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Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies


The reinforcement model of evolution argues that natural selection enhances pre-zygotic isolation between divergent populations or species by selecting against unfit hybrids1,2 or costly interspecific matings3. Reinforcement is distinguished from other models that consider the formation of reproductive isolation to be a by-product of divergent evolution4,5. Although theory has shown that reinforcement is a possible mechanism that can lead to speciation6,7,8, empirical evidence has been sufficiently scarce to raise doubts about the importance of reinforcement in nature6,9,10. Agrodiaetus butterflies (Lepidoptera: Lycaenidae) exhibit unusual variability in chromosome number. Whereas their genitalia and other morphological characteristics are largely uniform, different species vary considerably in male wing colour, and provide a model system to study the role of reinforcement in speciation. Using comparative phylogenetic methods, we show that the sympatric distribution of 15 relatively young sister taxa of Agrodiaetus strongly correlates with differences in male wing colour, and that this pattern is most likely the result of reinforcement. We find little evidence supporting sympatric speciation: rather, in Agrodiaetus, karyotypic changes accumulate gradually in allopatry, prompting reinforcement when karyotypically divergent races come into contact.

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Figure 1: Changes in male wing coloration along the phylogeny of Agrodiaetus.
Figure 2: Changes in male wing coloration between Agrodiaetus sister clades as a function of their genetic distance.
Figure 3: Age–range correlation plot.
Figure 4: Agrodiaetus karyotypic diversity strongly correlates with nucleotide divergence ( R 2 = 0.820; P < 0.002).


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We thank A. J. Berry, J. A. Coyne, S. V. Edwards, J. R. Morris and R. Vila for their advice during the preparation of this manuscript. C. Bilgin, J. Coleman, F. Fernández-Rubio, G. Grigorjev, J. Jubany, C. Ibánez, R. Martínez, M. L. Munguira, C. Sekercioglu, C. Stefanescu, M. A. Travassos, R. Vila and V. Zurilina helped with collecting specimens, and J. Coleman and R. Vila assisted with sequencing in the laboratory. W. H. Piel and A. Monteiro helped us to measure wing ultraviolet reflectance. This research was supported by three collecting grants from the Putnam Expeditionary Fund of the Museum of Comparative Zoology, Harvard University; a National Science Foundation Doctoral Dissertation Improvement Grant to N.P.K.; National Science Foundation and Baker Foundation grants to N.E.P.; Milton Fund grants to D.H. and J.B.P.; a Burroughs Wellcome Fund grant to J.B.P.; and grants from the Russian Foundation for Basic Research, and the Russian Federal Programs “Universities of Russia” and “Leading Scientific Schools” to V.A.L.

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Correspondence to Naomi E. Pierce.

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The sequences have been deposited in GenBank; see Supplementary Appendix 1 for details. Reprints and permissions information is available at The authors declare no competing financial interests.

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Supplementary Notes

This file contains Supplementary Methods, Supplementary Figures S1-S7, Supplementary Tables S1-S5, Supplementary Discussion and additional references. (PDF 4253 kb)

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Lukhtanov, V., Kandul, N., Plotkin, J. et al. Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies. Nature 436, 385–389 (2005).

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