Plant Systematics and Evolution

, Volume 303, Issue 9, pp 1145–1160 | Cite as

Geographic genetic structure of Iberian columbines (gen. Aquilegia)

  • Jose L. GarridoEmail author
  • Julio M. Alcántara
  • Pedro J. Rey
  • Mónica Medrano
  • Javier Guitián
  • María C. Castellanos
  • Jesús M. Bastida
  • Rafael Jaime
  • Carlos M. Herrera
Original Article


Southern European columbines (genus Aquilegia) are involved in active processes of diversification, and the Iberian Peninsula offers a privileged observatory to witness the process. Studies on Iberian columbines have provided significant advances on species diversification, but we still lack a complete perspective of the genetic diversification in the Iberian scenario. This work explores how genetic diversity of the genus Aquilegia is geographically structured across the Iberian Peninsula. We used Bayesian clustering methods, principal coordinates analyses, and NJ phenograms to assess the genetic relationships among 285 individuals from 62 locations and detect the main lineages. Genetic diversity of Iberian columbines consists of five geographically structured lineages, corresponding to different Iberian taxa. Differentiation among lineages shows particularly complex admixture patterns at Northeast and highly homogeneous toward Northwest and Southeast. This geographic genetic structure suggests the existence of incomplete lineage sorting and interspecific hybridization as could be expected in recent processes of diversification under the influence of quaternary postglacial migrations. This scenario is consistent with what is proposed by the most recent studies on European and Iberian columbines, which point to geographic isolation and divergent selection by habitat specialization as the main diversification drivers of the Iberian Aquilegia complex.


AFLP Aquilegia Diversification Iberian Peninsula Quaternary postglacial migrations Spatial genetic structure 



Authors thank D. Guzmán and A.R. Larrinaga for his invaluable aid while sampling at Pyrenees. At Sierras de Segura y Cazorla, A. Benavente helped us finding locations and S. Arenas assisted in the field. We also benefited from the helpful advice of P. Bazaga on laboratory procedures. M.C. Martinell provided DNA extract from Avp. We also thank the Remote Sensing and Geographic Information Systems Laboratory of EBD (LAST-EBD). Bioinformatic STRUCTURE analyses were carried out on the (earlier times) freely available Bioportal ( This work was partly supported by Grant BOS2003-03979-C02-01, BOS2003-03979-C02-02, and CGL2006-01355/BOS from Ministerio Ciencia y Tecnología. During part of this work JLG was granted by the postdoctoral program (EX2003-0376) of Ministerio Educación, Cultura y Deporte, and by the Severo Ochoa Program for Centres of Excellence in R+D+I (SEV-2012-0262) of Ministerio de Economía y Competitividad.

Compliance with Ethical Standards

Human and animal rights

The authors declare that the research included in this article accomplishes with the ethical standards of the journal and with all legal requirements regarding samples collection. All authors have been informed and consent with this submission. Founding sources have been declared both in Acknowledgements section as well as in the online submission form. The research does not involve human participants and/or animals.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

606_2017_1428_MOESM1_ESM.pdf (29 kb)
Supplementary material 1 (PDF 29 kb)
606_2017_1428_MOESM2_ESM.pdf (57 kb)
Supplementary material 2 (PDF 57 kb)
606_2017_1428_MOESM3_ESM.pdf (113 kb)
Supplementary material 3 (PDF 112 kb)
606_2017_1428_MOESM4_ESM.pdf (129 kb)
Supplementary material 4 (PDF 128 kb)


  1. Alcántara JM, Bastida JM, Rey PJ (2010) Linking divergent selection on vegetative traits to environmental variation and phenotypic diversification in the Iberian columbines (Aquilegia). J Evol Biol 23:1218–1233. doi: 10.1111/j.1420-9101.2010.01981.x CrossRefPubMedGoogle Scholar
  2. Alcántara JM, Jaime R, Bastida JM, Rey PJ (2014) The role of genetic constraints on the diversification of Iberian taxa of the genus Aquilegia (Ranunculaceae). Biol J Linn Soc 111:252–261. doi: 10.1111/bij.12215 CrossRefGoogle Scholar
  3. Bastida JM, Alcántara JM, Rey PJ, Vargas P, Herrera CM (2010) Extended phylogeny of Aquilegia: the biogeographical and ecological patterns of two simultaneous but contrasting radiations. Pl Syst Evol 284:171–185. doi: 10.1007/s00606-009-0243-z CrossRefGoogle Scholar
  4. Bastida JM, Rey PJ, Alcántara JM (2014) Plant performance and morpho-functional differentiation in response to edaphic variation in Iberian columbines: cues for range distribution? J Pl Ecol 7:403–412. doi: 10.1093/jpe/rtt046 CrossRefGoogle Scholar
  5. Bennett KD, Tzedakis PC, Willis KJ (1991) Quaternary refugia of North European trees. J Biogeogr 18:103–115. doi: 10.2307/2845248 CrossRefGoogle Scholar
  6. Bittkau C, Comes HP (2005) Evolutionary processes in a continental island system: molecular phylogeography of the Aegean Nigella arvensis alliance (Ranunculaceae) inferred from chloroplast DNA. Molec Ecol 14:4065–4083. doi: 10.1111/j.1365-294X.2005.02725.x CrossRefGoogle Scholar
  7. Bonin A, Ehrich D, Manel S (2007) Statistical analysis of amplified fragment length polymorphism data: a toolbox for molecular ecologists and evolutionists. Molec Ecol 16:3737–3758. doi: 10.1111/j.1365-294X.2007.03435.x CrossRefGoogle Scholar
  8. Castellanos MC, Alcántara JM, Rey PJ, Bastida JM (2011) Intra-population comparison of vegetative and floral trait heritabilities estimated from molecular markers in wild Aquilegia populations. Molec Ecol 20:3513–3524. doi: 10.1111/j.1365-294X.2011.05094.x Google Scholar
  9. Comes HP, Kadereit JW (2003) Spatial and temporal patterns in the evolution of the flora of the European Alpine System. Taxon 52:451–462CrossRefGoogle Scholar
  10. Corander J, Marttinen P (2006) Bayesian identification of admixture events using multi-locus molecular markers. Molec Ecol 15:2833–2843. doi: 10.1111/j.1365-294X.2006.02994.x CrossRefGoogle Scholar
  11. Corander J, Waldmann P, Sillanpää MJ (2003) Bayesian analysis of genetic differentiation between populations. Genetics 163:367–374PubMedPubMedCentralGoogle Scholar
  12. Cottrell JE, Krystufek V, Tabbener HE, Milner AD, Connolly T, Sing L, Fluch S, Burg K, Lefèvre F, Achard P, Bordács S, Gebhardt K, Vornam B, Smulders MJM, Broeck AHV, Slycken JV, Storme V, Boerjan W, Castiglione S, Fossati T, Alba N, Agúndez D, Maestro C, Notivol E, Bovenschen J, Dam BC (2005) Postglacial migration of Populus nigra L.: lessons learnt from chloroplast DNA. Forest Ecol Managem 206:71–90. doi: 10.1016/j.foreco.2004.10.052 CrossRefGoogle Scholar
  13. Cullen J, Heywood VH (1964) Aquilegia. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA (eds) Flora Europea 1, Cambridge University Press, Cambridge, pp 238–240Google Scholar
  14. Díaz T (1986) Aquilegia L. In: Castroviejo S (ed) Flora ibérica: plantas vasculares de la Península Ibérica e Islas Baleares. Real Jardín Botánico de Madrid (CSIC), Madrid, pp 376–387Google Scholar
  15. Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genet Resources 4:359–361CrossRefGoogle Scholar
  16. Edh K, Widen B, Ceplitis A (2007) Nuclear and chloroplast microsatellites reveal extreme population differentiation and limited gene flow in the Aegean endemic Brassica cretica (Brassicaceae). Molec Ecol 16:4972–4983. doi: 10.1111/j.1365-294X.2007.03585.x CrossRefGoogle Scholar
  17. Ehrich D (2006) AFLPdat: a collection of R functions for convenient handling of AFLP data. Molec Ecol Notes 6:603–604. doi: 10.1111/j.1471-8286.2006.01380.x CrossRefGoogle Scholar
  18. Ehrich D, Gaudeul M, Assefa A, Koch MA, Mummenhoff K, Nemomissa S, Consortium I, Brochmann C (2007) Genetic consequences of Pleistocene range shifts: contrast between the Arctic, the Alps and the East African mountains. Molec Ecol 16:2542–2559. doi: 10.1111/j.1365-294X.2007.03299.x CrossRefGoogle Scholar
  19. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molec Ecol 14:2611–2620. doi: 10.1111/j.1365-294X.2005.02553.x CrossRefGoogle Scholar
  20. Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587PubMedPubMedCentralGoogle Scholar
  21. Falush D, Stephens M, Pritchard JK (2007) Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molec Ecol Notes 7:574–578. doi: 10.1111/j.1471-8286.2007.01758.x CrossRefGoogle Scholar
  22. Felsenstein J (2005) PHYLIP (phylogeny inference package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, SeattleGoogle Scholar
  23. Fior S, Li MG, Oxelman B, Viola R, Hodges SA, Ometto L, Varotto C (2013) Spatiotemporal reconstruction of the Aquilegia rapid radiation through next-generation sequencing of rapidly evolving cpDNA regions. New Phytol 198:579–592. doi: 10.1111/nph.12163 CrossRefPubMedGoogle Scholar
  24. Garrido JL, Fenu G, Mattana E, Bacchetta G (2012) Spatial genetic structure of Aquilegia taxa endemic to the island of Sardinia. Ann Bot (Oxford) 109:953–964. doi: 10.1093/aob/mcs011 CrossRefGoogle Scholar
  25. Herrera CM, Bazaga P (2009) Quantifying the genetic component of phenotypic variation in unpedigreed wild plants: tailoring genomic scan for within-population use. Molec Ecol 18:2602–2614. doi: 10.1111/j.1365-294X.2009.04229.x CrossRefGoogle Scholar
  26. Hodges SA (1997) Floral nectar spurs and diversification. Int J Pl Sci 158:81–88CrossRefGoogle Scholar
  27. Hodges SA, Arnold ML (1994) Columbines - a geographically widespread species flock. Proc Natl Acad Sci USA 91:5129–5132CrossRefPubMedPubMedCentralGoogle Scholar
  28. Hodges SA, Derieg NJ (2009) Adaptive radiations: from field to genomic studies. Proc Nat Acad Sci USA 106:9947–9954CrossRefPubMedPubMedCentralGoogle Scholar
  29. Holland BR, Clarke AC, Meudt HM (2008) Optimizing automated AFLP scoring parameters to improve phylogenetic resolution. Syst Biol 57:347–366. doi: 10.1080/10635150802044037 CrossRefPubMedGoogle Scholar
  30. Hubisz MJ, Falush D, Stephens M, Pritchard JK (2009) Inferring weak population structure with the assistance of sample group information. Molec Ecol Res 9:1322–1332. doi: 10.1111/j.1755-0998.2009.02591.x CrossRefGoogle Scholar
  31. Jaime R, Rey PJ, Alcántara JM, Bastida JM (2013) Glandular trichomes as an inflorescence defence mechanism against insect herbivores in Iberian columbines. Oecologia 172:1051–1060. doi: 10.1007/s00442-012-2553-z CrossRefPubMedGoogle Scholar
  32. Jaime R, Serichol C, Alcántara JM, Rey PJ (2014) Differences in gas exchange contribute to habitat differentiation in Iberian columbines from contrasting light and water environments. Pl Biol 16:354–364. doi: 10.1111/plb.12064 CrossRefGoogle Scholar
  33. Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics 23:1801–1806. doi: 10.1093/bioinformatics/btm233 CrossRefPubMedGoogle Scholar
  34. Joly S, McLenachan PA, Lockhart PJ (2009) A Statistical approach for distinguishing hybridization and incomplete lineage sorting. Amer Naturalist 174:E54–E70CrossRefGoogle Scholar
  35. Kadereit JW, Griebeler EM, Comes HP (2004) Quaternary diversification in European alpine plants: pattern and process. Philos Trans Ser B 359:265–274. doi: 10.1098/rstb.2003.1389 CrossRefGoogle Scholar
  36. Kropf M, Kadereit JW, Comes HP (2003) Differential cycles of range contraction and expansion in European high mountain plants during the Late Quaternary: insights from Pritzelago alpina (L.) O. Kuntze (Brassicaceae). Molec Ecol 12:931–949. doi: 10.1046/j.1365-294X.2003.01781.x CrossRefGoogle Scholar
  37. Krzanowski WJ (1990) Principles of multivariate analysis. Clarendon, OxfordGoogle Scholar
  38. Lega M, Fior S, Li M, Leonardi S, Varotto C (2014) Genetic drift linked to heterogeneous landscape and ecological specialization drives diversification in the alpine endemic columbine Aquilegia thalictrifolia. J Heredity 105:542–554. doi: 10.1093/jhered/esu028 CrossRefGoogle Scholar
  39. Lo Presti RM, Oberprieler C (2011) The central Mediterranean as a phytodiversity hotchpotch: phylogeographical patterns of the Anthemis secundiramea group (Compositae, Anthemideae) across the Sicilian Channel. J Biogeogr 38:1109–1124. doi: 10.1111/j.1365-2699.2010.02464.x CrossRefGoogle Scholar
  40. Machado CA, Kliman RM, Markert JA, Hey J (2002) Inferring the history of speciation from multilocus DNA sequence data: the case of Drosophila pseudoobscura and close relatives. Molec Biol Evol 19:472–488. doi: 10.1093/oxfordjournals.molbev.a004103 CrossRefPubMedGoogle Scholar
  41. Maddison WP, Knowles LL (2006) Inferring phylogeny despite incomplete lineage sorting. Syst Biol 55:21–30. doi: 10.1080/10635150500354928 CrossRefPubMedGoogle Scholar
  42. Magri D, Vendramin GG, Comps B, Dupanloup I, Geburek T, Gömöry D, Latalowa M, Litt T, Paule L, Roure JM, Tantau I, van der Knaap OW, Petit RJ, de Beaulieu JL (2006) A new scenario for the Quaternary history of European beech populations: palaeobotanical evidence and genetic consequences. New Phytol 171:199–221. doi: 10.1111/j.1469-8137.2006.01740.x CrossRefPubMedGoogle Scholar
  43. Martín-Bravo S, Valcárcel V, Vargas P, Luceño M (2010) Geographical speciation related to Pleistocene range shifts in the western Mediterranean mountains (Reseda sect. Glaucoreseda, Resedaceae). Taxon 59:466–482Google Scholar
  44. Martinell MC, Rovira A, Blanché C, Bosch M (2011) Shift towards autogamy in the extremely narrow endemic Aquilegia paui and comparison with its widespread close relative A. vulgaris (Ranunculaceae). Pl Syst Evol 295:73–82. doi: 10.1007/s00606-011-0463-x CrossRefGoogle Scholar
  45. Medail F, Diadema K (2009) Glacial refugia influence plant diversity patterns in the Mediterranean Basin. J Biogeogr 36:1333–1345. doi: 10.1111/j.1365-2699.2008.02051.x CrossRefGoogle Scholar
  46. Medrano M, Herrera CM (2008) Geographical structuring of genetic diversity across the whole distribution range of Narcissus longispathus, a habitat-specialist, Mediterranean narrow endemic. Ann Bot (Oxford) 102:183–194. doi: 10.1093/aob/mcn086 CrossRefGoogle Scholar
  47. Medrano M, Castellanos C, Herrera CM (2006) Comparative floral and vegetative differentiation between two European Aquilegia taxa along a narrow contact zone. Pl Syst Evol 262:209–224. doi: 10.1007/s00606-006-0473-2 CrossRefGoogle Scholar
  48. Mereda P, Hodalova I, Kucera J, Zozomova-Lihova J, Letz DR, Slovak M (2011) Genetic and morphological variation in Viola suavis s.l. (Violaceae) in the western Balkan Peninsula: two endemic subspecies revealed. Syst Biodivers 9:211–231. doi: 10.1080/14772000.2011.603903 CrossRefGoogle Scholar
  49. Meudt HM, Clarke AC (2007) Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends Pl Sci 12:106–117. doi: 10.1016/j.tplants.2007.02.001 CrossRefGoogle Scholar
  50. Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590PubMedPubMedCentralGoogle Scholar
  51. Nei M (1987) Molecular Evolutionary Genetics. Columbia University Press, New YorkGoogle Scholar
  52. Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273CrossRefPubMedPubMedCentralGoogle Scholar
  53. Nieto Feliner G (2014) Patterns and processes in plant phylogeography in the Mediterranean Basin. A review. Perspect Pl Ecol Evol Syst 16:265–278. doi: 10.1016/j.ppees.2014.07.002 CrossRefGoogle Scholar
  54. Nold R (2003) Columbines. Aquilegia, Paraquilegia and Semiaquilegia. Timber Press, CambridgeGoogle Scholar
  55. Olalde M, Herrán A, Espinel S, Goicoechea PG (2002) White oaks phylogeography in the Iberian Peninsula. Forest Ecol Managem 156:89–102. doi: 10.1016/S0378-1127(01)00636-3 CrossRefGoogle Scholar
  56. Pamilo P, Nei M (1988) Relationships between gene trees and species trees. Molec Biol Evol 5:568–583. doi: 10.1093/oxfordjournals.molbev.a040517 PubMedGoogle Scholar
  57. Parks M, Cronn R, Liston A (2009) Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biol 7:84. doi: 10.1186/1741-7007-7-84 CrossRefPubMedPubMedCentralGoogle Scholar
  58. Prazmo W (1965) Cytogenetic studies on the genus Aquilegia. III. Inheritance of the traits distinguishing different complexes in the genus Aquilegia. Acta Soc Bot Poloniae 34:403–437. doi: 10.5586/asbp.1965.031 CrossRefGoogle Scholar
  59. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedPubMedCentralGoogle Scholar
  60. Ro KE, McPheron BA (1997) Molecular phylogeny of the Aquilegia group (Ranunculaceae) based on internal transcribed spacers and 5.8S nuclear ribosomal DNA. Biochem Syst Ecol 25:445–461. doi: 10.1016/S0305-1978(97)00029-X CrossRefGoogle Scholar
  61. Rosenberg NA (2004) DISTRUCT: a program for the graphical display of population structure. Molec Ecol Notes 4:137–138. doi: 10.1046/j.1471-8286.2003.00566.x CrossRefGoogle Scholar
  62. Rymer PD, Manning JC, Goldblatt P, Powell MP, Savolainen V (2010) Evidence of recent and continuous speciation in a biodiversity hotspot: a population genetic approach in southern African gladioli (Gladiolus; Iridaceae). Molec Ecol 19:4765–4782.  10.1111/j.1365-294X.2010.04794.x CrossRefGoogle Scholar
  63. Schlüter PM, Harris SA (2006) Analysis of multilocus fingerprinting data sets containing missing data. Molec Ecol Notes 6:569–572. doi: 10.1111/j.1471-8286.2006.01225.x CrossRefGoogle Scholar
  64. Schönswetter P, Tribsch A (2005) Vicariance and dispersal in the alpine perennial Bupleurum stellatum L. (Apiaceae). Taxon 54:725–732CrossRefGoogle Scholar
  65. Strand AE, Milligan BG, Pruitt CM (1996) Are populations islands? Analysis of chloroplast DNA variation in Aquilegia. Evolution 50:1822–1829. doi: 10.2307/2410739 CrossRefPubMedGoogle Scholar
  66. Swofford DL (2002) PAUP*. Phylogenetic analysis using parsimony (*and other methods).Version 4. Sinauer Associates, Sunderland, MassachusettsGoogle Scholar
  67. Taberlet P, Fumagalli L, Wust-Saucy AG, Cosson JF (1998) Comparative phylogeography and postglacial colonization routes in Europe. Molec Ecol 7:453–464. doi: 10.1046/j.1365-294x.1998.00289.x CrossRefGoogle Scholar
  68. Taylor RJ (1967) Interspecific hybridization and its evolutionary significance in genus Aquilegia. Brittonia 19:374–390. doi: 10.2307/2805535 CrossRefGoogle Scholar
  69. Thompson JD (2005) Plant Evolution in the Mediterranean. Oxford University Press, New YorkCrossRefGoogle Scholar
  70. Vekemans X (2002) AFLP-SURV version 1.0. Distributed by the author, Laboratoire de Génétique et Ecologie Végétale, Université Libre de Bruxelles, BruxellesGoogle Scholar
  71. Vos P, Hogers R, Bleeker M, Reijans M, Vandelee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP - a New Technique for DNA-Fingerprinting. Nucl Acids Res 23:4407–4414. doi: 10.1093/nar/23.21.4407 CrossRefPubMedPubMedCentralGoogle Scholar
  72. Wang RL, Wakeley J, Hey J (1997) Gene flow and natural selection in the origin of Drosophila pseudoobscura and close relatives. Genetics 147:1091–1106PubMedPubMedCentralGoogle Scholar
  73. Wang AL, Yang MH, Liu JQ (2005) Molecular phylogeny, recent radiation and evolution of gross morphology of the rhubarb genus Rheum (Polygonaceae) inferred from chloroplast DNA trnL-F sequences. Ann Bot (Oxford) 96:489–498. doi: 10.1093/aob/mci201 CrossRefGoogle Scholar
  74. Whitfield JB, Lockhart PJ (2007) Deciphering ancient rapid radiations. Trends Ecol Evol 22:258–265. doi: 10.1016/j.tree.2007.01.012 CrossRefPubMedGoogle Scholar
  75. Whittall JB, Hodges SA (2007) Pollinator shifts drive increasingly long nectar spurs in columbine flowers. Nature 447:U706–U712CrossRefGoogle Scholar
  76. Whittall JB, Medina-Marino A, Zimmer EA, Hodges SA (2006) Generating single-copy nuclear gene data for a recent adaptive radiation. Molec Phylogen Evol 39:124–134. doi: 10.1016/j.ympev.2005.10.010 CrossRefGoogle Scholar
  77. Whittemore AT (1997) Aquilegia. In: Morin NR (ed) Flora of North America. Oxford University Press, New YorkGoogle Scholar

Copyright information

© Springer-Verlag Wien 2017

Authors and Affiliations

  • Jose L. Garrido
    • 1
    Email author
  • Julio M. Alcántara
    • 2
  • Pedro J. Rey
    • 2
  • Mónica Medrano
    • 1
  • Javier Guitián
    • 4
  • María C. Castellanos
    • 3
  • Jesús M. Bastida
    • 2
  • Rafael Jaime
    • 2
  • Carlos M. Herrera
    • 1
  1. 1.Depto. Ecología EvolutivaEstación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (EBD-CSIC)SevilleSpain
  2. 2.Depto. Biología Animal, Biología Vegetal y EcologíaUniversity of JaénJaénSpain
  3. 3.School of Life SciencesUniversity of SussexBrightonUK
  4. 4.Depto. BotánicaUniversity of Santiago de CompostelaSantiago de CompostelaSpain

Personalised recommendations