Sex chromosome systems tend to be highly conserved and knowledge about their evolution typically comes from macroevolutionary inferences. 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 hybridization (array-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 hybridization (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-homologs of Z-linked orthologs and characterized their molecular differentiation. Our results suggest that the multiple sex chromosome system evolved in a common ancestor of these three Leptidea species as a result of dynamic genome reshuffling through repeated rearrangements between the sex chromosomes and autosomes, including translocations and fissions. Thus, the sex chromosome turnover could not play a role in reproductive isolation between the Leptidea species studied. However, we suggest that subsequent species-specific rearrangements of multiple sex chromosomes, along with different rates of neo-W chromosome degeneration and significantly increased number of Z-linked genes could accelerate the accumulation of genetic incompatibilities between populations and promote their divergence resulting in speciation.

README.txt
This text file contains description of all files associated with this Dryad repository.

L_juvernica_non_redundant_transcripts.fas
This fasta file contains a dataset of 30057 non-redundant transcripts of L. juvernica produced by the EvidentialGene pipeline. 

L_juvernica_HaMStR_orthologs.fas
This fasta file contains sequences of 4398 L. juvernica orthologs identified by HaMStR search using the Lepidoptera core ortholog set by Kawahara and Breinholt (2014. Proc. R. Soc. B 281: 20140970). This dataset was used to design the Agilent custom microarray.

L_juvernica_array_design.zip
This archive contains design of the Agilent microarray with 37909 probes which was used for array-CGH. 

L_juvernica_CGH_log_ratio_values.csv
This tab-delimited file contains results of array-CGH performed in L. juvernica, i.e. log2(M/F) values for four replicas and their mean. 

L_juvernica_linkage_and_chromosome_assignment_in_Bmori_and_Mcinxia.csv
This tab-delimited file contains information on linkage of L. juvernica orthologs as determined by array-CGH along with their Bombyx mori and Melitaea cinxia ortholog IDs and chromosome assingments.