Skip to main content

Genome size variation and morphological differentiation within Ranunculus parnassifolius group (Ranunculaceae) from calcareous screes in the Northwest of Spain

Abstract

Ranunculus parnassifolius is an orophilous plant distributed throughout Central and Southwestern Europe (Alps, Pyrenees and Cantabrian Mountains). Its evolutionary history and taxonomy are often complicated, having been little studied before now. The purpose of this article is to present flow cytometry measurements and multivariate morphometric analyses to ascertain cytotype distribution patterns and the morphological differentiation of R. parnassifolius s.l. from calcareous screes in the Northwest of Spain. DNA ploidy level and morphometric analysis were determined for plants of R. parnassifolius s.l. using flow cytometry (112 individuals) and multivariate analysis (152 individuals). Specimens were collected in eight localities in the Northwest of the Iberian Peninsula. Different sample preservation methods (fresh, frozen, and herbarium specimens) were employed as well as the use of various buffers and internal standards, in order to test the reproducibility of DNA flow cytometry. Three ploidy levels were detected in the study area (diploid, tetraploid, and pentaploid), and mixed-cytotype populations were also found. The mean nuclear DNA content of the R. parnassifolius group ranged from 7.43 ± 0.185 to 7.63 ± 0.339 pg/2C in diploids and from 15.09 ± 0.161 to 15.85 ± 0.587 pg/2C in tetraploids. The analysis of the monoploid genome sizes (1Cx) did not reveal a clear difference among cytotypes. These results suggest low intraspecific variation, at least among the populations studied. In addition, a comparison of different DNA reference standards was conducted. A new value for the chicken genome size was used as internal reference standard (2C = 3.14 ± 0.155 pg), with similar results found using both animal and plant standards (Pisum sativum and Solanum lycopersicum). Finally, herbarium vouchers and frozen tissue were proved to be suitable for DNA ploidy level measurements. This study provided a first assessment of C values in the R. parnassifolius group using flow cytometry. The weak morphological distinction of the cytotypes and the existence of mixed-cytotype populations in the Northwest of Spain are reported here for the first time. The different distribution pattern of the two cytotypes is discussed.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Bennett MD, Leitch IJ (2004) Angiosperm DNA C-values database (release 5.0, Dec. 2004). http://www.kew.org/cvalues/homepage.html. Accessed 27 Oct 2008

  • Bouxin G (2005) Ginkgo, a multivariate analysis package. J Veg Sci 16:355–359

    Article  Google Scholar 

  • Bueno Sánchez A, Fernández Casado MA, Fernández Prieto JA (1992) A new subspecies of Ranunculus parnassiifolius L. (Ranunculaceae) from the Cantabrian Mountains, Spain. Bot J Linn Soc 109:359–367

    Article  Google Scholar 

  • Chen JF, Staub JE, Adelberg J, Lewis S, Kunkle B (2002) Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis. Euphytica 123:315–322

    Article  Google Scholar 

  • Chinnusamy V, Zhu J, Zhu J-K (2007) Cold stress regulation of gene expression in plants. Trends Plant Sci 12(10):444–451

    PubMed  Article  CAS  Google Scholar 

  • Cook CDK, Grau J, López González G (1986) Ranunculus L. Sect. Ranuncella (Spach) Freyn. In: Castroviejo S, Laínz M, López González G, Montserrat P, Muñoz Garmendia F, Paiva J, Villar L (eds) Flora Iberica, vol I. Lycopodiaceae-Papaveraceae. Real Jardín Botánico, C.S.I.C., Madrid, pp 279–371

  • Creber HMC, Davies MS, Francis D, Walker HD (1994) Variation in DNA C value in natural populations of Dactylis glomerata L. New Phytol 128:555–561

    Article  CAS  Google Scholar 

  • de Candolle AP (1817) Regni vegetabilis systema naturale, sive ordines, genera et species plantarum secundum methodi naturalis normas digestarum et descriptarum, vol 1. Sumptibus Sociorum Treuttel & Würtz, Paris

  • Doležel J, Göhde W (1995) Sex determination in dioecious plants Melandrium album and M rubrum using high-resolution flow cytometry. Cytometry 19:103–106

    PubMed  Article  Google Scholar 

  • Doležel J, Binarová P, Lucretti S (1989) Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant 31(2):113–120

    Article  Google Scholar 

  • Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plant 85:625–631

    Article  Google Scholar 

  • Doležel J, Greilhuber J, Lucretti S, Meister A, Lysák MA, Nardi L, Obermayer R (1998) Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann Bot 82 (Suppl. A):17–26

    Article  Google Scholar 

  • Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry Part A 51:127–128

    Article  Google Scholar 

  • Doležel J, Greilhuber J, Suda J (2007a) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2(9):2233–2244

    PubMed  Article  CAS  Google Scholar 

  • Doležel J, Greilhuber J, Suda J (2007b) Flow cytometry with plants: an overview. In: Doležel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells. Analysis of genes chromosomes and genomes. Wiley-VCH, Weinheim, pp 41–65

    Google Scholar 

  • Emshwiller E (2002) Ploidy levels among species in the ‘Oxalis tuberosa Alliance’ as inferred by flow cytometry. Ann Bot 89:741–753

    PubMed  Article  Google Scholar 

  • Escudero A, Gavilán R, Rubio A (1994) Una breve revisión de técnicas de análisis multivariantes aplicables en fitosociología. Bot Complut 19:9–38

    Google Scholar 

  • Fernández Prieto JA (1983) Aspectos geobotánicos de la Cordillera Cantábrica. An Jard Bot Madr 39(2):489–513

    Google Scholar 

  • Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051

    PubMed  Article  CAS  Google Scholar 

  • Goepfert D (1974) Karyotypes and DNA content in species of Ranunculus L. and related genera. Bot Not 127:464–489

    CAS  Google Scholar 

  • Gower JC (1971) A general coefficient of similarity and some of its properties. Biometrics 27(4):857–871

    Article  Google Scholar 

  • Greilhuber J (2005) Intraspecific variation in genome size in angiosperms: identifying its existence. Ann Bot 95:91–98

    PubMed  Article  CAS  Google Scholar 

  • Greilhuber J (2008) Cytochemistry and C-values: the less-well-known world of nuclear DNA amounts. Ann Bot 101:791–804

    PubMed  Article  CAS  Google Scholar 

  • Greilhuber J, Doležel J, Lysák MA, Bennett MD (2005) The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Ann Bot 95:255–260

    PubMed  Article  CAS  Google Scholar 

  • Greilhuber J, Temsch EM, Loureiro JCM (2007) Nuclear DNA content measurement. In: Doležel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells. Analysis of genes chromosomes and genomes. Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim, pp 67–101

    Google Scholar 

  • Guinea López E (1953) Geografía botánica de Santander. Publicaciones de la Excelentísima Diputación Provincial de Santander (Imprenta Provincial de Santander), Santander

  • Hardie DC, Gregory TR, Hebert PDN (2002) From pixels to picograms: a beginners’ guide to genome quantification by Feulgen image analysis densitometry. J Histochem Cytochem 50(6):735–749

    PubMed  CAS  Google Scholar 

  • Hopping ME (1993) Preparation and preservation of nuclei from plant tissues for quantitative DNA analysis by flow cytometry. N Z J Bot 31:391–401

    Google Scholar 

  • Hörandl E, Paun O, Johansson JT, Lehnebach C, Armstrong T, Chen L, Lockhart P (2005) Phylogenetic relationships and evolutionary traits in Ranunculus s.l. (Ranunculaceae) inferred from ITS sequence analysis. Mol Phylogenet Evol 36:305–327

    PubMed  Article  CAS  Google Scholar 

  • Jalas J, Suominen J (eds) (1989) Atlas Florae Europaeae. Distribution of vascular plants in Europe. 8. Nymphaeaceae to Ranunculaceae. The Committee for Mapping the Flora of Europe and Societas Biologica Fennica Vanamo, Helsinki

  • Johnston JS, Bennett MD, Rayburn AL, Galbraith DW, Price HJ (1999) Reference standards for determination of DNA content of plant nuclei. Amer J Bot 86(5):609–613

    Article  CAS  Google Scholar 

  • Knight CA, Molinari NA, Petrov DA (2005) The large genome constraint hypothesis: evolution, ecology, and phenotype. Ann Bot 95:177–190

    PubMed  Article  CAS  Google Scholar 

  • Kolář F, Štech M, Trávníček P, Rauchová J, Urfus T, Vít P, Kubešová M, Suda J (2009) Towards resolving the Knautia arvensis agg (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales. Ann Bot 103:963–974

    PubMed  Article  Google Scholar 

  • Kron P, Suda J, Husband BC (2007) Applications of flow cytometry to evolutionary and population biology. Annu Rev Ecol Evol Syst 38:847–876

    Article  Google Scholar 

  • Küpfer P (1969) Recherches cytotaxinomiques sur la flore des montagnes de la Péninsule Ibérique. Bull Soc Neuchâtel Sci Nat 92:31–48

    Google Scholar 

  • Küpfer P (1974) Reserches sur les liens de parenté entre la flore orophile des Alpes et celle des Pyrénées. Boissiera 23:1–322

    Google Scholar 

  • Laínz M (1976) Aportaciones al conocimiento de la Flora Cántabro-Astur, XI. Bol Inst Estud Ast Supl Ci 22:3–44

    Google Scholar 

  • Lim KY, Souckova-Skalicka K, Sarasan V, Clarkson JJ, Chase MW, Kovarik A, Leitch AR (2006) A genetic appraisal of a new synthetic Nicotiana tabacum (Solanaceae) and the Kostoff synthetic tobacco. Am J Bot 93(6):875–883

    Article  CAS  Google Scholar 

  • Loureiro J, Rodriguez E, Doležel J, Santos C (2006a) Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Ann Bot 98:515–527

    PubMed  Article  CAS  Google Scholar 

  • Loureiro J, Rodriguez E, Doležel J, Santos C (2006b) Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann Bot 98:679–689

    PubMed  Article  CAS  Google Scholar 

  • Loureiro J, Kopecký D, Castro S, Santos C, Silveira P (2007) Flow cytometric and cytogenetic analyses of Iberian Peninsula Festuca spp. Pl Syst Evol 269:89–105

    Article  Google Scholar 

  • Loureiro J, Doležel J, Greilhuber J, Santos C, Suda J (2008) Plant flow cytometry-far beyond the stone age. Cytometry Part A 73:579–580

    Article  Google Scholar 

  • Lucretti S, Nardi L, Nisini PT, Moretti F, Gualberti G, Doležel J (1999) Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle. Methods Cell Sci 21:155–166

    PubMed  Article  CAS  Google Scholar 

  • Mandáková T, Münzbergová Z (2008) Morphometric and genetic differentiation of diploid and hexaploid populations of Aster amellus agg in a contact zone. Pl Syst Evol 274:155–170

    Article  Google Scholar 

  • Nakamura D, Tiersch TR, Douglass M, Chandler RW (1990) Rapid identification of sex in birds by flow cytometry. Cytogenet Cell Genet 53:201–205

    PubMed  Article  CAS  Google Scholar 

  • Nava H (1988) Flora y vegetación orófila de los Picos de Europa. Ruizia 6:3–243

    Google Scholar 

  • Nsabimana A, van Staden J (2006) Ploidy investigation of bananas (Musa spp.) from the National Banana Germplasm Collection at Rubona-Rwanda by flow cytometry. S Afr J Bot 72:302–305

    Article  Google Scholar 

  • Otto F (1990) DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In: Crissman HA, Darzynkiewicz Z (eds) Methods in cell biology (vol 33). Academic Press, New York, pp 105–110

    Google Scholar 

  • Ozenda P, Borel JL, Borel JL (2000) An ecological map of Europe: why and how? C R Acad Sci Sér 3 Sci Vie 323:983–994

    CAS  Google Scholar 

  • Paun O, Stuessy TF, Hörandl E (2006) The role of hybridization, polyploidization and glaciation in the origin and evolution of the apomictic Ranunculus cassubicus complex. New Phytol 171:223–236

    PubMed  Article  CAS  Google Scholar 

  • Perný M, Kolarčik V, Majeský L, Mártonfi P (2008) Cytogeography of the Phleum pratense group (Poaceae) in the Carpathians and Pannonia. Bot J Linn Soc 157:475–485

    Article  Google Scholar 

  • Pfosser M, Amon A, Lelley T, Heberle-Bors E (1995) Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21:387–393

    PubMed  Article  CAS  Google Scholar 

  • Rivas Martínez S (2007) Mapa de series, geoseries y geopermaseries de vegetación de España (memoria del mapa de vegetación potencial de España). Parte I. Itinera Geobot 17:1–436

    Google Scholar 

  • Rivas Martínez S, Díaz TE, Fernández Prieto JA, Loidi J, Penas A (1984) La vegetación de la alta montaña cantábrica. Los Picos de Europa. Ediciones Leonesas, León

  • Rosenbaumová R, Plačková I, Suda J (2004) Variation in Lamium subg Galeobdolon (Lamiaceae): insights from ploidy levels, morphology and isozymes. Pl Syst Evol 244:219–244

    Article  Google Scholar 

  • Rothmaler W (1934) Species novae vel nomina nova florae hispanicae. Bol Soc Esp Hist Nat 34(2–3):147–155

    Google Scholar 

  • Sliwinska E (2006) Nuclear DNA content analysis of plant seeds by flow cytometry. In: Robinson JP, Darzynkiewicz Z, Dean PN, Orfao A, Rabinovitch PS, Stewart CC, Tanke HJ, Wheeless LL (eds) Current protocols in cytometry. Wiley, New York, pp 7.29.1–7.29.13

  • Smith JB, Bennett MD (1975) DNA variation in the genus Ranunculus. Heredity 35(2):231–239

    Article  Google Scholar 

  • Soltis DE, Soltis PS, Schemske DW, Hancock JF, Thompson JN, Husband BC, Judd WS (2007) Autopolyploidy in angiosperms: have we grossly underestimated the number of species? Taxon 56(1):13–30

    Google Scholar 

  • Španiel S, Marhold K, Hodálová I, Lihová J (2008) Diploid and tetraploid cytotypes of Centaurea stoebe (Asteraceae) in central Europe: morphological differentiation and cytotype distribution patterns. Folia Geobot 43(2):131–158

    Article  Google Scholar 

  • Ståhlberg D, Hedrén M (2008) Systematics and phylogeography of the Dactylorhiza maculata complex (Orchidaceae) in Scandinavia: insights from cytological, morphological and molecular data. Pl Syst Evol 273:107–132

    Article  Google Scholar 

  • Suda J, Trávníček P (2006a) Estimation of relative nuclear DNA content in dehydrated plant tissues by flow cytometry. In: Robinson JP, Darzynkiewicz Z, Dobrucki J, Hyun W, Nolan J, Orfao A, Rabinovitch P (eds) Current protocols in cytometry. Wiley, New York, pp 7.30.1–7.30.14

  • Suda J, Trávníček P (2006b) Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry—new prospects for plant research. Cytometry Part A 69:273–280

    Article  Google Scholar 

  • Suda J, Kron P, Husband BC, Trávníček P (2007a) Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In: Doležel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells. Analysis of genes chromosomes and genomes. Wiley-VCH, Weinheim, pp 103–130

    Google Scholar 

  • Suda J, Weiss-Schneeweiss H, Tribsch A, Schneeweiss GM, Trávníček P, Schönswetter P (2007b) Complex distribution patterns of di-, tetra- and hexaploid cytotypes in the European high mountain plant Senecio carniolicus (Asteraceae). Am J Bot 94(8):1391–1401

    Article  Google Scholar 

  • Sugiyama S (1998) Differentiation in competitive ability and cold tolerance between diploid and tetraploid cultivars in Lolium perenne. Euphytica 103:55–59

    Article  Google Scholar 

  • Sugiyama S, Yamaguchi K, Yamada T (2002) Intraspecific phenotypic variation associated with nuclear DNA content in Lolium perenne L. Euphytica 128:145–151

    Article  CAS  Google Scholar 

  • Tamura M (1993) Ranunculaceae. In: Kubitzki K, Rohwer JG, Bittrich V (eds) The families and genera of vascular plants. 2. Flowering plants. Dicotyledons, magnoliid, hamamelid, and caryophyllid families. Springer, Berlin, pp 563–583

    Google Scholar 

  • Tutin TG (1964) Ranunculus L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA (eds) Flora Europaea. vol 1. Lycopodiaceae to Platanaceae. Cambridge University Press, Cambridge, pp 223–238

  • Tutin TG, Akeroyd JR (1993) Ranunculus L. In: Tutin TG, Burges NA, Chater AO, Edmondson JR, Heywood VH, Moore DM, Valentine DH, Walters SM, Webb DA (eds) Flora Europaea. vol 1. Psilotaceae to Platanaceae, 2nd edn. Cambridge University Press, Cambridge, pp 269–286

    Google Scholar 

  • Vuille C, Küpfer P (1985) Aposporie chez le R. parnassifolius L. I. Étude cytoembryologique. Bull Soc Neuchâtel Sci Nat 108:123–134

    Google Scholar 

  • Ziman SN, Keener CS (1989) A geographical analysis of the family Ranunculaceae. Ann Missouri Bot Gard 76:1012–1049

    Article  Google Scholar 

  • Zonneveld BJM (2008) The systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae). Pl Syst Evol 275:109–132

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We acknowledge the technical assistance of Ana Salas and Marta Alonso Guervós (Flow Cytometry Area, Scientific-Technical Services, University of Oviedo). Seeds of tomato cv. ‘Stupicke’ and peas cv. ‘Ctirad’ were supplied by Dr. J. Doležel (Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic). We express our gratitude to Anu Väinölä, Frantisek Krahulec, Elvira Hörandl, Eva M. Temsch, and Miguel de Cáceres for their encouragement and their helpful comments. We also express our gratitude to the curators and directors of all herbaria consulted. This research was funded by the Spanish Ministerio de Educación y Ciencia (CGL2006-11743). E. Cires is supported by a predoctoral grant by the University of Oviedo (UNOV-06-BECDOC-2).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Eduardo Cires.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cires, E., Cuesta, C., Peredo, E.L. et al. Genome size variation and morphological differentiation within Ranunculus parnassifolius group (Ranunculaceae) from calcareous screes in the Northwest of Spain. Plant Syst Evol 281, 193–208 (2009). https://doi.org/10.1007/s00606-009-0201-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00606-009-0201-9

Keywords

  • Flow cytometry
  • Genome size
  • Iberian Peninsula
  • Multivariate morphometric analysis
  • Ploidy levels
  • Ranunculus parnassifolius