Evolutionary Biology

, Volume 45, Issue 4, pp 425–436 | Cite as

Quantification of Reproductive Isolating Barriers Between Two Naturally Hybridizing Killifish Species

  • Ruthie E. Barbas
  • Matthew R. GilgEmail author
Research Article


Understanding the relative importance of various reproductive barriers to the early stages of speciation is an essential question in evolutionary biology. The closely related killifishes Fundulus heteroclitus and F. grandis occasionally hybridize in a small region in coastal Northeastern Florida showing that while barriers to reproduction exist, they are incomplete. The objective of this study was to elucidate barriers to reproduction between F. heteroclitus and F. grandis in the lab, as well as to quantify their strengths and relative contributions to reproductive isolation. Pre-zygotic (mating and fertilization) and post-zygotic (hatching) barriers were investigated by performing a variety of choice and no-choice laboratory mating experiments. Under no-choice conditions, barriers to mating had the greatest influence on hybrid production in F. grandis, whereas hatching barriers contributed to the majority of reproductive isolation in F. heteroclitus. Under choice conditions, however, pre-zygotic barriers had the greatest influence on hybrid production in both species. The total reproductive isolation that was observed in females of each species was stronger in F. heteroclitus than in F. grandis, and was nearly complete in F. heteroclitus females under choice conditions and was of moderate strength in F. grandis females. These results reveal an asymmetry in the potential gene flow between these two species, with F. grandis being more likely to hybridize than F. heteroclitus in the absence of environmental influences. No-choice backcrosses were also conducted and showed that at least some F1 hybrids are fertile. The observation that pre-zygotic barriers tend to be stronger than post-zygotic barriers in the early stages of speciation is consistent with similar studies in other organisms.


Reproductive isolation Speciation Hybridization Fundulus 



We would like to thank the Lerner-Gray Memorial Fund of the American Museum of Natural History, the UNF Graduate School, and the UNF Coastal Biology Program for providing funding for this research. We thank Carlos Barbas, Jennifer Raabe, Victor Senf, Veronica Logue and Leigh Jordan for their help with collecting and caring for animals. Additionally, we would like to thank Dr. Kelly Smith and Dr. Eric Johnson for their comments on previous versions of this work and to Dr. Elena Buzaianu for statistical suggestions.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11692_2018_9460_MOESM1_ESM.doc (93 kb)
Supplementary material 1 (DOC 93 KB)


  1. Able, K., & Hagan, S. (2003). Impact of common reed, Phragmites australis, on essential fish habitat: Influence on reproduction, embryological development, and larval abundance of Mummichog (Fundulus heteroclitus). Estuaries, 26, 40–50.CrossRefGoogle Scholar
  2. Able, K., & Hata, D. (1984). Reproductive behavior in the Fundulus heteroclitus-F. grandis complex. Copeia, 1984, 820–825.CrossRefGoogle Scholar
  3. Berdan, E. L., & Fuller, R. C. (2012). A test for environmental effects on behavioral isolation in two species of killifish. Evolution, 66, 3224–3237.CrossRefGoogle Scholar
  4. Bernardi, G., & Powers, D. A. (1995). Phylogenetic relationships among nine species from the genus Fundulus (Cyprinodontiformes, Fundulidae) inferred from sequences of the cytochrome B gene. Copeia, 1995, 469–473.CrossRefGoogle Scholar
  5. Case, T., & Taper, M. (2000). Interspecific competition, environmental gradients, gene flow, and the coevolution of species’ borders. The American Naturalist, 155, 583–605.CrossRefGoogle Scholar
  6. Coyne, J. A., & Orr, A. H. (2004). Speciation. Sunderland: Sinauer Associates, Inc.Google Scholar
  7. Coyne, J. A., & Orr, H. A. (1989). Patterns of speciation in Drosophila. Evolution, 43, 362–381.CrossRefGoogle Scholar
  8. Coyne, J. A., & Orr, H. A. (1998). The evolutionary genetics of speciation. Philosophical Transactions of the Royal Society of London B, 353, 287–305.CrossRefGoogle Scholar
  9. Dobzhansky, T. (1937). Genetics and the origin of species. New York: Columbia University Press.Google Scholar
  10. Dobzhansky, T. (1940). Speciation as a stage in evolutionary divergence. The American Naturalist, 74, 312–321.CrossRefGoogle Scholar
  11. Ellis, W., & Bell, S. (2004). Conditional use of mangrove habitats by fishes: Depth as a due to avoid predators. Estuaries, 27, 966–976.CrossRefGoogle Scholar
  12. Elmer, K. R., Lehtonen, T. K., & Meyer, A. (2009). Color assortative mating contributes to sympatric divergence of neotropical cichlid fish. Evolution, 63, 2750–2757.CrossRefGoogle Scholar
  13. Foster, N. R. (1967). Trends in the evolution of reproductive behavior in killifishes. In: F. M. Bayer et al., (Eds.), Proceedings of the international conference on tropical oceanography (pp. 549–566). Miami, FL: University of Miami Institute of Marine Sciences.Google Scholar
  14. Fricke, C., & Arnqvist, G. (2004). Conspecific sperm precedence in flour beetles. Animal Behaviour, 67, 729–732.CrossRefGoogle Scholar
  15. Galleher, S. N., Gilg, M. R., & Smith, K. J. (2010). Comparison of larval thermal maxima between Fundulus heteroclitus and F. grandis. Fish Physiology and Biochemistry, 36, 731–740.CrossRefGoogle Scholar
  16. Galleher, S. N., Gonzalez, I., Gilg, M. R., & Smith, K. J. (2009). Larvae and juvenile Fundulus heteroclitus abundance and distribution in Northeast Florida salt marshes. Southeastern Naturalist, 8, 495–502.CrossRefGoogle Scholar
  17. Geyer, L. B., & Palumbi, S. R. (2005). Conspecific sperm precedence in two species of tropical sea urchins. Evolution, 59, 97–105.CrossRefGoogle Scholar
  18. Gonzalez, I., Levin, M., Jermanus, S., Watson, B., & Gilg, M. R. (2009). Genetic assessment of species ranges in Fundulus heteroclitus and F. grandis in northeastern Florida salt marshes. Southeastern Naturalist, 8, 227–243.CrossRefGoogle Scholar
  19. Grady, J. M., Coykendall, D. K., Collette, B. B., & Quattro, J. M. (2001). Taxonomic diversity, origin, and conservation status of Bermuda killifishes (Fundulus) based on mitochondrial cytochrome b phylogenies. Conservation Genetics, 2, 41–52.CrossRefGoogle Scholar
  20. Gregorio, O., Berdan, E. L., Kozak, G. M., & Fuller, R. C. (2012). Reinforcement of male mate preference in sympatric killifish species Lucania goodie and Lucania parva. Behavioral Ecology and Sociobiology, 66, 1429–1436.CrossRefGoogle Scholar
  21. Hipperson, H., Dunning, L. T., Baker, W. J., Butlin, R. K., Hutton, I., Papadopulos, A. S. T., et al. (2016). Ecological speciation in sympatric palms: 2. Pre- and post-zygotic isolation. Journal of Evolutionary Biology, 29, 2143–2156.CrossRefGoogle Scholar
  22. Howard, D., Gregory, P., Chu, J., & Cain, M. (1998). Conspecific sperm precedence is an effective barrier to hybridization between closely related species. Evolution, 52, 511–516.CrossRefGoogle Scholar
  23. Howard, D. J. (1993). Reinforcement: Origin, dynamics and fate of an evolutionary hypothesis. In R. G. Harrison (Ed.), Hybrid zones and the evolutionary process (pp. 46–69). New York: Oxford University Press.Google Scholar
  24. Hsiao, S., & Meier, A. (1989). Comparison of semilunar cycles of spawning activity in Fundulus grandis and F. heteroclitus held under constant laboratory conditions. Journal of Experimental Zoology, 252, 213–218.CrossRefGoogle Scholar
  25. Hsiao, S. M., Limesand, S. W., & Wallace, R. A. (1996). Semilunar follicular cycle of an intertidal fish: The Fundulus model. Biology of Reproduction, 54, 809–818.CrossRefGoogle Scholar
  26. Hsiao, S. -M., & Meier, A. H. (1986). Spawning cycles of the Gulf killifish, Fundulus grandis, in closed circulation systems. Journal of Experimental Zoology, 240, 105–112.CrossRefGoogle Scholar
  27. Jordan, D., & Evermann, B. (1898). The fishes of North and Middle America: A descriptive catalogue of the species of fish-like vertebrates found in the waters of North America, north of the Isthmus of Panama, 47th ed. US Government Printing Office, Washington, District of Columbia.Google Scholar
  28. Kneib, R. (1986). The role of Fundulus heteroclitus in salt marsh trophic dynamics. American Zoologist, 26, 259–269.CrossRefGoogle Scholar
  29. Kneib, R. T. (1984). Patterns in the utilization of the intertidal salt marsh by larvae and juveniles of Fundulus heteroclitus (Linnaeus) and Fundulus luciae (Baird). Journal of Experimental Marine Biology and Ecology, 83, 41–51.CrossRefGoogle Scholar
  30. Lackey, A. C. R., & Boughman, J. W. (2017). Evolution of reproductive isolation in stickleback fish. Evolution, 71, 357–372.CrossRefGoogle Scholar
  31. Laurie, C. C. (1997). The weaker sex is heterogametic: 75 years of Haldane’s rule. Genetics, 147, 937–951.PubMedPubMedCentralGoogle Scholar
  32. Lowry, D. B., Modliszewski, J. L., Wright, K. M., Wu, C. A., & Willis, J. H. (2008). The strength and genetic basis of reproductive isolating barriers in flowering plants. Philosophical Transactions of the Royal Society of London B, 363, 3009–3021.CrossRefGoogle Scholar
  33. Ludlow, A. M., & Magurran, A. E. (2006). Gametic isolation in guppies (Poecilia reticulata). Proceedings of the Royal Society of London B, 273, 2477–2482.CrossRefGoogle Scholar
  34. Martin, M. D., & Mendelson, T. C. (2016). The accumulation of reproductive isolation in early stages of divergence supports a role for sexual selection. Journal of Evolutionary Biology, 29, 676–689.CrossRefGoogle Scholar
  35. Martin, M. D., & Mendelson, T. C. (2018). Hybrid sterility increases with genetic distance in snubnose darters (Percidae: Etheostoma). Environmental Biology of Fishes, 101, 215–221.CrossRefGoogle Scholar
  36. Martin, N. H., & Willis, J. H. (2007). Ecological divergence associated with mating system causes nearly complete reproductive isolation between sympatric Mimulus species. Evolution, 61, 68–82.CrossRefGoogle Scholar
  37. Martin, N. H., & Willis, J. H. (2010). Geographical variation in postzygotic isolation and its genetic basis within and between two Mimulus species. Philosophical Transactions of the Royal Society of London B, 365, 2469–2478.CrossRefGoogle Scholar
  38. Martín-Coello, J., Benavent-Corai, J., Roldan, E. R. S., & Gomendio, M. (2009). Sperm competition promotes asymmetries in reproductive barriers between closely related species. Evolution, 63, 613–623.CrossRefGoogle Scholar
  39. Matsubayashi, K. W., & Katakura, H. (2009). Contribution of multiple isolating barriers to reproductive isolation between a pair of phytophagous ladybird beetles. Evolution, 63, 2563–2580.CrossRefGoogle Scholar
  40. Mayr, E. (1940). Speciation phenomena in birds. The American Naturalist, 74, 249–278.CrossRefGoogle Scholar
  41. Mayr, E. (1942). Systematics and the origin of species, from the viewpoint of a zoologist. Cambridge: Harvard University Press.Google Scholar
  42. Mayr, E. (2000). The biological species concept. In Q. D. Wheeler & R. Meier (Eds.), Species concepts and phylogenetic theory: A debate (pp. 17–29). New York: Columbia University Press.Google Scholar
  43. Mendelson, T. C. (2003). Sexual isolation evolves faster than hybrid inviability in a diverse and sexually dimorphic genus of fish (Percidae: Etheostoma). Evolution, 57, 317–327.CrossRefGoogle Scholar
  44. Mendelson, T. C., Imhoff, V. E., & Venditti, J. J. (2007). The accumulation of reproductive barriers during speciation: Postmating barriers in two behaviorally isolated species of darters (Percidae: Etheostoma). Evolution, 61, 2596–2606.CrossRefGoogle Scholar
  45. Muller, H. J. (1939). Reversibility in evolution considered from the standpoint of genetics. Biological Reviews, 14, 261–280.CrossRefGoogle Scholar
  46. Muller, H. J. (1942). Isolating mechanisms, evolution, and temperature. Biology Symposium, 6, 71–125.Google Scholar
  47. Naisbit, R. E., Jiggins, C. D., Linares, M., Salazar, C., & Mallet, J. (2002). Hybrid sterility, Haldane’s rule and speciation in Heliconius cydno and H. melpomene. Genetics, 161, 1517–1526.PubMedPubMedCentralGoogle Scholar
  48. Newman, H. (1907). Spawning behavior and sexual dimorphism in Fundulus heteroclitus and allied fish. Biological Bulletin, 12, 314–348.CrossRefGoogle Scholar
  49. Nosil, P. (2012). Ecological speciation. New York: Oxford University Press.CrossRefGoogle Scholar
  50. Orr, H. A. (1987). Genetics of male and female sterility in hybrids of Drosophila pseudoobscura and D. persimilis. Genetics, 116, 555–563.PubMedPubMedCentralGoogle Scholar
  51. Orti, G., Bell, M. A., Reimchen, T. E., & Meyer, A. (1994). Global survey of mitochondrial DNA sequences in the threespine Stickleback: Evidence for recent migrations. Evolution, 48, 608–622.CrossRefGoogle Scholar
  52. Ostevik, K. L., Andrew, R. L., Otto, S. P., & Rieseberg, L. H. (2016). Multiple reproductive barriers separate recently diverged sunflower ecotypes. Evolution, 70, 2322–2335.CrossRefGoogle Scholar
  53. Panhuis, T. M., Butlin, R., Zuk, M., & Tregenza, T. (2001). Sexual selection and speciation. Trends in Ecology & Evolution, 16, 364–371.CrossRefGoogle Scholar
  54. Pombi, M., Kengne, P., Gimonneau, G., Tene-Fossog, B., Ayala, D., Kamdem, C., et al. (2017). Dissecting functional components of reproductive isolation among closely related sympatric species of the Anopheles gambiae complex. Evolutionary Applications, 10, 1102–1120.CrossRefGoogle Scholar
  55. Presgraves, D. C. (2010). Darwin and the origin of interspecific genetic incompatibilities. The American Naturalist, 176, S45–S60.CrossRefGoogle Scholar
  56. Ramsey, J., Bradshaw, H. D., & Schemske, D. W. (2003). Components of reproductive isolation between the monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae). Evolution, 57, 1520–1534.CrossRefGoogle Scholar
  57. Reynolds, J., & Gross, M. (1992). Female mate preference enhances offspring growth and reproduction in a fish, Poecilia reticulata. Proceedings of the Royal Society of London B, 250, 57–62.CrossRefGoogle Scholar
  58. Rundle, H. D., & Schluter, D. (1998). Reinforcement of stickleback mate preferences: Sympatry breeds contempt. Evolution, 52, 200–208.CrossRefGoogle Scholar
  59. Salzburger, W., Niederstätter, H., Brandstätter, A., Berger, B., Parson, W., Snoeks, J., et al. (2006). Colour-assortative mating among populations of Tropheus moorii, a cichlid fish from Lake Tanganyika, East Africa. Proceedings of the Royal Society of London B, 273, 257–66.CrossRefGoogle Scholar
  60. Schluter, D. (2001). Ecology and the origin of species. Trends in Ecology & Evolution, 16, 372–380.CrossRefGoogle Scholar
  61. Seehausen, O., van Alphen, J., & Witte, F. (1997). Cichlid fish diversity threatened by eutrophication that curbs sexual selection. Science, 277, 1808–1811.CrossRefGoogle Scholar
  62. Seehausen, O., & Van Alphen, J. J. M. (1998). The effect of male coloration on female mate choice in closely related Lake Victoria cichlids (Haplochromis nyererei complex). Behavioral Ecology and Sociobiology, 42, 1–8.CrossRefGoogle Scholar
  63. Sobel, J. M., & Chen, G. F. (2014). Unification of methods for estimating the strength of reproductive isolation. Evolution, 68, 1511–1522.CrossRefGoogle Scholar
  64. Sobel, J. M., Chen, G. F., Watt, L. R., & Schemske, D. W. (2010). The biology of speciation. Evolution, 64, 295–315.CrossRefGoogle Scholar
  65. Taylor, M., Leach, G., & DiMichele, L. (1979). Lunar Spawning Cycle in the Mummichog, Fundulus heteroclitus (Pisces: Cyprinodontidae). Copeia 1979, 291–297.CrossRefGoogle Scholar
  66. Vigueira, P. A., Schaefer, J. F., Duvernell, D. D., & Kreiser, B. R. (2007). Tests of reproductive isolation among species in the Fundulus notatus (Cyprinodontiformes: Fundulidae) species complex. Evolutionary Ecology, 22, 55–70.CrossRefGoogle Scholar
  67. Whitehead, A. (2010). The evolutionary radiation of diverse osmotolerant physiologies in killifish (Fundulus sp.). Evolution, 64, 2070–2085.PubMedGoogle Scholar
  68. Williams, T. H., & Mendelson, T. C. (2014). Quantifying reproductive barriers in a sympatric pair of darter species. Evolutionary Biology, 41, 212–220.CrossRefGoogle Scholar
  69. Zeng, L. W., & Singh, R. S. (1993). The genetic basis of Haldane’s rule and the nature of asymmetric hybrid male sterility among Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. Genetics, 134, 251–260.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiologyUniversity of North FloridaJacksonvilleUSA

Personalised recommendations