We report the discovery of a neo-sex chromosome in the monarch butterfly, Danaus plexippus, and several of its close relatives. Z-linked scaffolds in the D. plexippus genome assembly were identified via sex-specific differences in Illumina sequencing coverage. Additionally, a majority of the D. plexippus genome assembly was assigned to chromosomes based on counts of one-to-one orthologs relative to the butterfly Melitaea cinxia (with replication using two other lepidopteran species), in which genome scaffolds have been mapped to linkage groups. Sequencing coverage-based assessments of Z linkage combined with homology-based chromosomal assignments provided strong evidence for a Z-autosome fusion in the Danaus lineage, involving the autosome homologous to chromosome 21 in M. cinxia Coverage analysis also identified three notable assembly errors resulting in chimeric Z-autosome scaffolds. Cytogenetic analysis further revealed a large W chromosome that is partially euchromatic, consistent with being a neo-W chromosome. The discovery of a neo-Z and the provisional assignment of chromosome linkage for >90% of D. plexippus genes lays the foundation for novel insights concerning sex chromosome evolution in this female-heterogametic model species for functional and evolutionary genomics.

Department of Ecology and Evolutionary Biology University of Kansas Lawrence Kansas 66046

Faculty of Science University of South Bohemia in České Budéjovice 370 05 České Budějovice Czech Republic

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

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Sex-biased gene content is associated with sex chromosome turnover in Danaini butterflies

Degenerated, Undifferentiated, Rearranged, Lost: High Variability of Sex Chromosomes in Geometridae (Lepidoptera) Identified by Sex Chromatin

Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in Leptidea wood-white butterflies

Patterns of Sex Chromosome Differentiation in Spiders: Insights from Comparative Genomic Hybridisation

Absence of W Chromosome in Psychidae Moths and Implications for the Theory of Sex Chromosome Evolution in Lepidoptera

Sex Chromosome Turnover in Moths of the Diverse Superfamily Gelechioidea

Insights into the Structure of the Spruce Budworm (Choristoneura fumiferana) Genome, as Revealed by Molecular Cytogenetic Analyses and a High-Density Linkage Map