Sex-chromosome systems tend to be highly conserved and knowledge about their evolution typically comes from macroevolutionary inference. Rapidly evolving complex sex-chromosome systems represent a rare opportunity to study the mechanisms of sex-chromosome evolution at unprecedented resolution. Three cryptic species of wood-white butterflies-Leptidea juvernica, L. sinapis and L. reali-have each a unique set of multiple sex-chromosomes with 3-4 W and 3-4 Z chromosomes. Using a transcriptome-based microarray for comparative genomic hybridisation (CGH) and a library of bacterial artificial chromosome (BAC) clones, both developed in L. juvernica, we identified Z-linked Leptidea orthologs of Bombyx mori genes and mapped them by fluorescence in situ hybridisation (FISH) with BAC probes on multiple Z chromosomes. In all three species, we determined synteny blocks of autosomal origin and reconstructed the evolution of multiple sex-chromosomes. In addition, we identified W homologues of Z-linked orthologs and characterised their molecular differentiation. Our results suggest that the multiple sex-chromosome system evolved in a common ancestor as a result of dynamic genome reshuffling through repeated rearrangements between the sex chromosomes and autosomes, including translocations, fusions and fissions. Thus, the initial formation of neo-sex chromosomes could not have played a role in reproductive isolation between these Leptidea species. However, the subsequent species-specific fissions of several neo-sex chromosomes could have contributed to their reproductive isolation. Then, significantly increased numbers of Z-linked genes and independent neo-W chromosome degeneration could accelerate the accumulation of genetic incompatibilities between populations and promote their divergence resulting in speciation.

Biology Centre of the Czech Academy of Sciences Institute of Entomology Branišovská 31 370 05 České Budějovice Czech Republic

Faculty of Science University of South Bohemia Branišovská 1760 370 05 České Budějovice Czech Republic

Genomics Core Facility European Molecular Biology Laboratory Heidelberg Germany

Institut de Biologia Evolutiva Pg Marítim de la Barceloneta 37 08003 Barcelona Spain

Institute of Experimental Botany of the Czech Academy of Sciences Centre of the Region Hana for Biotechnological and Agricultural Research Šlechtitelů 31 779 00 Olomouc Czech Republic

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