The evolution of polymorphism in the warning coloration of the Amazonian poison frog Adelphobates galactonotus


While intraspecific variation in aposematic signals can be selected for by different predatory responses, their evolution is also contingent on other processes shaping genetic variation. We evaluate the relative contributions of selection, geographic isolation, and random genetic drift to the evolution of aposematic color polymorphism in the poison frog Adelphobates galactonotus, distributed throughout eastern Brazilian Amazonia. Dorsal coloration was measured for 111 individuals and genetic data were obtained from 220 individuals at two mitochondrial genes (mtDNA) and 7963 Single Nucleotide Polymorphisms (SNPs). Four color categories were described (brown, blue, yellow, orange) and our models of frog and bird visual systems indicated that each color was distinguishable for these taxa. Using outlier and correlative analyses we found no compelling genetic evidence for color being under divergent selection. A time-calibrated mtDNA tree suggests that the present distribution of dorsal coloration resulted from processes occurring during the Pleistocene. Separate phylogenies based on SNPs and mtDNA resolved the same well supported clades, each containing different colored populations. Ancestral character state analysis provided some evidence for evolutionary transitions in color type. Genetic structure was more strongly associated with geographic features, than color category, suggesting that the distribution of color is explained by localized processes. Evidence for geographic isolation together with estimates of low effective population size implicates drift as playing a key role in color diversification. Our results highlight the relevance of considering the neutral processes involved with the evolution of traits with important fitness consequences.

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  1. Amézquita A, Ramos Ó, González MC, Rodríguez C, Medina I, Simões PI et al. (2017) Conspicuousness, color resemblance, and toxicity in geographically diverging mimicry: the pan-Amazonian frog Allobates femoralis. Evolution 71:1039–1050

  2. Anderson S, Prager M (2006) Quantifying colors. In: McGraw KJ, Hill GE (eds) Bird Coloration, Vol I. Harvard University Press, Cambridge, MA, pp 41–89

  3. Bagnara JT, Fernandez PJ, Fujii R (2007) On the blue coloration of vertebrates. Pigment Cell Res 20:14–26

  4. Bagnara JT, Matsumoto J (2007) Comparative anatomy and physiology of pigment cells in nonmammalian tissues. In: Norlund J, Boissy R, Hearing V, King R, Oetting W, Ortonne JP (eds) The pigmentary system: physiology and pathophysiology, Blackwell Publishing Ltd, Oxford, UK, pp 11–59

  5. Barrett RDH, Schluter D (2008) Adaptation from standing genetic variation. Trends Ecol Evol 23:8–44

  6. Bell RC, Zamudio KR (2012) Sexual dichromatism in frogs: natural selection, sexual selection and unexpected diversity. Proc Biol Sci 279:4687–4693

  7. Benestan L, Quinn BK, Maaroufi H, Laporte M, Clark FK, Greenwood SJ et al. (2016) Seascape genomics provides evidence for thermal adaptation and current-mediated population structure in American lobster (Homarus americanus). Mol Ecol 25:5073–5092

  8. Bouckaert RR (2010) DensiTree: making sense of sets of phylogenetic trees. Bioinformatics 26:1372–1373

  9. Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu CH, Xie D et al. (2014) BEAST 2: a software platform for bayesian evolutionary analysis. PLoS Comput Biol 10:1–6

  10. Boul KE, Funk WC, Darst CR, Cannatella DC, Ryan MJ (2007) Sexual selection drives speciation in an Amazonian frog. Proc Biol Sci 274:399–406

  11. Brown JL, Twomey E (2009) Complicated histories: three new species of poison frogs of the genus Ameerega (Anura: Dendrobatidae) from north-central Peru. Zootaxa: 2049:1–38

  12. Bryant D, Bouckaert R, Felsenstein J, Rosenberg NA, Roychoudhury A (2012) Inferring species trees directly from biallelic genetic markers: bypassing gene trees in a full coalescent analysis. Mol Biol Evol 29:1917–1932

  13. Catchen J, Hohenlohe Pa, Bassham S, Amores A, Cresko WA (2013) Stacks: an analysis tool set for population genomics. Mol Ecol 22:3124–3140

  14. Che J, Chen H-M, Yang J-X, Jin J-Q, Jiang K, Yuan Z-Y et al. (2012) Universal COI primers for DNA barcoding amphibians. Mol Ecol Resour 12:247–258

  15. Cheng H, Sinha A, Cruz FW, Wang X, Edwards RL, D’Horta FM et al. (2013) Climate change patterns in Amazonia and biodiversity. Nat Commun 4:1411

  16. Chouteau M, Angers B (2012) Wright’s shifting balance theory and the diversification of aposematic signals PLoS ONE 7:e34028

  17. Clarke RT, Rothery P, Raybould AF (2002) Confidence limits for regression relationships between distance matrices: Estimating gene flow with distance. J Agric Biol Environ Stat 7:361–372

  18. Clement M, Posada D, Crandall KA (2000) TCS: a computer program to estimate gene genealogies. Mol Ecol 9:1657–1659

  19. Comeault AA, Noonan BP (2011) Spatial variation in the fitness of divergent aposematic phenotypes of the poison frog, Dendrobates tinctorius. J Evol Biol 24:1374–1379

  20. Cunningham CW, Omland KE, Oakley TH (1998) Reconstructing ancestral character states, a critical reappraisal. TREE 13:361–366

  21. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772

  22. Darst CR, Cummings ME (2006) Predator learning favors mimicry of a less-toxic model in poison frogs. Nature 440:208–211

  23. Devlin B, Roeder K (1999) Genomic control for association studies. Biometrics 55:997–1004

  24. Do C, Waples RS, Peel D, Macbeth GM, Tillett BJ, Ovenden JR (2014) NeEstimator v2: re-implementation of software for the estimation of contemporary effective population size (Ne) from genetic data. Mol Ecol Resour 14:209–214

  25. Drummond AJ, Rambaut A (2007) Bayesian evolutionary analysis by sampling trees. In: BMC Evolut Biol, Vol 7, 7:214.

  26. Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29:1969–1973

  27. Duchêne S, Lanfear R (2015) Phylogenetic uncertainty can bias the number of evolutionary transitions estimated from ancestral state reconstruction methods. J Exp Zool (Mol Dev Evol) 324:517–524

  28. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform 5:113

  29. Endler JA (1990) On the measurement and classification of colors in studies of animal colour patterns. Biol J Linn Soc 41:315–332

  30. Endler JA (1992) Signals, signal directions and the direction of evolution. Am Nat 139:125–153

  31. Endler JA, Greenwood JJD (1988) Frequency-dependent predation, crypsis and aposematic coloration [and Discussion]. Philos Trans R Soc B Biol Sci 319:505–523

  32. Endler JA, Mappes J (2004) Predator mixes and the conspicuousness of aposematic signals. Am Nat 4:532–547

  33. Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:186–194

  34. Exnerová A, Svádová K, Fouá P, Fučíková E, Ježová D, Niederlová A et al. (2008) European birds and aposematic Heteroptera: review of comparative experiments. Bull Insectol 61:163–165

  35. Ferreira A, Jehle R, Stow AJ, Lima AP (2018) Soil and forest structure predicts large scale patterns of occurrence and local abundance of a widespread Amazonian frog. PeerJ 26.

  36. Frichot E, François O (2015) LEA: an R package for landscape and ecological association studies (B O’Meara, Ed.). Methods Ecol Evol 6:925–929

  37. Frichot E, Schoville SD, Bouchard G, Francois O (2013) Testing for associations between loci and environmental gradients using latent factor mixed models. Mol Biol Evol 30:1687–1699

  38. Frichot E, Schoville SD, De Villemereuil P, Gaggiotti OE, François O (2015) Detecting adaptive evolution based on association with ecological gradients: orientation matters! Heredity 115:22–28

  39. Gehara M, Summers K, Brown JL (2013) Population expansion, isolation and selection: novel insights on the evolution of color diversity in the strawberry poison frog. Evol Ecol 27:797–824

  40. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704

  41. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

  42. Hofreiter M, Schöneberg T (2010) The genetic and evolutionary basis of colour variation in vertebrates. Cell Mol Life Sci 67:2591–603

  43. Hoogmoed MS, Avila-Pires TCS (2012) Inventory of color polymorphism in populations of Dendrobates galactonotus (Anura: Dendrobatidae), a poison frog endemic to Brazil. Phyllomedusa 11:95–115

  44. Jaccoud D, Peng K, Feinstein D, Kilian a (2001) Diversity arrays: a solid state technology for sequence information independent genotyping. Nucleic Acids Res 29:E25

  45. Jiggins CD, Naisbit RE, Coe RL, Mallet J (2001) Reproductive isolation caused by colour pattern mimicry. Nature 411:302–305

  46. Jombart T, Ahmed I (2011) adegenet 1.3-1: new tools for the analysis of genome-wide SNP. Data Bioinform 27:3070–3071

  47. Kaefer IL, Tsuji-Nishikido BM, Mota EP, Farias IP, Lima AP (2013) The early stages of speciation in Amazonian forest frogs: phenotypic conservatism despite strong genetic structure. Evol Biol 40:228–245

  48. Kang C, Sherratt TN, Kim YE, Shin Y, Moon J, Song U et al. (2017) Differential predation drives the geographical divergence in multiple traits in aposematic frogs. Behav Ecol 28:1122–1130

  49. Kemp DJ, Herberstein ME, Fleishman LJ, Endler JA, Bennett ATD, Dyer AG et al. (2015) An integrative framework for the appraisal of coloration in nature. Am Nat 185:705–724

  50. Kemppainen P, Knight CG, Sarma DK, Hlaing T, Prakash A, Maung Maung YN et al. (2015) Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions, local adaptation and geographic structure. Mol Ecol Resour 15:1031–1045

  51. Kilian A, Wenzl P, Huttner E, Carling J, Xia L, Blois H, et al. (2012) Diversity arrays technology: a generic genome profiling technology on open platforms. In: Pompanon F, Bonin A (eds) Data production and analysis in population genomics SE - 5, Methods in Molecular Biology. Berlin, German: Springer, Vol 888, pp 67–89

  52. Lambert SM, Geneva AJ, Luke Mahler D, Glor RE (2013) Using genomic data to revisit an early example of reproductive character displacement in Haitian Anolis lizards. Mol Ecol 22:3981–95

  53. Lawrence JP, Rojas B, Fouquet A, Mappes J, Blanchette A, Saporito RA, Bosque RJ, Courtois EA, Noonan BP (2019) Weak warning signals can persist in the absence of gene flow. PNAS 38:19037–19045

  54. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452

  55. Lindström L, Alatalo R, Mappes J, Riipi M, Vertainen L (1999) Can aposematic signals evolve by gradual change? Nature 397:249–251

  56. Maia R, Eliason CM, Bitton P-P, Doucet SM, Shawkey MD (2013) pavo: an R package for the analysis, visualization and organization of spectral data (A Tatem, Ed.). Methods Ecol Evol 4:906–913

  57. Mann ME, Cummings ME (2012) poison frog colors are honest signals of toxity, particularly for bird predators. Am Nat 179:E1–E14

  58. Maslin MA, Burns SJ (2000) Reconstruction of the Amazon Basin effective moisture availability over the past 14,000 years. Science (80-) 20:2285–2287

  59. Master TL (1999) Predation by Rufous Motmot on Black-and-Green Poison Dart Frog. Wilson Bull 111:439–440

  60. McLean CA, Stuart-Fox D (2014) Geographic variation in animal colour polymorphisms and its role in speciation. Biol Rev 89:860–873

  61. Medina I, Wang IJ, Salazar C, Amézquita A (2013) Hybridization promotes color polymorphism in the aposematic harlequin poison frog, Oophaga histrionica. Ecol Evol 3:4388–400

  62. Mueller RL (2006) Evolutionary rates, divergence dates, and the performance of mitochondrial genes in Bayesian phylogenetic analysis. Syst Biol 55:289–300

  63. Noonan BP, Comeault AA (2009) The role of predator selection on polymorphic aposematic poison frogs. Biol Lett 5:51–54

  64. Noonan BP, Gaucher P (2006) Refugial isolation and secondary contact in the dyeing poison frog Dendrobates tinctorius. Mol Ecol 15:4425–4435

  65. O’Leary SJ, Hice LA, Feldheim KA, Frisk MG, McElroy AE, Fast MD et al. (2013) Severe inbreeding and small effective number of breeders in a formerly abundant marine fish. PLoS ONE 8:2–9

  66. Paradis E, Schliep K (2018) ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics 35:526–528

  67. Paluh DJ, Hantak MM, Saporito RA (2014) A test of aposematism in the dendrobatid poison frog oophaga pumilio: the importance of movement in clay model experiments. J Herpetol 48:249–254

  68. Palumbi SR (1996) Nucleic acids II: the polymerase chain reaction. In: Hillis DM, Moritz C, Mable BK eds. Molecular systematics. Sinauer Associates, Inc, Sunderland, Massachusetts, pp 205–247

  69. Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinform 28:2537–2539

  70. Petroli CD, Sansaloni CP, Carling J, Steane DA, Vaillancourt RE, Myburg AA et al. (2012) Genomic characterization of DArT markers based on high-density linkage analysis and physical mapping to the Eucalyptus genome. PLoS ONE 7:e44684

  71. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959

  72. R Core Team (2014) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria

  73. Raj A, Stephens M, Pritchard JK (2014) FastSTRUCTURE: variational inference of population structure in large SNP data sets. Genetics 197:573–589

  74. Rambaut A, Suchard M, Xie D, Drummond A (2014). Tracer v1.6

  75. Ravinet M, Westram A, Johannesson K, Butlin R, André C, Panova M (2016) Shared and nonshared genomic divergence in parallel ecotypes of Littorina saxatilis at a local scale. Mol Ecol 25:287–305

  76. Rellstab C, Gugerli F, Eckert AJ, Hancock AM, Holderegger R (2015) A practical guide to environmental association analysis in landscape genomics. Mol Ecol 24:4348–4370

  77. Revell LJ (2012) phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol 3:217–223

  78. Revell LJ (2013) Two new graphical methods for mapping trait evolution on phylogenies. Methods Ecol Evolution 4:754–759

  79. Richards-Zawacki CL, Yeager J, Bart HPS (2013) No evidence for differential survival or predation between sympatric color morphs of an aposematic poison frog. Evol Ecol 27:783–795

  80. Rojas D, Stow A, Amézquita A, Simões PI, Lima AP (2015) No predatory bias with respect to colour familiarity for the aposematic Adelphobates galactonotus (Anura: Dendrobatidae). Behaviour 152:1637–1657

  81. Row JR, Knick ST, Oyler-McCance SJ, Lougheed SC, Fedy BC (2017) Developing approaches for linear mixed modeling in landscape genetics through landscape-directed dispersal simulations. Ecol Evol 7:3751–3761

  82. Rudh A, Rogell B, Hȍglund J (2007) Non-gradual variation in color morphs of the strawberry poison frog Dendrobates pumilio: genetic and geographical isolation suggests a role for selection in maintaining polymorphism. Mol Ecol 16:4284–4294

  83. Rudh A, Breed MF, Qvarnström A (2013) Does aggression and explorative behaviour decrease with lost warning coloration? Biol J Linn Soc 108:116–126

  84. Rudh A, Rogell B, Håstad O, Qvarnström A (2011) Rapid population divergence linked with co-variation between coloration and sexual display in strawberry poison frogs. Evolution 65:1271–1282

  85. Sansaloni C, Petroli C, Jaccoud D, Carling J, Detering F, Grattapaglia D et al. (2011) Diversity Arrays Technology (DArT) and next-generation sequencing combined: genome-wide, high throughput, highly informative genotyping for molecular breeding of Eucalyptus. BMC Proc 5:P54

  86. Santos JC, Cannatella DC (2011) Phenotypic integration emerges from aposematism and scale in poison frogs. Proc Natl Acad Sci USA 108:6175–6180

  87. Santos JC, Coloma LA, Summers K, Caldwell JP, Ree R, Cannatella DC (2009) Amazonian amphibian diversity is primarily derived from late Miocene Andean lineages. PLoS Biol 7:0448–0461

  88. Sefc KM, Mattersdorfer K, Zielgelbecker A, Neuhuttler N, Steiner O, Goessler O, Koblmuller (2017) Shifting barriers and phenotypic diversification by hybridization. Ecol Lett 20:651–662

  89. Siddiqi A, Cronin TW, Loew ER, Vorobyev M, Summers K (2004) Interspecific and intraspecific views of color signals in the strawberry poison frog Dendrobates pumilio. J Exp Biol 207:2471–2485

  90. Simões PI, Lima AP, Farias IP (2012) Restricted natural hybridization between two species of litter frogs on a threatened landscape in southwestern Brazilian Amazonia. Conserv Genet 13:1145–1159

  91. Simões PI, Stow A, Hödl W, Amézquita A, Farias IP, Lima AP (2014) The value of including intraspecific measures of biodiversity in environmental impact surveys is highlighted by the Amazonian brilliant-thighed frog (Allobates femoralis). Trop Conserv Sci 7:811–828

  92. Summers K, Clough ME (2001) The evolution of coloration and toxicity in the poison frog family (Dendrobatidae). Proc Natl Acad Sci USA 98:6227–6232

  93. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis Version 6.0. Mol Biol Evol 30:2725–2729

  94. Tazzyman SJ, Iwasa Y (2010) Sexual selection can increase the effect of random genetic drift—a quantitative genetic model of polymorphism in Oophaga pumilio, the strawberry poison-dart frog. Evolution 64:1719–1728

  95. Templeton AR, Crandall KA, Sing CF (1992) A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132:619–633

  96. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

  97. Trask AE, Bignal EM, McCracken DI, Piertney SB, Reid JM (2017) Estimating demographic contributions to effective population size in an age-structured wild population experiencing environmental and demographic stochasticity. J Anim Ecol 86:1082–1093

  98. de Villemereuil P, Frichot É, Bazin É, François O, Gaggiotti OE (2014) Genome scan methods against more complex models: when and how much should we trust them? Mol Ecol 23:2006–2019

  99. Vonhof HB, Kaandorp RJG (2011). Climate variation in Amazonia during the neogene and the quaternary. In: Hoorn C, Wesselingh F (eds) Amazonia: landscape and species evolution, Wiley Online Books. Wiley-Blackwell Publishing Ltd: Oxford, UK, pp 199–210

  100. Vorobyev M, Brandt R, Peitsch D, Laughlin S, Menzel R (2001) Colour thresholds and receptor noise: behaviour and physiology compared. Vis Res 41:639–653

  101. Vorobyev M, Osorio D (1998) Receptor noise as a determinant of receptor thresholds. Proc R Soc B Biol Sci 265:351–358

  102. Wang IJ, Summers K (2010) Genetic structure is correlated with phenotypic divergence rather than geographic isolation in the highly polymorphic strawberry poison-dart frog. Mol Ecol 19:447–458

  103. Whitlock MC, Lotterhos KE (2015) Reliable detection of loci responsible for local adaptation: inference of a null model through trimming the distribution of FST. Am Nat 186:S24–S36

  104. Wright S (1932) The roles of mutation, inbreeding, cross breeding and selection in evolution. In: Proceedings of the 6th International Congress on Genetics 1:356–366

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We thank the Editor and two anonymous reviewers for insightful comments that greatly improved this manuscript. Estação Científica Ferreira Penna and ICMBio provided valuable support and facilities at ICMBio Caxiuanã Station. R.M. Brabo, P.M. Brabo, M.R. da Silva, J.M.P. dos Santos, J.L.F. da Costa, E.P de Souza, J.N.B. Carneiro, A.P. Farias, M.F.M. Pedroso, J.A. da Costa Filho, G. O. da Silva, C.A. Lopes for field assistance. Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) provided collection permits (# 36135-1). Financial support was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Project 472198/2011-4) and Programa Ciência sem Fronteiras (Project 401327/2012-4). Support was also provided by a PVE fellowship to AS (312315/2012-0), SWE fellowship to DR (200292/2014-5), a PDJ fellowship for PIS (151409/2013-7), PhD fellowship from CNPq (141886/2012-9) to DR, and a BEV fellowship from CNPq (170211/2012-6) to AA, a research fellowship (312674/2013-9) to TCSAP. PM receives an Academy of Finland postdoctoral fellowship (grant # 316294).

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DR, AS, and APL designed the project. DR, APL, AS, and PIS collected samples. TCSA-P and MSH contribued useful discussion. TCSA-P, MSH and YOCB contributed samples and useful discussion. DR and AA measured, analyzed and interpreted the color data. AS, PIS, ILK, PM and RD analyzed and interpreted genetic data. DR and AS wrote the manuscript with contributions from all co-authors.

Correspondence to Adam Stow.

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Rojas, D., Lima, A.P., Momigliano, P. et al. The evolution of polymorphism in the warning coloration of the Amazonian poison frog Adelphobates galactonotus. Heredity (2019).

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