Evolutionary Ecology

, Volume 33, Issue 5, pp 671–686 | Cite as

Epigenomic changes in the túngara frog (Physalaemus pustulosus): possible effects of introduced fungal pathogen and urbanization

  • Mark J. GarciaEmail author
  • Sofía Rodríguez-Brenes
  • Ashley Kobisk
  • Laurie Adler
  • Michael J. Ryan
  • Ryan C. Taylor
  • Kimberly L. Hunter
Original Paper


Amphibian populations are being threatened by human related activities including the spread of the fungal pathogen, Batrachochytrium dendrobatidis (Bd) and urbanization. With growing losses in global amphibian biodiversity, it is essential to document how amphibian populations are responding to rapid environmental changes. While most evolutionary processes, e.g. changes in allelic frequencies, may be too slow to allow adequate response to environmental changes, epigenetic modifications can rapidly translate environmental changes into adaptive phenotypic responses. Epigenetic modifications come in multiple, non-exclusive forms, the most notable being DNA methylation. Here we sought to examine variation in the frequency of DNA methylation among four túngara frog populations distributed across Gamboa, Panama; which vary in both their level of fungal presence/prevalence and urbanization. DNA samples were collected from amplexed (male–female) pairs and frequency of DNA methylation was analyzed using a methylation-sensitive amplified fragment length polymorphism protocol. We found significant variation in DNA methylation among populations, and correlations between Bd infection status and methylation patterns. Urbanization, however, had no influences on the frequency of DNA methylation. These data suggest epigenetic modifications are substantially flexible across fine-scale, environmental gradients and there appears to be possible biologically relevant links between DNA methylation and Bd infection status. Our results provide a basis for future work investigating the causal role epigenetics have in mediating phenotypic response to human-induced, environmental changes.


Túngara frog Batrachochytrium dendrobatidis Epigenetics Urbanization DNA methylation 



We thank Salisbury University for providing funding for equipment. We are grateful to Matthew Murphy, Paul Swim, and Tyler Bowling for their assistance with the MS-AFLP procedure. Rosalind Ludovici and Derek Coss assisted with data analysis. We would also like to thank two anonymous reviewers for their input during the drafting of this work.

Compliance with ethical standards

Conflict of interest

The authors declare they have no conflict of interests.

Ethical standards

Handling and toe clipping were performed in accordance with The American Society of Ichthyology and Herpetologists’ “Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research.” All experiments were conducted under Salisbury University’s Institutional Care and Use Committee (IACUC Protocol # SU 0036).

Supplementary material

10682_2019_10001_MOESM1_ESM.docx (82 kb)
Supplementary material 1 (DOCX 82 kb)


  1. Asplen MK, Anfora G, Biondi A et al (2015) Invasion biology of spotted wing Drosophila (Drosophila suzukii): a global perspective and future priorities. J Pest Sci 88:469–494Google Scholar
  2. Becker G, Zamudio KR (2011) Tropical amphibian populations experience higher disease risk in natural habitats. PNAS 108(24):9893–9898Google Scholar
  3. Bender J (2004) DNA methylation and epigenetics. Annu Rev Plant Biol 55(1):41–68Google Scholar
  4. Berger L, Speare R, Daszak P et al (1998) Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. PNAS 95(15):9031–9036Google Scholar
  5. Bird A (2007) Perceptions of epigenetics. Nature 447:396–398Google Scholar
  6. Bossdorf O, Richards CL, Pigliucci M (2008) Epigenetics for ecologists. Ecol Lett 11(2):106–115Google Scholar
  7. Burggren WW, Crews D (2014) Epigenetics in comparative biology: why we should pay attention. J Integr Comp Biol 54(1):7–20Google Scholar
  8. Ellison A, Lopez CMR, Moran P et al (2015) Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates. Proc R Soc B 282(1819):20151900Google Scholar
  9. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14(8):2611–2620Google Scholar
  10. Foust CM, Preite V, Schrey AW et al (2016) Genetic and epigenetic differences associated with environmental gradients in replicate populations of two salt marsh perennials. Mol Ecol 25(8):1639–1652Google Scholar
  11. Gao L, Geng Y, Li B et al (2010) Genome-wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation. Plant Cell Environ 33(11):1820–1827Google Scholar
  12. Greally JM (2018) A user's guide to the ambiguous word 'epigenetics'. Nat Rev Mol Cell Biol 19(4):207–208Google Scholar
  13. Gridi-Papp M, Rand AS, Ryan MJ (2006) Animal communication: complex call production in the túngara frog. Nature 441:38Google Scholar
  14. Halfwerk W, Jones PL, Taylor RC et al (2014) Risky ripples allow bats and frogs to eavesdrop on a multisensory sexual display. Science 343(6169):413–416Google Scholar
  15. Halfwerk W, Blaas M, Kramer L et al (2019) Adaptive changes in sexual signaling in response to urbanization. Nat Ecol Evol 3(3):374–383Google Scholar
  16. Hammond SA, Nelson CJ, Helbing CC (2016) Environmental influences on the epigenomes of herpetofauna and fish. Biochem Cell Biol 94:95–100Google Scholar
  17. Herrera CM, Bazaga P (2010) Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis. New Phytol 187(3):867–876Google Scholar
  18. Herrera CM, Bazaga P (2011) Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory. Mol Ecol 20(8):1675–1688Google Scholar
  19. Herrera CM, Bazaga P (2016) Genetic and epigenetic divergence between disturbed and undisturbed subpopulations of a Mediterranean shrub: a 20-year field experiment. Ecol Evol 187(3):3832–3847Google Scholar
  20. Hof C, Araujo MB, Jetz W, Rahbeck C (2014) Additive threats from pathogens, climate and land-use change for global amphibian diversity. Nat Lett 480:516–519Google Scholar
  21. Holland BR, Clarke AC, Meudt HM (2008) Optimizing automated AFLP scoring parameters to improve phylogenetic resolution. Syst Biol 57(3):347–366Google Scholar
  22. Hu J, Barrett RD (2017) Epigenetics in natural animal populations. J Evol Biol 30:1612–1632Google Scholar
  23. Jackson ST, Sax DF (2010) Balancing biodiversity in a changing environment: extinction debt, immigration credit and species turnover. Trends Ecol Evol 25(3):153–160Google Scholar
  24. Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33:245–254Google Scholar
  25. James TY, Toledo LF, Rödder D et al (2015) Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research. Ecol Evol 5(18):4079–4097Google Scholar
  26. Jirtle RL, Skinner MK (2007) Environmental epigenomics and disease susceptibility. Nat Rev Genet 8:253–262Google Scholar
  27. Johannes F, Porcher E, Teixeira FK et al (2009) Assessing the impact of transgenerational epigenetic variation on complex traits. PLoS Genet 5(6):e1000530Google Scholar
  28. Johnson PTJ, Mckenzie VJ, Peterson AC et al (2011) Regional decline of an iconic amphibian associated with elevation, land-use change, and invasive species. Conserv Biol 25(3):556–566Google Scholar
  29. Jombart T, Ahmed I (2011) adegenet 1.3-1: New tools for the analysis of genome-wide SNP data. Bioinformatics 27:3070–3071Google Scholar
  30. Jombart T, Devillard S, Balloux F (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet 11:94Google Scholar
  31. Kolby J, Daszak P (2016). The emerging amphibian fungal disease, chytridiomycosis: a key example of the global phenomenon of wildlife emerging infectious diseases. In: Scheld W, Hughes J, Whitley R (eds) Emerging infections, vol 10. ASM Press, Washington, DCGoogle Scholar
  32. Koo TK, Li MY (2016) A guideline of selecting and reporting intraclass correlation coefficients for reliability research. J Chiropr Med 15:155–163Google Scholar
  33. Kosch TA, Bataille A, Didinger C et al (2016) MHC selection dynamics in pathogen-infected túngara frog (Physalaemus pustulosus) populations. Biol Lett 12:20160345Google Scholar
  34. Lampert KP, Rand AS, Mueller UG, Ryan MJ (2003) Fine-scale genetic pattern and evidence for sex-biased dispersal in the túngara frog Physalaemus pustulosus. Mol Ecol 12(12):3325–3334Google Scholar
  35. Lankau R, Jørgensen PS, Harris DJ, Sih A (2011) Incorporating evolutionary principles into environmental management and policy. Evol Appl 4(2):315–325Google Scholar
  36. Lappalainen T, Greally JM (2017) Associating cellular epigenetic models with human phenotypes. Nat Rev Genet 18(7):441–451Google Scholar
  37. Ledon-Rettig CC, Richards CL, Martin LB (2013) Epigenetics for behavioral ecologists. Behav Ecol 24:311–324Google Scholar
  38. Liebl AL, Schrey AW, Richards CL, Martin LB (2013) Patterns of DNA methylation throughout a range expansion of an introduced songbird. Integr Comp Biol 53(2):351–358Google Scholar
  39. Liu S, Sun K, Jiang T et al (2012) Natural epigenetic variation in the female great roundleaf bat (Hipposideros armiger) populations. Mol Gene Genomics 287(8):643–650Google Scholar
  40. McNew SM, Beck D, Sadler-Riggleman I et al (2017) Epigenetic variation between urban and rural populations of Darwin’s finches. BMC Evol Biol 17:183Google Scholar
  41. Nilsson EE, Skinner MK (2015) Environmentally induced epigenetic transgenerational inheritance of disease susceptibility. Transl Res 165(1):12–17Google Scholar
  42. Nori J, Lemes P, Urbina-Cardona N et al (2015) Amphibian conservation, land-use changes and protected areas: a global overview. Biol Conserv 191:367–374Google Scholar
  43. Palumbi S (2001) Humans as the world’s greatest evolutionary force. Science 293(5536):1786–1790Google Scholar
  44. Paradis E (2010) Pegas: an R package for population genetics with an integrated-modular approach. Bioinformatics 26(3):419–420Google Scholar
  45. Pereira HM, Leadley PW, Proença V et al (2010) Scenarios for global biodiversity in the twenty-first century. Science 330(6010):1496–1501Google Scholar
  46. Perez-Figueroa A (2013) MSAP: a tool for the statistical analysis of methylation-sensitive amplified polymorphism data. Mol Ecol Resour 13(3):522–527Google Scholar
  47. Phillips BL, Puschendorf R (2013) Do pathogens become more virulent as they spread? Evidence from the amphibian declines in Central America. Proc R Soc B Biol Sci 280(1766):20131290Google Scholar
  48. Platt A, Gugger PF, Pellegrini M, Sork VL (2015) Genome-wide signature of local adaptation linked to variable CpG methylation in oak populations. Mol Ecol 24(15):3823–3830Google Scholar
  49. Pounds JA, Crump ML (1994) Declines and climate amphibian the case disturbance: toad and the of the golden frog harlequin. Conserv Biol 8(1):72–85Google Scholar
  50. Pounds JA, Fogden MPL, Savage JM, Gorman GC (1997) Tests of null models for amphibian declines on a tropical mountain. Conserv Biol 11(6):1307–1322Google Scholar
  51. R Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.
  52. Robertson BA, Rehage JS, Sih A (2013) Ecological novelty and the emergence of evolutionary traps. Trends Ecol Evol 28(9):552–560Google Scholar
  53. Robinson B, Pfennig D (2013) Inducible competitors and adaptive diversification. Curr Zool 59(4):537–552Google Scholar
  54. Rodríguez-Brenes S, Rodriguez D, Ibá R, Ryan MJ (2016) Spread of amphibian chytrid fungus across lowland populations of túngara frogs in Panamá. PLoS ONE 11(5):e0155745Google Scholar
  55. Ryan MJ (1985) The túngara frog, a study in sexual selection and communication. University of Chicago Press, ChicagoGoogle Scholar
  56. Savage AE, Terrell KA, Gratwicke B et al (2016) Reduced immune function predicts disease susceptibility in frogs infected with a deadly fungal pathogen. Conserv Physiol 4(1):cow011Google Scholar
  57. Schrey AW, Alvarez M, Foust CM et al (2013) Ecological epigenetics: beyond MS-AFLP. Integr Comp Biol 53(2):340–350Google Scholar
  58. Sih A (2013) Understanding variation in behavioural responses to human-induced rapid environmental change: a conceptual overview. Anim Behav 85(5):1077–1088Google Scholar
  59. Sih A, Stamps J, Yang LH, McElreath R, Ramenofsky M (2010) Behavior as a key component of integrative biology in a human-altered world. Integr Comp Biol 50(6):934–944Google Scholar
  60. Skinner MK (2014) Environmental stress and epigenetic transgenerational inheritance. BMC Med 12(153):1–5Google Scholar
  61. Smith TA, Martin MD, Nguyen M, Mendelson TC (2016) Epigenetic divergence as a potential first step in darter speciation. Mol Ecol 25(8):1883–1894Google Scholar
  62. Strimmer K (2008) fdrtool: a versatile R package for estimating local and tail area-based false discovery rates. Bioinformatics 24(12):1461–1462Google Scholar
  63. Taylor RC, Ryan MJ (2013) Interactions of multisensory components perceptually rescue túngara frog mating signals. Science 341(6143):273–274Google Scholar
  64. Vaiserman A (2015) Epidemiologic evidence for association between adverse environmental exposures in early life and epigenetic variation: a potential link to disease susceptibility? Clin Epigenet 7(1):96Google Scholar
  65. Weaver ICG, Cervoni N, Champagne FA et al (2004) Epigenetic programming by maternal behavior. Nat Neurosci 7(8):847–854Google Scholar
  66. Wenzel MA, Piertney SB (2014) Fine-scale population epigenetic structure in relation to gastrointestinal parasite load in red grouse (Lagopus lagopus scotica). Mol Ecol 23(17):4256–4273Google Scholar
  67. Whitfield PE, Gardner T, Vives SP et al (2002) Biological invasion of the Indo-Pacific lionfish Pterois volitans along the Atlantic coast of North America. Mar Ecol Prog Ser 235:289–297Google Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Mark J. Garcia
    • 1
    • 3
    Email author
  • Sofía Rodríguez-Brenes
    • 2
  • Ashley Kobisk
    • 3
  • Laurie Adler
    • 3
  • Michael J. Ryan
    • 2
    • 4
  • Ryan C. Taylor
    • 3
    • 4
  • Kimberly L. Hunter
    • 3
  1. 1.Department of EntomologyUniversity of KentuckyLexingtonUSA
  2. 2.Department of Integrative BiologyUniversity of TexasAustinUSA
  3. 3.Department of Biological SciencesSalisbury UniversitySalisburyUSA
  4. 4.Smithsonian Tropical Research InstituteBalboa AnconPanama

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