Distinguishing between hybrid introgression and incomplete lineage sorting causing incongruence among gene trees in that they exhibit topological differences requires application of statistical approaches that are based on biologically relevant models. Such study is especially challenging in hybrid systems, where usual vectors mediating interspecific gene transfers--hybrids with Mendelian heredity--are absent or unknown. Here we study a complex of hybridizing species, which are known to produce clonal hybrids, to discover how one of the species, Cobitis tanaitica, has achieved a pattern of mito-nuclear mosaic genome over the whole geographic range. We appplied three distinct methods, including the method using solely the information on gene tree topologies, and found that the contrasting mito-nuclear signal might not have resulted from the retention of ancestral polymorphism. Instead, we found two signs of hybridization events related to C. tanaitica; one concerning nuclear gene flow and the other suggested mitochondrial capture. Interestingly, clonal inheritance (gynogenesis) of contemporary hybrids prevents genomic introgressions and non-clonal hybrids are either absent or too rare to be detected among European Cobitis. Our analyses therefore suggest that introgressive hybridizations are rather old episodes, mediated by previously existing hybrids whose inheritance was not entirely clonal. Cobitis complex thus supports the view that the type of resulting hybrids depends on a level of genomic divergence between sexual species.

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics AS CR v v i Libechov Czech Republic

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics AS CR v v i Libechov Czech Republic; Ecological Genetics Research Unit Department of Biosciences University of Helsinki Helsinki Finland

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics AS CR v v i Libechov Czech Republic; Institute of Zoology Slovak Academy of Sciences Bratislava Slovakia

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics AS CR v v i Libechov Czech Republic; Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics of the ASCR v v i Prague Czech Republic

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics AS CR v v i Libechov Czech Republic; Life Science Research Centre Department of Biology and Ecology Faculty of Natural Sciences University of Ostrava Ostrava Czech Republic

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics AS CR v v i Libechov Czech Republic; Zoological Institute Evolutionary Biology University of Basel Basel Switzerland; Department of Zoology Faculty of Science Charles University Prague Prague Czech Republic

See more in PubMed

Arnold ML (2006) Evolution through genetic exchange. USA: Oxford University Press.

Gompert Z, Fordyce JA, Forister ML, Shapiro AM, Nice CC (2006) Homoploid Hybrid Speciation in an Extreme Habitat. Science 314: 1923–1925. PubMed

Mallet J (2005) Hybridization as an invasion of the genome. Trends Ecol Evol 229–237. PubMed

Mallet J (2007) Hybrid speciation. Nature 446: 279–283. PubMed

Jones JC, Perez-Sato J, Meyer A (2012) A phylogeographic investigation of the hybrid origin of a species of swordtail fish from Mexico. Mol Ecol 21: 2692–2712. PubMed

Sang T, Zhong Y (2000) Testing Hybridization Hypotheses Based on Incongruent Gene Trees. Syst Biol 49: 422–434. PubMed

Ballard JW, Whitlock MC (2004) The incomplete natural history of mitochondria. Mol Ecol 13: 729–744. PubMed

Peters JL, Zhuravlev Y, Fefelov I, Logie A, Omland KE (2007) Nuclear loci and coalescent methods support ancient hybridization as cause of mitochondrial paraphyly between gadwall and falcated duck (Anas spp.). Evolution 61: 1992–2006. PubMed

Joly S, McLenachan PA, Lockhart PJ (2009) A statistical approach for distinguishing hybridization and incomplete lineage sorting. Am Nat 174: E54–70. PubMed

Kuhner MK (2009) Coalescent genealogy samplers: windows into population history. Trends Ecol Evol 24: 86–93. PubMed PMC

Hudson RR, Turelli M (2003) Stochasticity overrules the “three-times rule”: genetic drift, genetic draft, and coalescence times for nuclear loci versus mitochondrial DNA. Evolution 57: 182–190. PubMed

Rannala B, Yang Z (2003) Bayes Estimation of Species Divergence Times and Ancestral Population Sizes Using DNA Sequences From Multiple Loci. Genetics 164: 1645–1656. PubMed PMC

Hey J (2006) Recent advances in assessing gene flow between diverging populations and species. Curr Opin Genet Dev 16: 592–596. PubMed

Hey J (2010) Isolation with Migration Models for More Than Two Populations. Mol Biol Evol 27: 905–920. PubMed PMC

Good JM, Hird S, Reid N, Demboski JR, Steppan SJ, et al. (2008) Ancient hybridization and mitochondrial capture between two species of chipmunks. Mol Ecol 17: 1313–1327. PubMed

Senn HV, Pemberton JM (2009) Variable extent of hybridization between invasive sika (Cervus nippon) and native red deer (C. elaphus) in a small geographical area. Mol Ecol 18: 862–876. PubMed

Bachtrog D, Thornton K, Clark A, Andolfatto P (2006) Extensive introgression of mitochondrial DNA relative to nuclear genes in the Drosophila yakuba species group. Evolution 60: 292–302. PubMed

Ropiquet A, Hassanin A (2006) Hybrid origin of the Pliocene ancestor of wild goats. Mol Phyl Evol 41: 395–404. PubMed

Gompert Z, Forister ML, Fordyce JA, Nice CC (2008) Widespread mito-nuclear discordance with evidence for introgressive hybridization and selective sweeps in Lycaeides . Mol Ecol 17: 5231–5244. PubMed

Takahata N (1989) Gene genealogy in three related populations: consistency probability between gene and population trees. Genetics 122: 957–966. PubMed PMC

Rosenberg NA (2002) The probability of topological concordance of gene trees and species trees. Theor l Pop Biol 2002 61: 225–247. PubMed

Currat M, Ruedi M, Petit RJ, Excoffier L (2008) The hidden side of invasions: massive introgression by local genes. Evolution 62: 1908–1920. PubMed

Petit RJ, Excoffier L (2009) Gene flow and species delimitation. Trends Ecol Evol 24: 386–393. PubMed

Wilson CC, Bernatchez L (2008) The ghost of hybrids past: fixation of arctic charr (Salvelinus alpinus) mitochondrial DNA in an introgressed population of lake trout (S. namaycush). Mol Ecol 7: 127–132.

Carson EW, Dowling TE (2006) Influence of hydrogeographic history and hybridization on the distribution of genetic variation in the pupfishes Cyprinodon atrorus and C. bifasciatus . Mol Ecol 15: 667–679. PubMed

Nevado B, Koblmüller S, Sturmbauer C, Snoeks J, Usano-Alemany J, et al. (2009) Complete mitochondrial DNA replacement in a Lake Tanganyika cichlid fish. Mol Ecol 18: 4240–4255. PubMed

Di Candia MR, Routman EJ (2007) Cytonuclear discordance across a leopard frog contact zone. Mol Phyl Evol 45: 564–575. PubMed

Dowling TE, Secor CL (1997) The role of hybridization and introgression in the diversification of animals. Ann Rev Ecol Syst 28: 593–619.

Seehausen O 2004 Hybridization and adaptive radiation. Trends Ecol Evol 19: 198–207. PubMed

Gerard D, Gibbs HL, Kubatko L (2011) Estimating hybridization in the presence of coalescence using phylogenetic intraspecific sampling. BMC Evol Biol 11: 291. PubMed PMC

Allendorf FW, Leary RF, Spruell P, Wenburg JK (2001) The problems with hybrids: setting conservation guidelines. Trends Ecol Evol 16: 613–622.

Meyer A, Salzburger W, Schartl M (2006) Hybrid origin of a swordtail species (Teleostei: Xiphophorus clemenciae) driven by sexual selection. Mol Ecol 15: 721–730. PubMed

Alves MJ, Coelho MM, Collares-Pereira MJ (1998) Diversity in the reproductive modes of females of the Rutilus alburnoides complex (Teleostei, Cyprinidae): a way to avoid the genetic constraints of uniparentalism. Mol Biol Evol 15: 1233–1242.

Spolsky C, Uzzell T (1984) Natural interspecies transfer of mitochondrial DNA in amphibians. Proc Natl Acad Sci 81: 5802–5805. PubMed PMC

Hotz H, Beerli P, Spolsky C (1992) Mitochondrial DNA reveals formation of nonhybrid frogs by natural matings between hemiclonal hybrids. Mol Biol Evol 9: 610–620. PubMed

Goddard KA, Schultz RJ (1993) Aclonal Reproduction by Polyploid Members of the Clonal Hybrid Species Phoxinus eos-neogaeus (Cyprinidae). Copeia 3: 650–660.

Saitoh K, Kim I-S, Lee E-H (2004) Mitochondrial Gene Introgression between Spined Loaches via Hybridogenesis. Zool Sci 21: 795–798. PubMed

Binet M-C, Angers B (2005) Genetic identification of members of the Phoxinus eos-neogaeus hybrid complex. J Fish Biol 67: 1169–1177.

Matos I, Sucena É, Machado MP, Gardner R, Inácio Â, et al. (2011) Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid Squalius alburnoides . BMC Genetics 12: 101. PubMed PMC

Vergilino R, Markova S, Ventura M, Manca M, Dufresne F (2011) Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers. Mol Ecol 20: 1191–207. PubMed

Schultz RJ (1969) Hybridization, unisexuality, and polyploidy in the teleost Poeciliopsis (Poeciliidae) and other vertebrates. Am Natur 103: 605–619.

Schartl M, Nanda I, Schlupp I, Wilde B, Epplen JT, et al. (1995) Incorporation of subgenomic amounts of DNA as compensation for mutational load in a gynogenetic fish. Nature 373: 68–71.

Stöck M, Lamatsch DK, Steinlein C, Epplen JT, Grosse WR, et al. (2002) A bisexually reproducing all-triploid vertebrate. Nat Genet 30: 325–328. PubMed

Morishima K, Yoshikawa H, Arai K (2008) Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage. Heredity 100: 581–586. PubMed

Christiansen DG, Reyer H-U (2009) From clonal to sexual hybrids: genetic recombination via triploids in all-hybrid populations of water frogs. Evolution 63: 1754–1768. PubMed

Bogart JP, Bi K, Fu J, Noble DWA, Niedzwiecki J (2007) Unisexual salamanders (genus Ambystoma) present a new reproductive mode for eukaryotes. Genome 50: 119–136. PubMed

Stöck M, Ustinova J, Betto-Colliard C, Schartl M, Moritz C, et al. (2012) Simultaneous Mendelian and clonal genome transmission in a sexually reproducing, all-triploid vertebrate. Proc R Soc London Ser B 279: 1293–1299. PubMed PMC

Toews DPL, Brelsford A (2012) The biogeography of mitochondrial and nuclear discordance in animals. Mol Ecol 21: 3907–3930. PubMed

Janko K, Culling MA, Rab P, Kotlik P (2005) Ice age cloning-comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation. Mol Ecol 14: 2991–3004. PubMed

Janko K, Flajšhans M, Choleva L, Bohlen J, Šlechtová V, et al. (2007) Diversity of European spined loaches (genus Cobitis L.): an update of the geographic distribution of the Cobitis taenia hybrid complex with a description of new molecular tools for species and hybrid determination. J Fish Biol 71: 387–408.

De Gelas K, Janko K, Volckaert FAM, De Charleroy D, Van Houdt JKJ (2008) Development of nine polymorphic microsatellite loci in the spined loach, Cobitis taenia, and cross-species amplification in the related species C. elongatoides, C. taurica and C. tanaitica . Mol Ecol Res 8: 1001–1003. PubMed

Janko K, Culling MA, Ráb P, Kotlík P (2005) Ice age cloning – comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation. Mol Ecol 14: 2991–3004. PubMed

Janko K, Kotusz J, De Gelas K, Slechtova V, Opoldusova Z, et al. (2012) Dynamic formation of asexual diploid and polyploid lineages: multilocus analysis of Cobitis reveals the mechanisms maintaining the diversity of clones. PloS ONE 7: e45384. PubMed PMC

Janko K, Bohlen J, Lamatsch D, Flajšhans M, Epplen JT, et al. (2007) The gynogenetic reproduction of diploid and triploid hybrid spined loaches (Cobitis: Teleostei), and their ability to establish successful clonal lineages—on the evolution of polyploidy in asexual vertebrates. Genetica 131: 185–194. PubMed

Choleva L, Janko K, De Gelas K, Bohlen J, Šlechtová V, et al. (2012) Synthesis of clonality and polyploidy in vertebrate animals by hybridization between two sexual species. Evolution 66: 2191–2203. PubMed

Buj I, Podnar M, Mrakovčić M, Choleva L, Šlechtová V, et al. (2008) Genetic diversity and phylogenetic relationships of spined loaches (genus Cobitis) in Croatia based on mtDNA and allozyme analyses. Folia Zool 57: 71–82.

Choleva L, Apostolou A, Ráb P, Janko K (2008) Making it on their own: sperm-dependent hybrid fishes (Cobitis) switch the sexual hosts and expand beyond the ranges of their original sperm donors. Philos Trans R Soc Lond B Biol Sci 363: 2911–2919. PubMed PMC

Saat TV (1991) Reproduction of the diploid and polyploid spinous loach (Cobitis, Teleostei). Oocyte maturation and fertilization in the triploid form. Soviet J Dev Biol 22: 332–338.

Vasil'ev VP, Akimova NV, Emel'yanova NG, Pavlov DA, Vasil'eva ED (2003) Reproductive capacities in the polyploid males of spined loaches from the unisexual–bisexual complex, occurred in the Moscow River. Folia Biol (Krakow) 51: 67–73. PubMed

Janko K, Kotlik P, Ráb P (2003) Evolutionary history of asexual hybrid loaches (Cobitis: Teleostei) inferred from phylogenetic analysis of mitochondrial DNA variation. J Evol Biol 16: 1280–1287. PubMed

Nadachowska K, Babik W (2009) Divergence in the Face of Gene Flow: The Case of Two Newts (Amphibia: Salamandridae). Mol Biol Evol 26: 829–841. PubMed

Liu L (2008) BEST: Bayesian estimation of species trees under the coalescent model. Bioinformatics 24: 2542–2543. PubMed

Yang Z, Rannala B (2010) Bayesian species delimitation using multilocus sequence data. Proc Natl Acad Sci 107: 9264–9269. PubMed PMC

Nielsen R, Wakeley J (2001) Distinguishing Migration From Isolation: A Markov Chain Monte Carlo Approach. Genetics 158: 885–896. PubMed PMC

Hey J, 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

Hey J (2010) The Divergence of Chimpanzee Species and Subspecies as Revealed in Multipopulation Isolation-with-Migration Analyses. Mol Biol Evol 27: 921–933. PubMed PMC

Cui R, Schumer M, Kruesi K, Walter R, Andolfatto P, et al. (2013) Phylogenomics reveals extensive reticulate evolution in Xiphophorus fishes. Evolution 67: 2166–2179. PubMed

Sequeira F, Sodré D, Ferrand N, Bernardi JA, Sampaio I, et al. (2011) Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers. BMC Evol Biol 11: 264. PubMed PMC

Slowinski JB, Guyer C (1989) Testing the Stochasticity of Patterns of Organismal Diversity: An Improved Null Model. Am Natur 134: 907–921.

Felsenstein J (2004) Inferring phylogenies. Sunderland, MA: Sinauer Associates. 457 p.

Buckley TR, Cordeiro M, Marshall DC, Simon C (2006) Differentiating between Hypotheses of Lineage Sorting and Introgression in New Zealand Alpine Cicadas (Maoricicada Dugdale). Syst Biol 55: 411–425. PubMed

Addison JA, Pogson GH (2009) Multiple gene genealogies reveal asymmetrical hybridization and introgression among strongylocentrotid sea urchins. Mol Ecol 18: 1239–1251. PubMed

Won Y-J, Hey J (2005) Divergence population genetics of chimpanzees. Mol Biol Evol 22: 297–307. PubMed

Slatkin M (2005) Seeing ghosts: the effect of unsampled populations on migration rates estimated for sampled populations. Mol Ecol 14: 67–73. PubMed

Gaggiotti OE (2011) Making inferences about speciation using sophisticated statistical genetics methods: look before you leap. Mol Ecol 20: 2229–2232. PubMed

Sousa VC, Grelaud A, Hey J (2011) On the nonidentifiability of migration time estimates in isolation with migration models. Mol Ecol 20: 3956–3962. PubMed PMC

Strasburg JL, Rieseberg LH (2011) Interpreting the estimated timing of migration events between hybridizing species. Mol Ecol 20: 2353–2366 14. PubMed

Niemiller ML, Nosil P, Fitzpatrick BM (2010) Corrigendum. Mol Ecol 19: 1513–15.

Moritz C, Brown WM, Densmore LD, Wright JW, Vyas D, et al.. (1989) Genetic diversity and the dynamics of hybrid parthenogenesis in Cnemidophorus (Teiidae) and Heteronotia (Gekkonidae). pp. 87–112 in Dawley RM and Bogart JP, eds. Evolution and ecology of unisexual vertebrates. Vol. 466. Albany, NY: New York State Museum.

Alves MJ, Coelho MM, Collares-Pereira MJ (2001) Evolution in action through hybridisation and polyploidy in an Iberian freshwater fish: a genetic review. Genetica 11: 375–385. PubMed

Plötner J, Uzzell T, Beerli P, Spolsky C, Ohst T, et al. (2008) Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs. J Evol Biol 21: 668–681. PubMed PMC

Sousa VC, Grelaud A, Hey J (2011) On the nonidentifiability of migration time estimates in isolation with migration models. Mol Ecol 20: 3956–3962. PubMed PMC

Mee JA, Taylor EB (2012) The cybrid invasion: widespread postglacial dispersal by Phoxinus (Pisces: Cyprinidae) cytoplasmic hybrids. Can J Zool 90: 577–584.

Vrijenhoek RC (1994) Unisexual fish: model systems for studying ecology and evolution. Annu Rev Ecol Evol Syst 25: 71–96.

Bogart JP, Bi K (2013) Genetic and genomic interactions of animals with different ploidy levels. Cytogenet Gen Res 140: 117–136. PubMed

Stenberg P, Saura A (2013) Meiosis and its deviations in polyploid animals. Cytogenet Gen Res 140: 185–203. PubMed

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. PubMed PMC

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.

Clark AG (1990) Inference of haplotypes from PCR-amplified samples of diploid populations. Mol Biol Evol 7: 111–122. PubMed

Nei M (1987) Molecular Evolutionary Genetics. New York: Columbia University Press.

Watterson GA (1975) On the number of segregating sites in genetical models without recombination. Theor Pop Biol 7: 256–276. PubMed

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

Hudson RR, Kaplan NL (1985) Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111: 147–164. PubMed PMC

Hudson RR, Kreitman M, Aguadé M (1987) A Test of Neutral Molecular Evolution Based on Nucleotide Data. Genetics 116: 153–159. PubMed PMC

Tachida H (2000) DNA evolution under weak selection. Gene 261: 3–9. PubMed

Presgraves DC (2005) Recombination Enhances Protein Adaptation in Drosophila melanogaster . Curr Biol 15: 1651–1656. PubMed

Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818. PubMed

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. PubMed

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Mol Biol Evol 24: 1596–1599. PubMed

Tajima F (1993) Simple Methods for Testing the Molecular Evolutionary Clock Hypothesis. Genetics 135: 599–607. PubMed PMC

Liu L, Pearl DK, Liu L (2007) Species trees from gene trees: Reconstructing Bayesian posterior distributions of a species phylogeny using estimated gene tree distributions. Syst Biol 56: 504–514. PubMed

Maddison WP, Maddison DR (2009) Mesquite: a modular system for evolutionary analysis. Version 2.6, available online: http://mesquiteproject.org.

Jennings WB, Edwards SV (2005) Speciational history of Australian grass finches (Poephila) inferred from thirty gene trees. Evolution 59: 2033–2047. PubMed

Tang Q, Freyhof J, Xiong B, Liu H (2008) Multiple invasions of Europe by East Asian cobitid loaches (Teleostei: Cobitidae). Hydrobiologia 605: 17–28.

Axelsson E, Smith NGC, Sundström H, Berlin S, Ellegren H (2004) Male-Biased Mutation Rate and Divergence in Autosomal, Z-Linked and W-Linked Introns of Chicken and Turkey. Mol Biol Evol 21: 1538–1547. PubMed

Sperm-dependent asexual species and their role in ecology and evolution

No evidence for accumulation of deleterious mutations and fitness degradation in clonal fish hybrids: Abandoning sex without regrets

Cytogenetic Characterization of Seven Novel satDNA Markers in Two Species of Spined Loaches (Cobitis) and Their Clonal Hybrids

Hybrid asexuality as a primary postzygotic barrier between nascent species: On the interconnection between asexuality, hybridization and speciation

Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei)