Supplementary Material for: Karyotypes versus Genomes: The Nymphalid Butterflies Melitaea cinxia, Danaus plexippus, and D. chrysippus

<p>The number of sequenced lepidopteran genomes is increasing rapidly. However, the corresponding assemblies rarely represent whole chromosomes and generally also lack the highly repetitive W sex chromosome. Knowledge of the karyotypes can facilitate genome assembly and further our understanding of sex chromosome evolution in Lepidoptera. Here, we describe the karyotypes of the Glanville fritillary <i>Melitaea cinxia</i> (n = 31), the monarch <i>Danaus plexippus</i> (n = 30), and the African queen <i>D. chrysippus</i> (2n = 60 or 59, depending on the source population). We show by FISH that the telomeres are of the (TTAGG)<sub>n</sub> type, as found in most insects. <i>M. cinxia</i> and <i>D. plexippus</i> have “conventional” W chromosomes which are heterochromatic in meiotic and somatic cells. In <i>D. chrysippus</i>, the W is inconspicuous. Neither telomeres nor W chromosomes are represented in the published genomes of <i>M. cinxia</i> and <i>D. plexippus</i>. Representation analysis in sequenced female and male <i>D. chrysippus</i> genomes detected an evolutionarily old autosome-Z chromosome fusion in <i>Danaus</i>. Conserved synteny of whole chromosomes, so called “macro synteny”, in Lepidoptera permitted us to identify the chromosomes involved in this fusion. An additional and more recent sex chromosome fusion was found in <i>D. chrysippus</i> by karyotype analysis and classical genetics. In a hybrid population between 2 subspecies, <i>D. c. chrysippus</i> and <i>D. c. dorippus</i>, the W chromosome was fused to an autosome that carries a wing colour locus. Thus, cytogenetics and the present state of genome data complement one another to reveal the evolutionary history of the species.</p>