, 130:241 | Cite as

Genetic differentiation and history of populations of the Italian treefrog Hyla intermedia: lack of concordance between mitochondrial and nuclear markers

  • Daniele Canestrelli
  • Andrea Verardi
  • Giuseppe Nascetti
Original Paper


The genetic differentiation among 33 populations of the Italian treefrog, Hyla intermedia (Anura: Hylidae), was investigated using both biparentally (23 allozyme loci) and maternally (partial mitochondrial cytochrome b gene) inherited markers. Two main population groups were evidenced by both markers, located north and south of the northern Apennines. However, the pattern of differentiation between these two groups was much less pronounced at allozymes than at mtDNA, leading to gene flow estimates that were 25 times lower at mitochondrial than at nuclear level. Also, the mtDNA divergence between the two groups was particularly marked for two cospecific lineages of anuran amphibians (the P-distance being on average 9.04%), while their average genetic distance at allozymes was comparatively low (D NEI = 0.07). This contrasting pattern of nuclear versus mitochondrial genetic variation is discussed in the context of: (1) marker specific selection, (2) secondary contact and sex-biased gene flow and (3) ancestral polymorphism and colonization from north to south. Finally we emphasize how, for population genetic studies, the use of multiple markers having distinct evolutionary properties can help unravel the existence of more complex evolutionary histories.


Allozymes Mitochondrial DNA Italian treefrog Population structure Discrepant genetic markers 



We are indebted to Fred Janzen for useful suggestions, which greatly improved a previous version of the manuscript. Sincere thanks are also due to Luciano Bullini, Roberta Cimmaruta and Francesca Zangari for useful discussions and suggestions, to Claudio Bagnoli and Paola Bellini for kind help during field sampling and manuscript preparation and to Mark Eltelton who reviewed the English. This work was funded by MIUR (Italian Ministry of University and of Scientific and Technological Research).


  1. Amorosi A, Colalongo ML, Fusco F (1999) Glacio-eustatic control of continental shallow marine cyclicity from late Quaternary deposits of the southeastern Pò plain, northern Italy. Quat Res 52:1–13CrossRefGoogle Scholar
  2. Andolfatto P (2001) Adaptive hitchhiking effects on genome variability. Curr Opin Genet Dev 11:635–641PubMedCrossRefGoogle Scholar
  3. Arnaud-Haond S, Monteforte M, Blanc F, Bonhomme F (2003) Evidence for male-biased effective sex ratio and recent step-by-step colonization in the bivalve Pinctada mazatlanica. J Evol Biol 16:790–796PubMedCrossRefGoogle Scholar
  4. Avise JC, Aquadro CF (1982) A comparative summary of genetic distances in the vertebrates. Evol Biol 15:151–185Google Scholar
  5. Avise JC, Neigel J, Arnold J (1984) Demographic influences on mitochondrial DNA lineage survivorship in animal populations. J Mol Evol 20:99–105PubMedCrossRefGoogle Scholar
  6. Avise JC (2004) Molecular markers, natural history and evolution, 2nd edn. Sinauer Associates, Sunderland, MAGoogle Scholar
  7. Avise JC (2000) Phylogeography: the history and formation of species. Harvard University Press, Cambridge, MAGoogle Scholar
  8. Ayala FJ, Powell JR, Tracey ML, Mourão CA, Pérez-Salas S (1972) Enzyme variability in the Drosophila willistoni group. IV. Genic variation in natural populations of Drosophila willistoni. Genetics 70:113–139PubMedGoogle Scholar
  9. Baer CF (1999) Among-locus variation in Fst: fish, allozymes and the Lewontin-Krakauer test revisited. Genetics 152:653–659PubMedGoogle Scholar
  10. Baker CS, Megrano-Gonzalez L, Calambokidis J, Perry A, Pichler F, Rosenbaum H, Straley JM, Urban-Ramirez J, Yamaguchi M, Von-Ziegesar O (1998). Population structure of nuclear and mitochondrial DNA variation among humpback whales in the North Pacific. Mol Ecol 7:695–707PubMedCrossRefGoogle Scholar
  11. Ballard JWO, Whitlock MC (2004) The incomplete natural history of mitochondria. Mol Ecol 13:729–744PubMedCrossRefGoogle Scholar
  12. Birky CW Jr, Maruyama T, Fuerst P (1983) An approach to population and evolutionary genetic theory for genes in mitochondria and chloroplasts, and some results. Genetics 103:513–527PubMedGoogle Scholar
  13. Birky CW Jr, Walsh JB (1988) Effects of linkage on rates of molecular evolution. Proc Natl Acad Sci USA 85:6414–6418PubMedCrossRefGoogle Scholar
  14. Borkin LJ (1999) Distribution of amphibians in North Africa, Europe, Western Asia, and the Former Soviet Union. In: Duellmann WE (ed) Patterns of distribution of amphibians: a global perspective. The Johns Hopkins University Press, Baltimore, MD, pp 329–420Google Scholar
  15. Brewer GJ, Sing CF (1970) An introduction to isozyme techniques. Academic Press, New York, LondonGoogle Scholar
  16. Broughton RE, Harrison RG (2003) Nuclear gene genealogies reveal historical, demographic, and selective factors associated with speciation in field crickets. Genetics 163:1389–1401PubMedGoogle Scholar
  17. Canestrelli D, Cimmaruta R, Costantini V, Nascetti G (2006) Genetic diversity and phylogeography of the Apennine yellow-bellied toad Bombina pachypus, with implications for conservation. Mol Ecol (in press)Google Scholar
  18. Cattani L (2003) Considerazioni floristiche sull’evoluzione degli ambienti. In: Guidi A, Piperno M (eds) Italia preistorica. Edizioni La Terza, Italy, pp 46–67Google Scholar
  19. Charlesworth B (1998) Measures of divergence between populations and the effect of forces that reduce variability. Mol Biol Evol 15:538–543PubMedGoogle Scholar
  20. Charlesworth B, Morgan MT, Charlesworth D (1993) The effect of deleterious mutations on neutral molecular variation. Genetics 134:1289–1303PubMedGoogle Scholar
  21. Cherty LM, Case SM, Wilson AC (1978) Frog perspective on the morphological difference between humans and chimpanzees. Science 200:209–211PubMedCrossRefGoogle Scholar
  22. Cremaschi M (2003) Mutamenti del clima nel Quaternario: le linee generali. In: Guidi A, Piperno M (eds) Italia preistorica. Edizioni La Terza, Italy, pp 3–15Google Scholar
  23. Cremaschi M (2003b) La penisola italiana nel Quaternario: aspetti geologici e geoarcheologici. In: Guidi A, Piperno M (eds) Italia preistorica. Edizioni La Terza, Italy, pp 15–39Google Scholar
  24. Crochet PA, Chen JZ, Pons JM, Lebreton JD, Hebert PDN, Bonhomme F (2003) Genetic differentiation at nuclear and mitochondrial loci among large white-headed gulls: sex biased interspecific gene flow? Evolution 57:2865–2878PubMedGoogle Scholar
  25. Delfino M, Bailon S (2000) Early Pleistocene herpetofauna from Cava Dell’Erba and Cava Pirro (Apulia, Southern Italy). Herp J 10:95–110Google Scholar
  26. Di Giovanni MV, Vlach MR, Giangiuliani G, Goretti E, Torricelli R (1998) Genetic analysis of the species of Sigara s. str. (Heteroptera, Corixidae) in the Italian Peninsula. Ital J Zool 65:393–397CrossRefGoogle Scholar
  27. Dubois A (1995) The valid scientific name of the Italian treefrog, with comments on the status of some early scientific names of Amphibia Anura, and on some articles of the Code concerning secondary homonyms. Dumerilia 2:55–71Google Scholar
  28. Edmonds CA, Lillie AS, Cavalli-Sforza LL (2004) Mutations arising in the wave front of an expanding population. Proc Natl Acad Sci USA 101:975–979PubMedCrossRefGoogle Scholar
  29. Fay JC, Wu CI (1999) A human population bottleneck can account for the discordance between patterns of mitochondrial versus nuclear dna variation. Mol Biol Evol 16:1003–1005PubMedGoogle Scholar
  30. Felsenstein J (1993) Phylogeny inference package (PHYLIP) Version 3.5c. University of Washington, SeattleGoogle Scholar
  31. Ferguson JWH (2002) On the use of genetic divergence for identifying species. Biol J Linn Soc 75:509–516CrossRefGoogle Scholar
  32. Giraudi C (2004) The Apennine glaciations in Italy. In: Ehlers J, Gibbard PL (eds) Quaternary glaciations—extent and chronology, part I: Europe. Developments in quaternary science, vol 2a. Elsevier Science, Amsterdam, pp 215–224Google Scholar
  33. Goudet J (1999) PCAGEN, a computer package, which performs principal component analysis (PCA) on gene frequency data. Available at Scholar
  34. Goudet J (2001) FSTAT, a program to estimate and test gene diversities and fixation indices (Version 2.9.3). Available at Scholar
  35. Goudet J, Perrin N, Waser P (2002) Tests for sex-biased dispersal using bi-parentally inherited genetic markers. Mol Ecol 11:1103–1114PubMedCrossRefGoogle Scholar
  36. Greenwood PJ (1980) Mating systems, philopatry and dispersal in birds and mammals. Anim Behav 28:1140–1162CrossRefGoogle Scholar
  37. Hudson RD, Coyne JA (2002) Mathematical consequences of the genealogical species concept. Evolution 56:1557–1565PubMedGoogle Scholar
  38. Hare MP, Avise JC (1998) Population structure in the American oyster as inferred by nuclear gene genealogies. Mol Biol Evol 15:119–128PubMedGoogle Scholar
  39. Hare MP (2001) Prospects for nuclear gene phylogeography. Trends Ecol Evol 16:700–706CrossRefGoogle Scholar
  40. Harris H (1966) Enzyme polymorphism in man. Proc R Soc Lond B 164:298–310PubMedCrossRefGoogle Scholar
  41. Harris H, Hopkinson DA (1976) Handbook of enzyme electrophoresis in human genetics. North-Holland Publishing Company Inc., AmsterdamGoogle Scholar
  42. Helbig AJ, Salomon M, Bensch S, Seibold I (2001) Male-biased gene flow across an avian hybrid zone: evidence from mitochondrial and microsatellite DNA. J Evol Biol 14:277–287CrossRefGoogle Scholar
  43. Hoelzer GA (1997) Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees revisited. Evolution 51:622–626CrossRefGoogle Scholar
  44. Jehle R, Arntzen JW (2002) Microsatellite markers in amphibian conservation genetics. Herp J 12:1–9Google Scholar
  45. Johnson JA, Toepfer JE, Dunn PO (2003) Contrasting patterns of mitochondrial and microsatellite population structure in fragmented populations of greater prairie-chickens. Mol Ecol 12:3335–3347PubMedCrossRefGoogle Scholar
  46. Kawamura T, Nishioka M (1977) Reproductive biology of Japanese anurans. In: Tylor DH, Guttman SI (eds) Reproductive biology of amphibians. Plenum Press, New York, pp 103–139Google Scholar
  47. Kimura M (1980) A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. J Mol Evol 16:111–120PubMedCrossRefGoogle Scholar
  48. Kocher TD, Thomas WK, Meyer A, Pääbo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA 86:6196–6200PubMedCrossRefGoogle Scholar
  49. Lampert KP, Rand AS, Mueller UG, Ryan MJ (2003) Fine-scale genetic pattern and evidence for sex-biased dispersal in the tungara frog, Physalaemus pustulosus. Mol Ecol 12:3325–3334PubMedCrossRefGoogle Scholar
  50. Lanza B (1983) Anfibi, Rettili. Guide per il riconoscimento delle specie animali delle acque interne italiane. CNR, ItalyGoogle Scholar
  51. Lee JE, Yang DE, Kim YR, Lee H, Lee HI, Yang SY, Lee HY (1999) Genetic relationships of Korean treefrogs (Amphibia; Hylidae) based on mitochondrial cytochrome b and 12S rRNA genes. Korean J Biol Sci 3:295–301Google Scholar
  52. Lee MSY (2003) Species concepts and species reality: salvaging a Linnean rank. J Evol Biol 16:179–188PubMedCrossRefGoogle Scholar
  53. Lewontin RC, Krakauer J (1973) Distribution of gene frequency as a test of the theory of the selective neutrality of polymorphisms. Genetics 74:175–195PubMedGoogle Scholar
  54. Marsh DM, Trenham PC (2000) Metapopulation dynamics and amphibian conservation. Cons Biol 15:40–49CrossRefGoogle Scholar
  55. Martínez-Solano I, Gonçalves H, Arntzen JW, García-París M (2004) Phylogenetic relationships and biogeography of midwife toads (Discoglossidae: Alytes). J Biogeog 31:603–618Google Scholar
  56. Maynard Smith J, Haigh J (1974) The hitchhiking effect of a favorable gene. Genet Res 23:23–35CrossRefGoogle Scholar
  57. Mazzotti S, Caramori G, Barbieri C (1999) Atlante degli Anfibi e dei Rettili dell’Emilia-Romagna. Quad Staz Ecol Civ Mus Stor Nat Ferrara 12:121Google Scholar
  58. Miola A, Albanese D, Valentini G, Corain L (2003) Pollen data for a biostratigraphy of LGM in the Venetian Pò plain. Ital J Quat Sci 16:21–25Google Scholar
  59. Monsen KJ, Blouin MS (2003) Genetic structure in a montane ranid frog: restricted gene flow and nuclear-mitochondrial discordance. Mol Ecol 12:3275–3286PubMedCrossRefGoogle Scholar
  60. Montuire S, Marcolini F (2002) Palaeoenvironmental significance of the mammalian faunas of Italy since the Pliocene. J Quat Sci 17:87–96CrossRefGoogle Scholar
  61. Moore WS (1995) Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nucelar-gene trees. Evolution 49:718–726CrossRefGoogle Scholar
  62. Moritz C, Schneider CJ, Wake DB (1992) Evolutionary relationships within Ensatina eschscholtzi complex confirm the ring species interpretation. Syst Zool 41:273–291Google Scholar
  63. Nascetti G, Capula M, Lanza M, Bullini L (1985) Ricerche elettroforetiche su anfibi della regione mediterranea: aspetti tassonomici ed evolutivi. Riassunti del IV congresso associazione Ghigi, p 45Google Scholar
  64. Nascetti G, Lanza B, Bullini L (1995) Genetic data for the specific status of the Italian treefrog (Amphibia: Anura: Hylidae). Amphib Reptil 16:215–227Google Scholar
  65. Nei M (1972) Genetic distance between populations. Am Nat 106:283–292CrossRefGoogle Scholar
  66. Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 83:583–589Google Scholar
  67. Neigel JE (2002). Is F ST obsolete? Cons Gen 3:167–173CrossRefGoogle Scholar
  68. Neigel JE, Avise AC (1986) Phylogenetic relationships of mitochondrial DNA under various demographic models of speciation. In: Karlin K, Nevo E (eds) Evolutionary processes and theory. Academic Press, New York, pp 515–534Google Scholar
  69. Paillette M (1967) Valeur taxonomique des émissions sonores chez les Hyla (Amphibiens, Anoures) de la faune française. CR Acad Sci Paris sér D 264:1626–1628Google Scholar
  70. Palumbi SR, Baker CS (1994) Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales. Mol Biol Evol 11:426–435PubMedGoogle Scholar
  71. Piel WH, Nutt KJ (2000) One species or several? Discordant patterns of geographic variation between allozymes and mtDNA sequences among spiders in the genus Metepeira (Araneae: Araneidae). Mol Phylogenet Evol 15:414–418PubMedCrossRefGoogle Scholar
  72. Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818PubMedCrossRefGoogle Scholar
  73. Poulik MD (1957) Starch gel electrophoresis in a discontinuous system of buffers. Nature 180:1477PubMedCrossRefGoogle Scholar
  74. Ravazzi C, Strick MR (1995) Vegetation change in a climatic cycle of Early Pleistocene age in the Leffe Basin (Northern Italy). Palaeogeog Palaeoclim Palaeoecol 117:105–122CrossRefGoogle Scholar
  75. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225CrossRefGoogle Scholar
  76. Richards CM, Moore WS (1996) A phylogeny for the African treefrog family Hyperoliidae based on mitochondrial DNA. Mol Phylogenet Evol 5:522–532PubMedCrossRefGoogle Scholar
  77. Ripplinger JI, Wagner RS (2004) Phylogeography of northern populations of the pacific treefrog, Pseudacris regilla. Northwest Nat 85:118–125Google Scholar
  78. Roe BA, Ma DP, Wilson RK, Wong JFH (1985) The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J Biol Chem 260:9759–9774PubMedGoogle Scholar
  79. Ron SR, Santos JC, Cannatella DC (2006) Phylogeny of the túngara frog genus Engystomops (=Physalaemus pustulosus species group; Anura: Leptodactylidae). Mol Phylogenet Evol 39:392–403PubMedCrossRefGoogle Scholar
  80. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  81. Schierup MH, Vekemans X, Charlesworth D (2000) The effect of subdivision on variation at multi-allelic loci under balancing selection. Genet Res Camb 76:51–62Google Scholar
  82. Schlotterer C (2003) Hitchhiking mapping—functional genomics from the population genetics perspective. Trends Genet 19:32–38PubMedCrossRefGoogle Scholar
  83. Schneider H (1974) Structure of mating calls and relationship of the European tree frogs (Hylidae, Anura). Oecologia 14:99–110CrossRefGoogle Scholar
  84. Schreiber E (1875) Herpetologia europaea. Druck and Verlag von Friedrich Vieweg and Sohn, Braunschweig, 639 p, Chap. XVIIGoogle Scholar
  85. Selander RK, Smith MH, Yang SY, Johnson WE, Gentry JB (1971) Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old-field mouse. In: Studies in genetics IV, vol 7103. University of Texas Publications, TX, pp 49–90Google Scholar
  86. Shaw KL (2002) Conflict between mitochondrial and nuclear DNA phylogenies of a recent species radiation: what mitochondrial reveals and conceals about modes of speciation in Hawaiian crickets. Proc Natl Acad Sci USA 99:16122–16127PubMedCrossRefGoogle Scholar
  87. Shaw CR, Prasad R (1970) Starch gel electrophoresis of enzymes - a compilation of recipes. Biochem Genet 4:297–320PubMedCrossRefGoogle Scholar
  88. Slatkin M, Wiehe T (1998) Genetic hitch-hiking in a subdivided population. Genet Res 71:155–160PubMedCrossRefGoogle Scholar
  89. Stefani F, Galli P, Crosa G, Zaccara S, Calamari D (2004) Alpine and Apennine barriers determining the differentiation of the rudd (Scardinius erythrophthalmus L.) in the Italian peninsula. Ecol Fresh Fish 13:168–175CrossRefGoogle Scholar
  90. Swofford DL, Selander RB (1999) BIOSYS-2: a computer program for the analysis of allelic variation in population genetics and biochemical systematics (Release 2.0). University of Illinois, Urbana, Champaign, ILGoogle Scholar
  91. Swofford DL (2003) PAUP*: phylogenetic analysis using parsimony (* and other methods). Beta, Version 4.0b 10. Sinauer Associates, Sunderland, MAGoogle Scholar
  92. Szymura JM, Spolsky C, Uzzell T (1985) Concordant change in mitochondrial and nuclear genes in a hybrid zone between two frog species (genus Bombina). Experientia 41:1469–1470CrossRefGoogle Scholar
  93. Tajima F (1993) Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135:599–607PubMedGoogle Scholar
  94. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  95. Ting TC, Tsaur SC, Wu CI (2000) The phylogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus. Proc Natl Acad Sci USA 97:5313–5316PubMedCrossRefGoogle Scholar
  96. Veith M (1996) Molecular markers and species delimitation: examples from the European batrachofauna. Amphib Reptil 17:303–314Google Scholar
  97. Weigt LA, Crawford AJ, Rand AS, Ryan MJ (2005) Biogeography of the tungara frog, Physalaemus pustulosus: a molecular perspective. Mol Ecol 14:3857–3876PubMedCrossRefGoogle Scholar
  98. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370CrossRefGoogle Scholar
  99. Whitlock MC, McCauley DE (1999) Indirect measures of gene flow and migration: FST not equal 1/(4Nm + 1). Heredity 82:117–125PubMedCrossRefGoogle Scholar
  100. Wright S (1951) The genetical structure of populations. Ann Eugen 15:323–353Google Scholar
  101. Zhang DX, Hewitt GM (2003) Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects. Mol Ecol 12:563–584PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Daniele Canestrelli
    • 1
  • Andrea Verardi
    • 2
  • Giuseppe Nascetti
    • 1
  1. 1.Dipartimento di Ecologia e Sviluppo Economico SostenibileUniversità della TusciaViterboItaly
  2. 2.Dipartimento di Genetica e Biologia Molecolare “Charles Darwin”Università degli Studi di Roma “La Sapienza”RomaItaly

Personalised recommendations