the Compositae. Biologiske Skrifter, 55, 343–374.Google Scholar
  24. Funk, V. A., Chan, R., & Keeley, S. C. (2004). Insights into the evolution of the tribe Arctoteae (Compositae: subfamily Cichorioideae s.s.) using trnL-F, ndhF, and ITS. Taxon, 53, 637–655.CrossRefGoogle Scholar
  25. Garnatje, T., Susanna, A., Garcia-Jacas, N., Vilatersana, R., & Vallès, J. (2005). A first approach to the molecular phylogeny of the genus Echinops (Asteraceae): Sectional delimitation and relationships with the genus Acantholepis. Folia Geobotanica, 40, 407–419.CrossRefGoogle Scholar
  26. Gemeinholzer, B., & Bachmann, K. (2005). Examining morphological and molecular diagnostic character states in Cichorium intybus L. (Asteraceae) and Cichorium spinosum L. Plant Systematics and Evolution, 253, 105–123.CrossRefGoogle Scholar
  27. Gemeinholzer, B., Dröge, G., Zetzsche, H., Haszprunar, G., Klenk, H.-P., Güntsch, A., et al. (2011). The DNA Bank Network: the start from a German initiative. Biopreservation and Biobanking, 9, 51–55.CrossRefGoogle Scholar
  28. Goertzen, L. R., Cannone, J. J., Gutell, R. R., & Jansen, R. K. (2003). ITS secondary structure derived from comparative analysis: implications for sequence alignment and phylogeny of the Asteraceae. Molecular Phylogenetics and Evolution, 29, 216–234.PubMedCrossRefGoogle Scholar
  29. Graur, D., & Martin, W. (2004). Reading the entrails of chickens: molecular timescales of evolution and the illusion of precision. Trends in Genetics, 20, 80–86.PubMedCrossRefGoogle Scholar
  30. Gustaffson, M. H. G., Pepper, A. S.-R., Albert, V. A., & Källersjö, M. (2001). Molecular phylogeny of the Barnadesioideae (Asteraceae). Nordic Journal of Botany, 21, 149–160.CrossRefGoogle Scholar
  31. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.Google Scholar
  32. Heads, M. (2011). Old taxa on young islands: a critique of the use of island age to date island-endemic clades and calibrate phylogenies. Systematic Biology, 60, 204–218.PubMedCrossRefGoogle Scholar
  33. Hochuli, P. A. (1978). Palynologische Untersuchungen im Oligozän und Untermiozän der Zentralen und Westlichen Paratethys. Beiträge zur Paläontologie von Österreich, 4, 1–132.Google Scholar
  34. Hugall, A. F., Foster, R., & Lee, M. S. Y. (2007). Calibration choice, rate smoothing, and the pattern of tetrapod diversification according to the long nuclear gene RAG-1. Systematic Biology, 56, 543–563.PubMedCrossRefGoogle Scholar
  35. Ivanov, D. A. (1997). Miocene palynomorphs from the Southern part of the Forecarpathian basin (Northwest Bulgaria). Flora Tertiaria Mediterranea, 6, 1–81.Google Scholar
  36. Ivanov, D. A., & Slavomirova, E. (2000). New palynological data on the Late Miocene flora and vegetation in Gotse-Delchev Basin (Southwestern Bulgaria). Review of the Bulgarian Geological Society, 61, 39–46.Google Scholar
  37. Karis, P. O., Eldenäs, P., & Källersjö, M. (2001). New evidence for the systematic position of Gundelia L. with notes on delimitation of Arctoteae (Asteraceae). Taxon, 50, 105–114.CrossRefGoogle Scholar
  38. Kay, K. M., Whittall, J. B., & Hodges, S. A. (2006). A survey of nuclear ribosomal internal transcribed spacer substitution rates across angiosperms: an approximate molecular clock with life history effects. BMC Evolutionary Biology, 6, 36.PubMedCrossRefGoogle Scholar
  39. Kilian, N., Gemeinholzer, B., & Lack, H. W. (2009). Tribe Cichorieae Lam. & DC. (1806). In V. A. Funk, A. Susanna, T. Stuessy, & R. J. Bayer (Eds.), Systematics, evolution, and biogeography of Compositae (pp. 343–383). Vienna: International Association for Plant Taxonomy.Google Scholar
  40. Kim, S.-C., Crawford, D. J., Francisco-Ortega, J., & Santos-Guerra, A. (1996). A common origin for woody Sonchus and five related genera in the Macaronesian islands: molecular evidence for extensive radiation. Proceedings of the National Academy of Sciences USA, 93, 7743–7748.CrossRefGoogle Scholar
  41. Kimball, R. T., & Crawford, D. J. (2004). Phylogeny of Coreopsideae (Asteraceae) using ITS sequences suggests lability in reproductive characters. Molecular Phylogenetics and Evolution, 33, 127–139.PubMedCrossRefGoogle Scholar
  42. Lavin, M., Herendeen, P. S., & Wojciechowski, M. F. (2005). Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the Tertiary. Systematic Biology, 54, 575–594.PubMedCrossRefGoogle Scholar
  43. Lee, J., Baldwin, B. G., & Gottlieb, L. D. (2002). Phylogeny of Stephanomeria and related genera (Compositae–Lactuceae) based on analysis of 18 S–26S nuclear rDNA ITS and ETS sequences. American Journal of Botany, 89, 160–168.PubMedCrossRefGoogle Scholar
  44. Lee, J., Baldwin, B. G., & Gottlieb, L. D. (2003). Phylogenetic relationships among the primarily North American genera of Cichorieae (Compositae) based on analysis of 18 S-26S nuclear rDNA ITS and ETS sequences. Systematic Botany, 28, 616–626.Google Scholar
  45. Mai, D. H. (1995). Tertiäre Vegetationsgeschichte Europas. Jena: Fischer.Google Scholar
  46. Marincovich, L., & Gladenkov, A. Y. (1999). Evidence for an early opening of the Bering Strait. Nature, 397, 149–151.CrossRefGoogle Scholar
  47. Marjanović, D., & Laurin, M. (2007). Fossils, molecules, divergence times, and the origin of lissamphibians. Systematic Biology, 56, 369–388.PubMedCrossRefGoogle Scholar
  48. Mavrodiev, E. V., Edwards, C. E., Albach, D. C., Gitzendanner, M. A., Soltis, P. S., & Soltis, D. E. (2004). Phylogenetic relationships in subtribe Scorzonerinae (Asteraceae: Cichorioideae: Cichorieae) based on ITS sequence data. Taxon, 53, 699–712.CrossRefGoogle Scholar
  49. Muller, J. (1981). Fossil pollen records of extant angiosperms. Botanical Review, 47, 1–146.CrossRefGoogle Scholar
  50. Nagy, E. (1969). Palynological elaborations on the Miocene layers of the Mecsek Mountains. Annales Instituti Geologici Publici Hungarici, 52, 287–648.Google Scholar
  51. Panero, J. L., & Funk, V. A. (2002). Toward a phylogenetic subfamilial classification for the Compositae (Asteraceae). Proceedings of the Biological Society of Washington, 115, 909–922.Google Scholar
  52. Pérez-Losada, M., Høeg, J. T., & Crandall, K. A. (2004). Unraveling the evolutionary radiation of the thoracican barnacles using molecular and morphological evidence: a comparison of several divergence time estimation approaches. Systematic Biology, 53, 244–264.PubMedCrossRefGoogle Scholar
  53. Posada, D., & Crandell, K. A. (1998). Modeltest: testing the model of DNA substitution. Bioinformatics, 14, 817–818.PubMedCrossRefGoogle Scholar
  54. Potts, R., & Behrensmeyer, A. K. (1992). Late Cenozoic terrestrial ecosystems. In A. K. Behrensmeyer, J. D. Damuth, W. A. DiMichele, R. Potts, H.-D. Sues, & S. L. Wing (Eds.), Terrestrial ecosystems through time (pp. 419–541). Chicago: The University of Chicago Press.Google Scholar
  55. Poux, C., Chevret, P., Huchon, D., De Jong, W. W., & Douzery, E. J. P. (2006). Arrival and diversification of caviomorph rodents and platyrrhine primates in South America. Systematic Biology, 55, 228–244.PubMedCrossRefGoogle Scholar
  56. Rambaut, A., & Drummond, A. J. (2003–2009). Tracer, Version 1.5.0, available from
  57. Rambaut, A., & Drummond, A. J. (2002–2010). TreeAnnotator, Version 1.6.1, available from
  58. Renner, S., & Zhang, L.-B. (2004). Biogeography of the Pistia clade (Araceae): based on chloroplast and mitochondrial DNA sequences and Bayesian divergence time inference. Systematic Biology, 53, 422–432.PubMedCrossRefGoogle Scholar
  59. Renner, S. S. (2005). Relaxed molecular clocks for dating historical plant dispersal events. Trends in Plant Science, 10, 550–558.PubMedCrossRefGoogle Scholar
  60. Rieseberg, L. H., & Soltis, D. E. (1991). Phylogenetic consequences of cytoplasmic gene flow in plants. Evolutionary Trends in Plants, 5, 65–84.Google Scholar
  61. Rivas-Carballo, M. R., Alonso-Gavilán, G., Valle, M. F., & Civis, J. (1994). Miocene palynology of the central sector of the Duero basin (Spain) in relation to palaeogeography and palaeoenvironment. Review of Palaeobotany and Palynology, 82, 251–264.CrossRefGoogle Scholar
  62. Robinson, H. (1994). Notes on the tribes Eremothamneae, Gundelieae, and Moquinieae, with comparisons of their pollen. Taxon, 43, 33–44.CrossRefGoogle Scholar
  63. Samuel, R., Gutermann, W., Stuessy, T. F., Ruas, C. F., Lack, H.-W., Tremetsberger, K., et al. (2006). Molecular phylogenetics reveals Leontodon (Asteraceae, Lactuceae) to be diphyletic. American Journal of Botany, 93, 1193–1205.PubMedCrossRefGoogle Scholar
  64. Samuel, R., Stuessy, T. F., Tremetsberger, K., Baeza, C. M., & Siljak-Yakovlev, S. (2003). Phylogenetic relationships among species of Hypochaeris (Asteraceae, Cichorieae) based on ITS, plastid trnL intron, trnL-F spacer, and matK sequences. American Journal of Botany, 90, 496–507.PubMedCrossRefGoogle Scholar
  65. Skvarla, J. J., & Larson, D. A. (1965). An electron microscopic study of pollen morphology in the Compositae with special reference to the Ambrosiinae. Grana Palynologica, 6, 210–269.CrossRefGoogle Scholar
  66. Susanna, A., Garcia-Jacas, N., Hidalgo, O., Vilatersana, R., & Garnatje, T. (2006). The Cardueae (Compositae) revisited: insights from ITS, trnL-trnF and matK nuclear and chloroplast DNA analysis. Annals of the Missouri Botanical Garden, 93, 150–171.CrossRefGoogle Scholar
  67. Swofford, D. L. (2003). PAUP*: Phylogenetic Analyses Using Parsimony (*and Other Methods), Version 4.0b10. Sunderland: Sinauer.Google Scholar
  68. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882.PubMedCrossRefGoogle Scholar
  69. Tiffney, B. H., & Manchester, S. R. (2001). The use of geological and paleontological evidence in evaluating plant phylogeographic hypotheses in the northern Hemisphere Tertiary. International Journal of Plant Sciences, 162(6 Suppl.), S3–S17.CrossRefGoogle Scholar
  70. Tomb, A. S. (1975). Pollen morphology in tribe Lactuceae (Compositae). Grana, 15, 79–89.Google Scholar
  71. Tremetsberger, K., Weiss-Schneeweiss, H., Stuessy, T., Samuel, R., Kadlec, G., Ortiz, M. Á., & Talavera, S. (2005). Nuclear ribosomal DNA and karyotypes indicate a NW African origin of South American Hypochaeris (Asteraceae, Cichorieae). Molecular Phylogenetics and Evolution, 35, 102–116.PubMedCrossRefGoogle Scholar
  72. Wen, J. (1999). Evolution of eastern Asian and eastern North American disjunct distributions in flowering plants. Annual Review of Ecology and Systematics, 30, 421–455.CrossRefGoogle Scholar
  73. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: a guide to methods and applications (pp. 315–322). San Diego: Academic.Google Scholar
  74. Whitton, J., Wallace, R. S., & Jansen, R. K. (1994). Phylogenetic relationships and patterns of character change in the tribe Lactuceae (Asteraceae) based on chloroplast DNA restriction site variation. Canadian Journal of Botany, 73, 1058–1073.CrossRefGoogle Scholar
  75. Wodehouse, R. P. (1935). Pollen grains: their structure, identification and significance in science and medicine. New York: McGraw-Hill.Google Scholar
  76. Won, H., & Renner, S. S. (2006). Dating dispersal and radiation in the gymnosperm Gnetum (Gnetales)—clock calibration when outgroup relationships are uncertain. Systematic Biology, 55, 610–622.PubMedCrossRefGoogle Scholar
  77. Xiang, Q.-Y., Soltis, D. E., Soltis, P. S., Manchester, S. R., & Crawford, D. J. (2000). Timing the eastern Asian-eastern North American floristic disjunction: molecular clock corroborates paleontological estimates. Molecular Phylogenetics and Evolution, 15, 462–472.PubMedCrossRefGoogle Scholar
  78. Zhao, Z., Skvarla, J. J., & Jansen, R. K. (2006). Mutisieae (Asteraceae) pollen ultrastructure atlas. Lundellia, 9, 51–76.Google Scholar

Copyright information

© Gesellschaft für Biologische Systematik 2012

Authors and Affiliations

  • Karin Tremetsberger
    • 1
    • 2
  • Birgit Gemeinholzer
    • 3
    • 4
  • Holger Zetzsche
    • 3
  • Stephen Blackmore
    • 5
  • Norbert Kilian
    • 3
  • Salvador Talavera
    • 2
  1. 1.Institute of Botany, Department of Integrative Biology and Biodiversity ResearchUniversity of Natural Resources and Life SciencesViennaAustria
  2. 2.Departamento de Biología Vegetal y Ecología, Facultad de BiologíaUniversidad de SevillaSevilleSpain
  3. 3.Botanic Garden and Botanical Museum Berlin-DahlemFreie Universität BerlinBerlinGermany
  4. 4.Justus-Liebig-Universität GießenAG Spezielle BotanikGießenGermany
  5. 5.Royal Botanic Garden EdinburghEdinburghUK

Personalised recommendations