Ancient origins, profound ecological divergence, and extensive hybridization make the fire-bellied toads Bombina bombina and B. variegata (Anura: Bombinatoridae) an intriguing test case of ecological speciation. Previous modeling has proposed that the narrow Bombina hybrid zones represent strong barriers to neutral introgression. We test this prediction by inferring the rate of gene exchange between pure populations on either side of the intensively studied Kraków transect. We developed a method to extract high confidence sets of orthologous genes from de novo transcriptome assemblies, fitted a range of divergence models to these data and assessed their relative support with analytic likelihood calculations. There was clear evidence for postdivergence gene flow, but, as expected, no perceptible signal of recent introgression via the nearby hybrid zone. The analysis of two additional Bombina taxa (B. v. scabra and B. orientalis) validated our parameter estimates against a larger set of prior expectations. Despite substantial cumulative introgression over millions of years, adaptive divergence of the hybridizing taxa is essentially unaffected by their lack of reproductive isolation. Extended distribution ranges also buffer them against small-scale environmental perturbations that have been shown to reverse the speciation process in other, more recent ecotypes.

Current Address Institute of Environmental Sciences Jagiellonian University Gronostajowa 7 30 387 Kraków Poland

Current Address Institute of Vertebrate Biology Academy of Sciences of the Czech Republic Brno Czech Republic

Department of Comparative Anatomy Institute of Zoology Jagiellonian University Gronostajowa 9 30 387 Kraków Poland

Institute of Evolutionary Biology University of Edinburgh Ashworth Laboratories Charlotte Auerbach Road Edinburgh EH9 3FL United Kingdom

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Evolution. 2016 Oct;70(10):2429. doi: 10.1111/evo.13044. PubMed

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Abrahamson, W. G. , and Blair C. P.. 2007. Sequential radiation through host‐race formation: herbivore diversity leads to diversity in natural enemies: Pp. 188–202 in Tilmon K. J., ed. Specialization, speciation, and radiation: the evolutionary biology of herbivorous insects. Chicago Univ. Press, Berkeley, CA.

Barker, M. S. , Dlugosch K. M., Dinh L., Challa R. S., Dracopoli N. C., King M. G., and Rieseberg L. H.. 2010. EvoPipes.net: bioinformatic tools for ecological and evolutionary genomics. Evol. Bioinforma. Online 6:143–149. PubMed PMC

Barton, N. H. 1979. Gene flow past a cline. Heredity 43:333–339.

Barton, N. H. 1983. Multilocus clines. Evolution 37:454–471. PubMed

Barton, N. H. 1986. The effects of linkage and density‐dependent regulation on gene flow. Heredity 57:415–426. PubMed

Barton, N. H. 2001. The role of hybridization in evolution. Mol. Ecol. 10:551–568. PubMed

Barton, N. H. , and Bengtsson B.‐O.. 1986. The barrier to genetic exchange between hybridising populations. Heredity 56:357–376. PubMed

Barton, N. H. , and Gale K. S.. 1993. Genetic analysis of hybrid zones Pp. 13–45 in Harrison R. G., ed. Hybrid zones and the evolutionary process. Oxford Univ. Press, Oxford.

Bolger, A. M. , Lohse M., and Usadel B.. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–21120. PubMed PMC

Bolnick, D. I. , and Near T. J.. 2005. Tempo of hybrid inviability in centrarchid fishes (Teleostei: Centrarchidae). Evolution 59:1754–1767. PubMed

Butlin, R. K. , Galindo J., and Grahame J. W.. 2008. Sympatric, parapatric or allopatric: the most important way to classify speciation? Philos. Trans. R. Soc. Lond. B Biol. Sci. 363:2997–3007. PubMed PMC

Cahais, V. , Gayral P., Tsagkogeorga G., Melo‐Ferreira J., Ballenghien M., Weinert L., Chiari Y., Belkhir K., Ranwez V., and Galtier N.. 2012. Reference‐free transcriptome assembly in non‐model animals from next‐generation sequencing data. Mol. Ecol. Resour. 12:834–845. PubMed

Chamberlain, A. J. , Vander Jagt C. J., Hayes B. J., Khansefid M., Marett L. C., Millen C. A., Nguyen T. T. T., and Goddard M. E.. 2015. Extensive variation between tissues in allele specific expression in an outbred mammal. BMC Genomics 16:993. PubMed PMC

Chevin, L.‐M. , Decorzent G., and Lenormand T.. 2014. Niche dimensionality and the genetics of ecological speciation. Evolution 68:1244–1256. PubMed

Chevreux, B. , Pfisterer T., Drescher B., Driesel A. J., Müller W. E. G., Wetter T., and Suhai S.. 2004. Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Res. 14:1147–1159. PubMed PMC

Coop, G. , and Ralph P.. 2012. Patterns of neutral diversity under general models of selective sweeps. Genetics 192:205–224. PubMed PMC

Coyne, J. A. , and Orr H. A.. 1989. Two rules of speciation Pp. 180–207 in Otte D. and Endler J. A., eds. Speciation and its consequences. Sinauer Associates, Sunderland, Massachusetts.

Coyne, J. A. , and Orr H. A. 1997. Patterns of speciation in Drosophila revisited. Evolution 51:295–303. PubMed

Coyne, J. A. , and Orr H. A. 2004. Speciation. Sinauer Associates, Sunderland, Massachusetts.

Darwin, C. 1859. On the origin of species by mean of natural selection, or the preservation of favoured races in the struggle for life. John Murray, London. PubMed PMC

Davey, J. W. , Hohenlohe P. A., Etter P. D., Boone J. Q., Catchen J. M., and Blaxter M. L.. 2011. Genome‐wide genetic marker discovery and genotyping using next‐generation sequencing. Nat. Rev. Genet. 12:499–510. PubMed

DePristo, M. A. , Banks E., Poplin R., Garimella K. V., Maguire J. R., Hartl C., Philippakis A. A., del Angel G., Rivas M. A., Hanna M., et al. 2011. A framework for variation discovery and genotyping using next‐generation DNA sequencing data. Nat. Genet. 43:491–498. PubMed PMC

De Wit, P. , Pespeni M. H., and Palumbi S. R.. 2015. SNP genotyping and population genomics from expressed sequences—current advances and future possibilities. Mol. Ecol. 24:2310–2323. PubMed

Dufresne, F. , and Jeffery N.. 2011. A guided tour of large genome size in animals: what we know and where we are heading. Chromosome Res. 19:925–938. PubMed

Dynesius, M. , and Jansson R.. 2014. Persistence of within‐species lineages: a neglected control of speciation rates. Evolution 68:923–934. PubMed

Faria, R. , Renaut S., Galindo J., Pinho C., Melo‐Ferreira J., Melo M., Jones F., Salzburger W., Schluter D., and Butlin R.. 2014. Advances in ecological speciation: an integrative approach. Mol. Ecol. 23:513–521. PubMed

Fijarczyk, A. , Nadachowska K., Hofman S., Litvinchuk S. N., Babik W., Stuglik M., Gollmann G., Choleva L., Cogalniceanu D., Vukov T., et al. 2011. Nuclear and mitochondrial phylogeography of the European fire‐bellied toads Bombina bombina and Bombina variegata supports their independent histories. Mol. Ecol. 20:3381–3398. PubMed

Futuyma, D. J. 1987. On the role of species in anagensis. Am. Nat. 130:465–473.

Gabaldón, T. , and Koonin E. V.. 2013. Functional and evolutionary implications of gene orthology. Nat. Rev. Genet. 14:360–366. PubMed PMC

Gavrilets, S. 2004. Fitness landscapes and the origin of species. Princeton Univ. Press, Princeton, New Jersey.

Gayral, P. , Melo‐Ferreira J., Glémin S., Bierne N., Carneiro M., Nabholz B., Lourenco J. M., Alves P. C., Ballenghien M., Faivre N., et al. 2013. Reference‐free population genomics from next‐generation transcriptome data and the vertebrate–invertebrate gap. PLoS Genet. 9:e1003457. PubMed PMC

Gollmann, G. 1987. Bombina bombina and B. variegata in the Mátra mountains (Hungary): new data on distribution and hybridization (Amhibia, Anura, Discoglossidae). Amphib.Reptil. 8:213–224.

Grabherr, M. G. , Haas B. J., Yassour M., Levin J. Z., Thompson D. A., Amit I., Adiconis X., Fan L., Raychowdhury R., Zeng Q., et al. 2011. Full‐length transcriptome assembly from RNA‐Seq data without a reference genome. Nat. Biotechnol. 29:644–652. PubMed PMC

Gregory, T. R. 2016. Animal genome size database. http://www.genomesize.com.

Harrison, R. G. , and Larson E. L.. 2016. Heterogeneous genome divergence, differential introgression, and the origin and structure of hybrid zones. Mol. Ecol 25:2454–2466. PubMed PMC

Hatfield, T. , and Schluter D.. 1999. Ecological speciation in sticklebacks: environment‐dependent hybrid fitness. Evolution 53:866–873. PubMed

Heger, A. , and Ponting C. P.. 2007. Evolutionary rate analyses of orthologs and paralogs from 12 Drosophila genomes. Genome Res. 17:1837–1849. PubMed PMC

Heled, J. , and Drummond A. J.. 2015. Calibrated birth–death phylogenetic time‐tree priors for Bayesian inference. Syst. Biol. 64:369–383. PubMed PMC

Hellsten, U. , Harland R. M., Gilchrist M. J., Hendrix D., Jurka J., Kapitonov V., Ovcharenko I., Putnam N. H., Shu S., Taher L., et al. 2010. The genome of the Western clawed frog Xenopus tropicalis . Science 328:633–636. PubMed PMC

Hendry, A. P. , Nosil P., and Rieseberg L. H.. 2007. The speed of ecological speciation. Funct. Ecol. 21:455–464. PubMed PMC

Hey, J. , and Nielsen R.. 2004. Multilocus methods for estimating population sizes, migration rates and divergence time, with applications to the divergence of Drosophila pseudoobscura and D. persimilis . Genetics 167:747–760. PubMed PMC

Huang, X. , and Madan A.. 1999. CAP3: a DNA sequence assembly program. Genome Res. 12:868–877. PubMed PMC

Huber, S. K. , De León L. F., Hendry A. P., Bermingham E., and Podos J.. 2007. Reproductive isolation of sympatric morphs in a population of Darwin's finches. Proc. R. Soc. Lond. B Biol. Sci. 274:1709–1714. PubMed PMC

Irisarri, I. , San Mauro D., Abascal F., Ohler A., Vences M., and Zardoya R.. 2012a. Data from: the origin of modern frogs (Neobatrachia) was accompanied by the acceleration in mitochondrial and nuclear substitution rates. Dryad Digital Repository, http://dx.doi.org/10.5061/dryad.3qd54. PubMed DOI PMC

Irisarri, I. , San Mauro D., Abascal F., Ohler A., Vences M., and Zardoya R.. 2012b. The origin of modern frogs (Neobatrachia) was accompanied by acceleration in mitochondrial and nuclear substitution rates. BMC Genomics 13:626. PubMed PMC

Jiggins, C. D. , Naisbit R. E., Coe R. L., and Mallet J.. 2001. Reproductive isolation caused by colour pattern mimicry. Nature 411:302–305. PubMed

Kent, W. J. 2002. BLAT—The BLAST‐like alignment tool. Genome Res. 12:656–664. PubMed PMC

Kleindorfer, S. , O'Connor J. A., Dudaniec R. Y., Myers S. A., Robertson J., and Sulloway F. J.. 2014. Species collapse via hybridization in Darwin's tree finches. Am. Nat. 183:325–341. PubMed

Kondrashov, A. 2003. Accumulation of Dobzhansky–Muller incompatibilties within a spatially structured population. Evolution 57:151–153. PubMed

Kristensen, D. M. , Wolf Y. I., Mushegian A. R., and Koonin E. V.. 2011. Computational methods for gene orthology inference. Brief. Bioinform. 12:379–391. PubMed PMC

Kruuk, L. E. B. , Gilchrist J. S., and Barton N. H.. 1999. Hybrid dysfunction in fire‐bellied toads (Bombina). Evolution 53:1611–1616. PubMed

Kumar, S. , and Blaxter M. L.. 2010. Comparing de novo assemblers for 454 transcriptome data. BMC Genomics 11:571. PubMed PMC

Langmead, B. , and Salzberg S. L.. 2012. Fast gapped‐read alignment with Bowtie 2. Nat. Methods 9:357–359. PubMed PMC

Li, B. , and Dewey C. N.. 2011. RSEM: accurate transcript quantification from RNA‐Seq data with or without a reference genome. BMC Bioinformatics 12:323. PubMed PMC

Li, L. , Stoeckert C. J., and Roos D. S.. 2003. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13:2178–2189. PubMed PMC

Linn, C. , Feder J. L., Nojima S., Dambroski H. R., Berlocher S. H., and Roelofs W.. 2003. Fruit odor discrimination and sympatric host race formation in Rhagoletis. Proc. Natl. Acad. Sci. USA 100:11490–11493. PubMed PMC

Lohse, K. , Chmelik M., Martin S. H., and Barton N. H.. 2016. Strategies for calculating blockwise likelihoods under the coalescent. Genetics 202:775–786. PubMed PMC

Lohse, K. , Harrison R. J., and Barton N. H.. 2011. A general method for calculating likelihoods under the coalescent process. Genetics 189:977–987. PubMed PMC

MacCallum, C. J. , Nürnberger B., Barton N. H., and Szymura J. M.. 1998. Habitat preference in the Bombina hybrid zone in Croatia. Evolution 52:227–239. PubMed

Mallet, J. 2008. Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363:2971–2986. PubMed PMC

Martin, M. 2011. Cutadapt removes adapter sequences from high‐throughput sequencing reads. EMBnet.journal 17:10–12.

McKenna, A. , Hanna M., Banks E., Sivachenko A., Cibulskis K., Kernytsky A., Garimella K., Altshuler D., Gabriel S., Daly M., et al. 2010. The genome analysis toolkit: a MapReduce framework for analyzing next‐generation DNA sequencing data. Genome Res. 20:1297–1303. PubMed PMC

Morescalchi, A. 1965. Osservazioni sulla cariologia di Bombina . Boll. Zool. 32:207–219.

Nagylaki, T. 1976. Clines with variable migration. Genetics 83:867–886. PubMed PMC

Nosil, P. 2012. Ecological speciation. Oxford Univ. Press, Oxford.

Nosil, P. , Harmon L. J., and Seehausen O.. 2009. Ecological explanations for (incomplete) speciation. Trends Ecol. Evol. 24:145–156. PubMed

Nürnberger, B. , Hofman S., Förg‐Brey B., Praetzel G., Maclean A., Szymura J. M., Abbott C. M., and Barton N. H.. 2003. A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae). Heredity 91:136–142. PubMed

Olmo, E. , Morescalchi A., Stingo V., and Odierna G.. 1982. Genome characteristics and the systematics of Discoglossidae (Amphibia, Salientia). Monit. ZoolItalNS 16:283–299.

Osborne, O. G. , Batstone T. E., Hiscock S. J., and Filatov D. A.. 2013. Rapid speciation with gene flow following the formation of Mt. Etna. Genome Biol. Evol. 5:1704–1715. PubMed PMC

Pabijan, M. , Wandycz A., Hofman S., Węcek K., Piwczyński M., and Szymura J. M.. 2013. Complete mitochondrial genomes resolve phylogenetic relationships within Bombina (Anura: Bombinatoridae). Mol. Phylogenet. Evol. 69:63–74. PubMed

Parra, G. , Bradnam K., and Korf I.. 2007. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23:1061–1067. PubMed

Price, T. D. , and Bouvier M. M.. 2002. The evolution of F1 postzygotic incompatibilities in birds. Evolution 56:2083–2089. PubMed

Rafińska, A. 1991. Reproductive biology of the fire‐bellied toads, Bombina bombina and B. variegata (Anura: Discoglossidae): egg size, clutch size and larval period length differences. Biol. J. Linn. Soc. 43:197–210.

Rage, J.‐C. , and Roček Z.. 2003. Evolution of anuran assemblages in the Tertiary and Quaternary of Europe, in the context of palaeoclimate and palaeogeography. Amphib.‐Reptil. 24:133–167.

Ramsey, J. , Bradshaw H. D., and Schemske D. W.. 2003. Components of reproductive isolation between the Monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae). Evolution 57:1520–1534. PubMed

Rasmussen, M. D. , and Kellis M.. 2012. Unified modeling of gene duplication, loss, and coalescence using a locus tree. Genome Res. 22:755–765. PubMed PMC

Reinius, B. , and Sandberg R.. 2015. Random monoallelic expression of autosomal genes: stochastic transcription and allele‐level regulation. Nat. Rev. Genet. 16:653–664. PubMed

Relyea, R. A. 2001. The relationship between predation risk and antipredator responses in larval anurans. Ecology 82:541–554.

Rice, W. R. , and Hostert E. E.. 1993. Laboratory experiments on speciation: what have we learned in 40 years? Evolution 47:1637–1653. PubMed

Roelants, K. , Haas A., and Bossuyt F.. 2011. Anuran radiations and the evolution of tadpole morphospace. Proc. Natl. Acad. Sci. 108:8731–8736. PubMed PMC

Rosenblum, E. B. , Sarver B. A. J., Brown J. W., Roches S. D., Hardwick K. M., Hether T. D., Eastman J. M., Pennell M. W., and Harmon L. J.. 2012. Goldilocks meets Santa Rosalia: an ephemeral speciation model explains patterns of diversification across time scales. Evol. Biol. 39:255–261. PubMed PMC

Rundle, H. D. , Nagel L., Boughman J. W., and Schluter D.. 2000. Natural selection and parallel speciation in sympatic sticklebacks. Science 287:306–308. PubMed

Sambatti, J. B. M. , Anderson J. L., Ortiz‐Barrientos D., Baack E. J., and Rieseberg L. H.. 2012. Reconciling extremely strong barriers with high levels of gene exchange in annual sunflowers. Evolution 66:1459–1473. PubMed

Sasa, M. M. , Chippindale P. T., and Johnson N. A.. 1998. Patterns of postzygotic isolation in frogs. Evolution 52:1811–1820. PubMed

Schemske, D. W. 2010. Adaptation and The Origin of Species . Am. Nat. 176:S4–S25. PubMed

Schmieder, R. , and Edwards R.. 2011. Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864. PubMed PMC

Seehausen, O. , Takimoto G., Roy D., and Jokela J.. 2008. Speciation reversal and biodiversity dynamics with hybridization in changing environments. Mol. Ecol. 17:30–44. PubMed

Servedio, M. R. , Doorn G. S. V., Kopp M., Frame A. M., and Nosil P.. 2011. Magic traits in speciation: “magic” but not rare? Trends Ecol. Evol. 26:389–397. PubMed

Shafer, A. B. A. , Gattepaille L. M., Stewart R. E. A., and Wolf J. B. W.. 2015. Demographic inferences using short‐read genomic data in an approximate Bayesian computation framework: in silico evaluation of power, biases and proof of concept in Atlantic walrus. Mol. Ecol. 24:328–345. PubMed

Slatkin, M. 1973. Gene flow and selection in a cline. Genetics 75:733–756. PubMed PMC

Sun, Y.‐B. , Xiong Z.‐J., Xiang X.‐Y., Liu S.‐P., Zhou W.‐W., Tu X.‐L., Zhong L., Wang L., Wu D.‐D., Zhang B.‐L., et al. 2015. Whole‐genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes. Proc. Natl. Acad. Sci. USA 112:E1257–E1262. PubMed PMC

Suyama, M. , Torrents D., and Bork P.. 2006. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 34:W609–W612. PubMed PMC

Szymura, J. M. 1976. Hybridization between discoglossid toads Bombina bombina and Bombina variegata in southern Poland as revealed by the electrophoretic technique. Z. Für Zool. Syst. Evol. 14:227–236.

Szymura, J. M. 1988. Regional differentiation between hybridizing species Bombina bombina (L.) and B. variegata (L.) (Salientia, Discoglossidae) in Europe. Rozprawy Habilitacyjne No. 147, Uniwersytet Jagielloński, Kraków.

Szymura, J. M. 1993. Analysis of hybrid zones with Bombina Pp. 261–289 in Harrison R. G., ed. Hybrid zones and the evolutionary process. Oxford Univ. Press, New York.

Szymura, J. M. 1996. Can the fire‐bellied toads Bombina bombina and Bombina variegata maintain their distinctness in the face of hybridization? Isr. J. Zool. 42:135–148.

Szymura, J. M. , and Barton N. H.. 1986. Genetic analysis of a hybrid zone between the fire‐bellied toads Bombina bombina and B variegata, near Cracow in Southern Poland. Evolution 40:1141–1159. PubMed

Szymura, J. M. , and Barton N. H. 1991. The genetic structure of the hybrid zone between the fire‐bellied toads Bombina bombina and B. variegata: comparison between transects and between loci. Evolution 45:237–261. PubMed

Szymura, J. M. , and Farana I.. 1978. Inheritance and linkage analysis of five enzyme loci in interspecific hybrids of toadlets, genus Bombina . Biochem. Genet. 16:307–319. PubMed

Taylor, E. B. , Boughman J. W., Groenenboom M., Sniatynski M., Schluter D., and Gow J. L.. 2006. Speciation in reverse: morphological and genetic evidence of the collapse of a three‐spined stickleback (Gasterosteus aculeatus) species pair. Mol. Ecol. 15:343–355. PubMed

Vines, T. H. , Köhler S. C., Thiel M., Ghira I. V., Sands T. R., MacCallum C. J., Barton N. H., and Nürnberger B.. 2003. The maintenance of reproductive isolation in a mosaic hybrid zone between the fire‐bellied toads Bombina bombina and B. variegata . Evolution 57:1876–1888. PubMed

Vonlanthen, P. , Bittner D., Hudson A. G., Young K. A., Müller R., Lundsgaard‐Hansen B., Roy D., Di Piazza S., Largiader C. R., and Seehausen O.. 2012. Eutrophication causes speciation reversal in whitefish adaptive radiations. Nature 482:357–362. PubMed

Vorndran, I. C. , Reichwaldt E., and Nürnberger B.. 2002. Does differential susceptibility to predation in tadpoles stabilize the Bombina hybrid zone? Ecology 83:1648–1659.

Wall, J. D. 2003. Estimating ancestral population sizes and divergence times. Genetics 163:395–404. PubMed PMC

Werner, E. E. , and Anholt B. R.. 1993. Ecological consequences of the trade‐off between growth and mortality rates mediated by foraging activity. Am. Nat. 142:242–272. PubMed

Yanchukov, A. , Hofman S., Szymura J. M., Mezhzherin S., Morozov‐Leonov S. Y., Barton N. H., and Nürnberger B.. 2006. Hybridization of Bombina bombina and B. variegata (Anura, Discoglossidae) at a sharp ecotone in Western Ukraine: comparisons across transects and over time. Evolution 60:583–600. PubMed

Yang, Z. 2007. PAML 4: Phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24:1586–1591. PubMed

Yeaman, S. , and Whitlock M. C.. 2011. The architecture of adaptation under migration‐selection balance. Evolution 65:1897–1911. PubMed

Zheng, Y. , Fu J., and Li S.. 2009. Toward understanding the distribution of Laurasian frogs: a test of Savage's biogeographical hypothesis using the genus Bombina . Mol. Phylogenet. Evol. 52:70–83. PubMed

A dense linkage map for a large repetitive genome: discovery of the sex-determining region in hybridizing fire-bellied toads (Bombina bombina and Bombina variegata)

Wolbachia affects mitochondrial population structure in two systems of closely related Palaearctic blue butterflies