Conservation Genetics

, Volume 13, Issue 4, pp 1145–1159 | Cite as

Restricted natural hybridization between two species of litter frogs on a threatened landscape in southwestern Brazilian Amazonia

  • Pedro Ivo Simões
  • Albertina P. Lima
  • Izeni P. Farias
Research Article


Natural hybridization between allopatric species following secondary contact has been poorly documented for Neotropical anurans inhabiting the Amazonian lowlands. We conducted a genetic survey across a contact zone between two species of litter frogs, Allobates hodli and Allobates femoralis (family Dendrobatidae), located on the left riverbank of the upper Madeira River, State of Rondônia, Brazil. We obtained tissue samples from 11 sampling sites on both riverbanks, covering approximately a 400 km long transect. We evaluated the genetic relationships between samples using haplotype networks and a distance-based phylogenetic tree obtained from a dataset of 16S rRNA mtDNA sequences. Estimates of genetic diversity, population structure, and the identification of sites where genetic admixture occurred were carried out by means of frequency-based methods and Bayesian inference on mtDNA and a set of four microsatellite loci, including samples collected throughout the study area. A reduced dataset including only microsatellite loci genotyped from samples on the left riverbank was applied in assignment tests for detecting levels of admixture at the contact zone and adjacent sampling sites, and for detecting and quantifying hybrid individuals. Our results suggest that genetic introgression between A. hodli and A. femoralis is restricted to the core area of the contact zone, where potential hybrids are less frequent than parental genotypes. Effects on the genetic variability of adjacent populations are only detected at sites located 1.5 km downstream and upstream of the core area, suggesting the existence of selection against hybrids, possibly mediated by postzygotic isolation mechanisms. The contact zone between A. femoralis and A. hodli is the first well delimited suture line between anuran species ever documented in the Brazilian Amazon. The settlement of two dams along the upper Madeira River poses an immediate threat to the gene flow and hybridization balance observed between the populations studied. Our results provided guidelines for a current monitoring program, aiming at the impacts of dams on this evolutionary system’s dynamics.


Amazon Madeira River Hybrid zone Genetic introgression Dendrobatidae Allobates femoralis 



We thank Walter Hödl, Daniel Rodrigues Santos, Pedro Rodrigues Santos, Adolfo Amézquita, and Iliana Medina for helping us during field work. We thank Mr. Bento Pereira da Silva for allowing us camping at his property for several occasions. We are grateful to Eva Ursprung and Robert Jehle for providing information on microsatellite primers and protocols. We thank Jeff Podos, José Manuel Padial, Marcelo Menin, Mario Cohn-Haft, Marina Anciães, José A. Alves Gomes, Tomas Hrbek, and Daniel Toffoli for suggestions and comments on earlier drafts of the manuscript. Conselho Nacional de Desenvolvimento Tecnológico (CNPq) provided funding for field excursions and laboratory analyses and equipment (CT-Amazônia/CT-Energia No. 13/2006; 470811/2006—Ed 02/2006 Universal; CNPq/CTAmazônia 575603/2008-9). Field work done between 2004 and 2005 received logistical support from Furnas Centrais Elétricas S.A. Collecting permits were provided by RAN-ICMBio/IBAMA (004/03-RAN; 131/04-RAN; 037/2007-RAN/; 13894-1/2009-RAN). Tissue collection permits were provided to CTGA-ICB/UFAM by deliberation nº 75 of August 26, 2004, by CGEN-IBAMA. P.I. Simões received a doctoral fellowship from CNPq from 2006-2010, while conducting this study.

Supplementary material

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Supplementary material 1 (DOC 27 kb)
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Supplementary material 2 (DOC 70 kb)
10592_2012_362_MOESM3_ESM.doc (102 kb)
Supplementary material 3 (DOC 102 kb)


  1. Allendorf FW, Luikart G (2007) Conservation and the genetics of populations. Blackwell, OxfordGoogle Scholar
  2. Amézquita A, Lima AP, Jehle R, Castellanos L, Ramos O, Crawford AJ, Gasser H, Hödl W (2009) Calls, colours, shapes, and genes: A multi-trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis. Biol J Linn Soc 98:826–838CrossRefGoogle Scholar
  3. Anderson EC, Thompson EA (2002) A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160:1217–1229PubMedGoogle Scholar
  4. Arnold ML, Bulger MR, Burke JM, Hempel AL, Williams JH (1999) Natural hybridization: how low can you go and still be important? Ecology 80(2):371–381CrossRefGoogle Scholar
  5. Barton NH, Hewitt GM (1985) Analysis of hybrid zones. Ann Rev Ecol Syst 16:133–148CrossRefGoogle Scholar
  6. Boecklen WJ, Howard DJ (1997) Genetic analysis of hybrid zones: numbers of markers and power resolution. Ecology 78(8):2611–2616CrossRefGoogle Scholar
  7. Brown J, Twomey E (2009) Complicated histories: three new species of poison frogs of the genus Ameerega (Anura: Dendrobatidae) from north-central Peru. Zootaxa 2049:1–38Google Scholar
  8. Burton RS, Ellison CK, Harrison JS (2006) The sorry state of F2 hybrids: Consequences of rapid mitochondrial DNA evolution in allopatric populations. Am Nat 168(Supplement):14–24CrossRefGoogle Scholar
  9. Clement M, Posada D, Crandall KA (2000) TCS: a computer program to estimate gene genealogies. Mol Ecol 9:1657–1659PubMedCrossRefGoogle Scholar
  10. Clemons KS (2007) Hydroeletric dams: transboundary environmental effects and international law. Fla State University Law Rev 36:487–536Google Scholar
  11. Corander J, Tang J (2007) Bayesian analysis of population structure based on linked molecular information. Math Biosci 205:19–31PubMedCrossRefGoogle Scholar
  12. Corander J, Marttinen P, Sirén J, Tang J (2008) Enhanced Bayesian modelling in BAPS software for learning genetic structures of populations. BMC Bioinforma 9:539CrossRefGoogle Scholar
  13. Coyne JA, Orr HA (2004) Speciation. Sinauer, SunderlandGoogle Scholar
  14. Darst CR, Cummings ME, Cannatella DC (2006) A mechanism for diversity in warning signals: Conspicuousness versus toxicity in poison frogs. PNAS 103(15):5852–5857Google Scholar
  15. Dawe KL, Shafer ABA, Herman TB, Stewart DT (2009) Diffusion of nuclear and mitochondrial genes across a zone of secondary contact in the maritime shrew, Sorex maritimensis: implications for the conservation of a Canadian endemic mammal. Conserv Genet 10:851–857CrossRefGoogle Scholar
  16. DeWoody JA, Shupp J, Kenefic L, Busch J, Murfitt L, Keim P (2004) Universal method for producing ROX-labeled size standards suitable for automated genotyping. Biotechniques 37:348–352PubMedGoogle Scholar
  17. DNPM—Departamento Nacional da Produção Mineral—Brasil (1978) Projeto RADAMBRASIL—Levantamento dos recursos naturais, Vol.18, Folha SC 20, Porto Velho: Geologia, geomorfologia, pedologia, vegetação e uso potencial da terra. Departamento Nacional da Produção Mineral, Rio de JaneiroGoogle Scholar
  18. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  19. Erdtmann LK, Simões PI, Mello AC, Lima AP (2011) Do natural differences in acoustic signals really interfere in conspecific recognition in the pan-Amazonian frog Allobates femoralis? Behaviour 148:485–500CrossRefGoogle Scholar
  20. Genovart M (2009) Natural hybridization and conservation. Biodivers Conserv 18:1435–1439Google Scholar
  21. Göd M, Franz A, Hödl W (2007) The influence of internote-interval variation of the advertisement call on the phonotactic behaviour in male Allobates femoralis (Dendrobatidae). Amphibia-Reptilia 28:227–234CrossRefGoogle Scholar
  22. Goudet J (2001) Fstat, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Available from
  23. Grant PR, Grant BR (1992) Hybridization of bird species. Science 256(5054):193–197PubMedCrossRefGoogle Scholar
  24. Grant PR, Grant BR (2002) Unpredictable evolution in a 30-year study of Darwin’s finches. Science 296:707–711PubMedCrossRefGoogle Scholar
  25. Grant T, Frost DR, Caldwell JP, Gagliardo R, Haddad CFB, Kok PJR, Means BD, Noonan BP, Schargel W, Wheeler WC (2006) Phylogenetic systematics of dart-poison frogs and their relatives (Anura: Athesphatanura: Dendrobatidae). Bull Am Mus Nat Hist 299:1–262CrossRefGoogle Scholar
  26. Haffer J (1997) Alternative models of vertebrate speciation in Amazonia: An overview. Biodivers Conserv 6:451–476CrossRefGoogle Scholar
  27. Haig SM, Mullins TD, Forsman ED, Trail PW, Wennerberg L (2004) Genetic identification of Spotted Owls, Barred Owls, and their hybrids: legal implication of hybrid identity. Conserv Biol 18:1347–1357CrossRefGoogle Scholar
  28. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95–98Google Scholar
  29. Hird S, Sullivan J (2009) Assessment of gene flow across a hybrid zone in red-tailed chipmunks (Tamias ruficaudus). Mol Ecol 18:3097–3109PubMedCrossRefGoogle Scholar
  30. Höbel G, Gerhardt HC (2003) Reproductive character displacement in the acoustic communication system of gree tree frogs (Hyla cinerea). Evolution 57(4):894–904PubMedGoogle Scholar
  31. Hödl W, Amézquita A, Narins PM (2004) The rôle of call frequency and the auditory papillae in phonotacticbehavior in male dart-poison frogs Epipedobates femoralis (Dendrobatidae). J Comparative Physiol A 190:823–829CrossRefGoogle Scholar
  32. Hofman S, Szymura JM (2007) Limited mitochondrial DNA introgression in a Bombina hybrid zone. Biol J Linn Soc 91:295–306CrossRefGoogle Scholar
  33. Hoorn C, Wesselingh FP (2010) Amazonia, landscape and species evolution. Blackwell, OxfordGoogle Scholar
  34. Hoskin CJ, Higgie H, McDonald KR, Moritz C (2005) Reinforcement drives rapid allopatric speciation. Nature 437:1353–1356PubMedCrossRefGoogle Scholar
  35. Jehle R, Gasser H, Amézquita A, Lima AP, Hödl W (2008) Ten polymorphic microsatellite loci for Allobates femoralis, an Amazonian dendrobatoid frog. Mol Ecol Resour 8(6):1326–1328PubMedCrossRefGoogle Scholar
  36. Jiggins CD, Mallet J (2000) Bimodal hybrid zones and speciation. Trends Ecol Evol 15(6):250–255PubMedCrossRefGoogle Scholar
  37. Keller B, Wolinska J, Manca M, Spaak P (2008) Spatial, environmental and anthropogenic effects on the taxon composition of hybridizing Daphnia. Philos Trans R Soc B 363:2943–2952CrossRefGoogle Scholar
  38. Laurance WF, Albernaz AKM, Fearnside PM, Vasconcelos HL, Ferreira LV (2004) Deforestation in Amazonia. Science 304:1109PubMedCrossRefGoogle Scholar
  39. Lemmon EM, Lemmon AR, Collins JT, Lee-Yaw JA, Cannatella DC (2007) Phylogeny-based delimitation of species boundaries and contact zones in the trilling chorus frogs (Pseudacris). Mol Phylogenet Evol 44:1068–1082PubMedCrossRefGoogle Scholar
  40. Levin DA, Francisco-Ortega J, Jansen RK (1996) Hybridization and the extinction of rare plant species. Conserv Biol 10(1):10–16CrossRefGoogle Scholar
  41. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452PubMedCrossRefGoogle Scholar
  42. Moritz C, Patton JL, Schneider CJ, Smith TB (2000) Diversification of rainforest faunas: an integrated molecular approach. Annu Rev Ecol Syst 31:533–563CrossRefGoogle Scholar
  43. Moritz C, Hoskin CJ, MacKenzie JB, Phillips BL, Tonione M, Silva N, VanDerWal J, Williams LE, Graham CH (2009) Identification and dynamics of a cryptic suture zone in tropical rainforest. Proc R Soc B 276:1235–1244PubMedCrossRefGoogle Scholar
  44. Palumbi SR (1996) Nucleic acids II: the polymerase chain reaction. In: Hillis DM, Moritz C, Mable BK (eds) Molecular systematics. Sinauer, Sunderland, pp 205–247Google Scholar
  45. Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295CrossRefGoogle Scholar
  46. Perz S, Brilhante S, Brown F, Caldas M, Ikeda S, Mendoza E, Overdevest C, Reis V, Reyes JF, Rojas D, Schmink M, Souza C, Walker R (2008) Road building, land use and climate change: prospects for environmental governance in the Amazon. Philos Trans R Soc B 363:1889–1895CrossRefGoogle Scholar
  47. Pfennig KS (2003) A test of alternative hypothesis for the evolution of reproductive isolation between spadefoot toads: support for the reinforcement hypothesis. Evolution 57(12):2842–2851PubMedGoogle Scholar
  48. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedGoogle Scholar
  49. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  50. 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(3):1–14CrossRefGoogle Scholar
  51. Sanz N, Araguas RM, Fernández R, Vera M, García-Marín JL (2009) Efficiency of markers and methods for detecting hybrids and introgression in stocked populations. Conserv Genet 10:225–236CrossRefGoogle Scholar
  52. Seehausen O (2004) Hybridization and adaptive radiation. Trends Ecol Evol 19(4):198–206PubMedCrossRefGoogle Scholar
  53. Seehausen O, Takimoto G, Roy D, Jokela J (2007) Speciation reversal and biodiversity dynamics with hybridization in changing environments. Mol Ecol 17(1):30–44PubMedCrossRefGoogle Scholar
  54. Simões PI, Lima AP, Magnusson WE, Hödl W, Amézquita A (2008) Acoustic and morphological differentiation in the frog Allobates femoralis: relationships with the upper Madeira River and other potential geological barriers. Biotropica 40(5):607–614CrossRefGoogle Scholar
  55. Simões PI, Lima AP, Farias IP (2010) The description of a cryptic species related to the pan-Amazonian frog Allobates femoralis (Boulenger 1883) (Anura: Aromobatidae). Zootaxa 2406:1–28Google Scholar
  56. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526PubMedGoogle Scholar
  57. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  58. 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 132:619–633PubMedGoogle Scholar
  59. Thompson JD, Higgins DG, Gibson TJ (1994) Improved sensitivity of profile searches through the use of sequence weights and gap excision. Comput Appl Biosci 10(1):19–29PubMedGoogle Scholar
  60. Vogel LS, Johnson SG (2008) Estimation of hybridization and introgression frequency in toads (Genus: Bufo) using DNA sequence variation at mitochondrial and nuclear loci. J Herpetol 42(1):61–75CrossRefGoogle Scholar
  61. Vorndran IC, Reichwaldt E, Nürnberger B (2002) Does differential susceptibility to predation in tadpoles stabilize the Bombina hybrid zone? Ecology 83(6):1648–1659CrossRefGoogle Scholar
  62. Wells KD (2007) The ecology and behavior of amphibians. The University of Chicago Press, ChicagoGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Pedro Ivo Simões
    • 1
  • Albertina P. Lima
    • 1
  • Izeni P. Farias
    • 2
  1. 1.Coordenação de Pesquisas em EcologiaInstituto Nacional de Pesquisas da AmazôniaManausBrazil
  2. 2.Departamento de Biologia, Laboratório de Evolução e Genética Animal (LEGAL)Universidade Federal do Amazonas (UFAM)ManausBrazil

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