Conservation Genetics

, Volume 13, Issue 1, pp 197–209 | Cite as

Population structure and genetic diversity of Rana dalmatina in the Iberian Peninsula

  • Vanessa Sarasola-Puente
  • María José Madeira
  • Alberto Gosá
  • Miguel Lizana
  • Benjamín Gómez-Moliner
Research Article


The increasing fragmentation of natural habitats may strongly affect patterns of dispersal and gene flow among populations, and thus alter evolutionary dynamics. We examined genetic variation at twelve microsatellite loci in the Agile frog (Rana dalmatina) from 22 breeding ponds in the Iberian Peninsula, the southwest limit of its range, where populations of this species are severely fragmented and are of conservation concern. We investigated genetic diversity, structure and gene flow within and among populations. Diversity as observed heterozygosities ranged from 0.257 to 0.586. The mean number of alleles was 3.6. Just one population showed a significant F IS value. Four populations show evidence of recent bottlenecks. Strong pattern of structure was observed due to isolation by distance and to landscape structure. The average degree of genetic differentiation among populations was F ST = 0.185. Three operational conservation units with metapopulation structure were identified. Additionally, there are some other isolated populations. The results reinforce the view that amphibian populations are highly structured even in small geographic areas. The knowledge of genetic structure pattern and gene flow is fundamental information for developing programmes for the preservation of R. dalmatina at the limits of its geographic distribution.


Rana dalmatina Microsatellites Metapopulation Conservation genetics Iberian Peninsula 



The authors are very grateful to Xabier Rubio for his essential assistance. Our research was supported by a project from the Basque Government. The authors acknowledge the permissions and financial assistance of the Department of Rural Development and the Environment from Navarra and the Basque Country. Very many thanks to Julia Günther and Heike Proehl who kindly sent us the European samples. Elisabeth Anderson revised the English version of the manuscript. Comments by two anonymous reviewers on an earlier draft greatly improved the manuscript.


  1. Allentoft ME, O’Brien J (2010) Global amphibian declines, loss of genetic diversity and fitness: a review. Diversity 2:47–71CrossRefGoogle Scholar
  2. Allentoft ME, Siegismund HR, Briggs L, Andersen LW (2009) Microsatellite analysis of the natterjack toad (Bufo calamita) in Denmark: populations are islands in a fragmented landscape. Conserv Genet 10:15–28CrossRefGoogle Scholar
  3. Beebee TJC (2005) Conservation genetics of amphibians. Heredity 95:423–427PubMedCrossRefGoogle Scholar
  4. Beerli P (2004) Effect of unsampled populations on the estimation of population sizes and migration rates between sampled populations. Mol Ecol 13(4):827–836PubMedCrossRefGoogle Scholar
  5. Beerli P (2006) Comparison of Bayesian and maximum-likelihood inference of population genetic parameters. Bioinformatics 22(3):341–345PubMedCrossRefGoogle Scholar
  6. Beerli P, Felsenstein J (2001) Maximum likelihood estimation of a migration matrix and effective population sizes in n subpopulations by using a coalescent approach. P Natl Acad Sci USA 98(8):4563–4568CrossRefGoogle Scholar
  7. Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F (1996–2004) GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Laboratoire genome, populations, interactions, CNRS UMR 5171, Université de Montpellier II, MontpellierGoogle Scholar
  8. Cornuet JM, Luikart G (1996) Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144(4):2001–2014PubMedGoogle Scholar
  9. Crandall KA, Bininda-Emonds ORP, Mace GM, Wayne RK (2000) Considering evolutionary processes in conservation biology. Trends Ecol Evol 15:290–295PubMedCrossRefGoogle Scholar
  10. Cushman SA (2006) Effects of habitat loss and fragmentation on amphibians: a review and prospectus. Biol Conserv 128(2):231–240CrossRefGoogle Scholar
  11. Ehrlich PR, Wilson EO (1991) Biodiversity studies: science and policy. Science 253(5021):758–762PubMedCrossRefGoogle Scholar
  12. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the softwar STRUCTURE: a simulation study. Mol Ecol 14:2611–2620PubMedCrossRefGoogle Scholar
  13. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform 1:47–50Google Scholar
  14. Ficetola GF, Garner TWJ, De Bernardi F (2007) Genetic diversity, but not hatcing success, is jointly affected by postglacial colonization and isolation in the threatened frog, Rana latastei. Mol Ecol 16:1787–1797PubMedCrossRefGoogle Scholar
  15. Frankham R (2005) Genetics and extinction. Biol Conserv 126(2):131–140CrossRefGoogle Scholar
  16. Gosá A (2002) Rana dalmatina Bonaparte, 1840. In: Pleguezuelos JM, Márquez R, Lizana M (eds) Atlas y libro rojo de los anfibios y reptiles de España. Dirección General de Conservación de la Naturaleza-Asociación Herpetológica española (2ª impresión), Madrid, pp 120–122Google Scholar
  17. Gosá A, Sarasola V (2009) Seguimiento y determinación de la situación de Rana dalmatina. Campaña 2009. Departamento de Desarrollo Rural y Medio Ambiente, Gobierno de Navarra, Inédito, p 23Google Scholar
  18. Goudet J (1995) FSTAT (Version 1.2): a computer program to calculate F-statistics. J Hered 86(6):485–486Google Scholar
  19. Guillot G, Mortier F, Estoup A (2005) GENELAND: a computer package for landscape genetics. Mol Ecol Notes 5(3):712–715CrossRefGoogle Scholar
  20. Guo SW, Thompson EA (1992) Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48(2):361–372PubMedCrossRefGoogle Scholar
  21. Hanski I (1998) Metapopulation dynamics. Nature 396(6706):41–49CrossRefGoogle Scholar
  22. Hauswaldt JS, Fuessel J, Guenther J, Steinfartz S (2008) Eight new tetranucleotide microsatellite loci for the Agile frog (Rana dalmatina). Mol Ecol Resour 8(6):1457–1459PubMedCrossRefGoogle Scholar
  23. Hitchings SP, Beebee TJC (1997) Genetic substructuring as a result of barriers to gene flow in urban Rana temporaria (common frog) populations: implications for biodiversity conservation. Heredity 79:117–127PubMedCrossRefGoogle Scholar
  24. Holderegger R, Wagner HH (2008) Landscape genetics. Bioscience 58:199–207CrossRefGoogle Scholar
  25. Jarne P, Lagoda PJL (1996) Microsatellites, from molecules to populations and back. Trends Ecol Evol 11(10):424–429PubMedCrossRefGoogle Scholar
  26. Johansson M, Primmer CR, Sahlsten J, Merila J (2005) The influence of landscape structure on occurrence, abundance and genetic diversity of the common frog, Rana temporaria. Global Change Biol 11:1664–1679CrossRefGoogle Scholar
  27. Knopp T, Merilä J (2009) Microsatellite variation and population structure of the moor frog (Rana arvalis) in Scandinavia. Mol Ecol 18:2996–3005PubMedCrossRefGoogle Scholar
  28. Krone A, Kühnel KD, Berger H (1997) Der springfrosch-ökologie und bestandssituation. In: Krone A, Kühnel KD, Berger H (eds) Sonderheft 2 der Rana, pp 309Google Scholar
  29. Lesbarrères D, Pagano A, Lodé T (2003) Inbreeding and road effect zone in a Ranidae: the case of Agile frog, Rana dalmatina Bonaparte, 1840. CR Biol 326:68–72CrossRefGoogle Scholar
  30. Lesbarrères D, Primmer CR, Lodé T, Merilä J (2006) The effects of 20 years of highway presence on the genetic structure of Rana dalmatina populations. Ecoscience 13:531–538CrossRefGoogle Scholar
  31. Lesica P, Allendorf FW (1995) When are peripheral populations valuable for conservation? Cons Biol 9:753–760CrossRefGoogle Scholar
  32. Luikart G, Allendorf FW, Cornuet JM, Sherwin WB (1998) Distortion of allele frequency distributions provides a test for recent population bottlenecks. J Hered 89(3):238–247PubMedCrossRefGoogle Scholar
  33. Manel S, Schwartz MK, Luikart G, Taberlet P (2003) Landscape genetics: combining landscape ecology and population genetics. Trends Ecol Evol 18:189–197CrossRefGoogle Scholar
  34. Mantel N (1967) Detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220PubMedGoogle Scholar
  35. Marsh DM, Trenham PC (2001) Metapopulation dynamics and amphibian conservation. Conserv Biol 15(1):40–49Google Scholar
  36. Monsen KJ, Blouin MS (2004) Extreme isolation by distance in a montane frog Rana cascadae. Conserv Genet 5(6):827–835CrossRefGoogle Scholar
  37. Moritz C (1994) Applications of mitochondrial-DNA analysis in conservation–a critical review. Mol Ecol 3(4):401–411CrossRefGoogle Scholar
  38. Newman RA, Squire T (2001) Microsatellite variation and fine-scale population structure in the wood frog (Rana sylvatica). Mol Ecol 10(5):1087–1100PubMedCrossRefGoogle Scholar
  39. Noël S, Ouellet M, Galois P, Lapointe FJ (2007) Impact of urban fragmentation on the genetic structure of the eastern red-backed salamander. Conserv Genet 8:599–606CrossRefGoogle Scholar
  40. Noss RF (1990) Can we maintain biological and ecological integrity. Conserv Biol 4(3):241–243CrossRefGoogle Scholar
  41. Palo JU, Schmeller DS, Laurila A, Primmer CR, Kuzmin SL, Merila J (2004) High degree of population subdivision in a widespread amphibian. Mol Ecol 13(9):2631–2644PubMedCrossRefGoogle Scholar
  42. Piry S, Luikart G, Cornuet JM (1999) BOTTLENECK: a computer program for detecting recent reductions in the effective population size using allele frequency data. J Hered 90(4):502–503CrossRefGoogle Scholar
  43. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2):945–959PubMedGoogle Scholar
  44. Purrenhage JL, Niewiarowski PH, Moore FBG (2009) Population structure of spotted salamanders (Ambystoma maculatum) in a fragmented landscape. Mol Ecol 18(2):235–247PubMedCrossRefGoogle Scholar
  45. Raymond M, Rousset F (1995) GENEPOP (version-1.2): population-genetics software for exact tests and ecumenicism. J Hered 86(3):248–249Google Scholar
  46. Reed DH, Frankham R (2003) Correlation between fitness and genetic diversity. Conserv Biol 17(1):230–237CrossRefGoogle Scholar
  47. Rowe G, Beebee TJC, Burke T (1999) Microsatellite heterozygosity, fitness and demography in natterjack toads Bufo calamita. Anim Conserv 2(2):85–92CrossRefGoogle Scholar
  48. Sarasola-Puente V, Beebee TJC, Gosá A, Gómez-Moliner BJ, Lizana M and Madeira MJ (2010) Characterization of ten polymorphic microsatellite loci in Rana dalmatina from enriched genomic libraries. In: Permanent genetic resources added to molecular ecology resources database 1 April 2010–31 May 2010. Mol Ecol Resour 10:1098–1105Google Scholar
  49. Shaffer G, Fellers GM, Magee A, Voss R (2000) The genetics of amphibian declines: population substructure and molecular differentiation in the Yosemite Toad, Bufo canorus (Anura, Bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data. Mol Ecol 9(3):245–257PubMedCrossRefGoogle Scholar
  50. Sjögren P (1991) Genetic variation in relation to demography of peripheral pool frog populations (Rana lessonae). Evol Ecol 5:248–271CrossRefGoogle Scholar
  51. Smith MA, Green DM (2005) Dispersal and the metapopulation paradigm in amphibian ecology and conservation: Are all amphibian populations metapopulations? Ecography 28(1):110–128CrossRefGoogle Scholar
  52. Spear SF, Peterson CR, Matocq MD, Storfer A (2005) Landscape genetics of the blotched tiger salamander (Ambystoma tigrinum melanostictum). Mol Ecol 14(8):2553–2564PubMedCrossRefGoogle Scholar
  53. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306(5702):1783–1786PubMedCrossRefGoogle Scholar
  54. Templeton AR, Shaw K, Routman E, Davis SK (1990) The genetic consequences of habitat fragmentation. Ann Mo Bot Garden 77(1):13–27CrossRefGoogle Scholar
  55. Treves S, Vilsen B, Chiozzi P, Andersen JP, Zorzato F (1992) Molecular-cloning, functional expression and tissue distribution of the cDNA-encoding frog skeletal-muscle calsequestrin. Biochem J 283:767–772PubMedGoogle Scholar
  56. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4(3):535–538CrossRefGoogle Scholar
  57. Vos CC, Antonisse-De Jong AG, Goedhart PW, Smulders MJM (2001) Genetic similarity as a measure for connectivity between fragmented populations of the moor frog (Rana arvalis). Heredity 86:598–608PubMedCrossRefGoogle Scholar
  58. Wang IJ (2009) Fine-scale population structure in a desert amphibian: landscape genetics of the black toad (Bufo exsul). Mol Ecol 18(18):3847–3856PubMedCrossRefGoogle Scholar
  59. Waples RS, Gaggiotti O (2006) What is a population? An empirical evaluation of some genetic methods for identifying the number of gene pools and their degree of connectivity. Mol Ecol 15(6):1419–1439PubMedCrossRefGoogle Scholar
  60. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population-structure. Evolution 38(6):1358–1370CrossRefGoogle Scholar
  61. Wright S (1978) Evolution and the genetics of populations: variability within and among natural populations, vol 4. University of Chicago Press, ChicagoGoogle Scholar
  62. Zeisset I, Beebee TJC (2001) Determination of biogeographical range: an application of molecular phylogeography to the European pool frog Rana lessonae. Proc R Soc B 268:933–938PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Vanessa Sarasola-Puente
    • 1
    • 2
    • 3
  • María José Madeira
    • 2
  • Alberto Gosá
    • 1
  • Miguel Lizana
    • 3
  • Benjamín Gómez-Moliner
    • 2
  1. 1.Observatory of HerpetologyAranzadi Society of SciencesDonostia-San SebastiánSpain
  2. 2.Departamento de Zoología y Biología Celular AnimalUniversidad del País VascoVitoria-GasteizSpain
  3. 3.Departamento de Biología AnimalUniversidad de SalamancaSalamancaSpain

Personalised recommendations