X chromosomes have long been thought to conserve the structure and gene content of the ancestral autosome from which the sex chromosomes evolved. We compared the recently evolved papaya sex chromosomes with a homologous autosome of a close relative, the monoecious Vasconcellea monoica, to infer changes since recombination stopped between the papaya sex chromosomes. We sequenced 12 V. monoica bacterial artificial chromosomes, 11 corresponding to the papaya X-specific region, and 1 to a papaya autosomal region. The combined V. monoica X-orthologous sequences are much shorter (1.10 Mb) than the corresponding papaya region (2.56 Mb). Given that the V. monoica genome is 41% larger than that of papaya, this finding suggests considerable expansion of the papaya X; expansion is supported by a higher repetitive sequence content of the X compared with the papaya autosomal sequence. The alignable regions include 27 transcriptencoding sequences, only 6 of which are functional X/V. monoica gene pairs. Sequence divergence from the V. monoica orthologs is almost identical for papaya X and Y alleles; the Carica-Vasconcellea split therefore occurred before the papaya sex chromosomes stopped recombining, making V. monoica a suitable outgroup for inferring changes in papaya sex chromosomes. The papaya X and the hermaphrodite-specific region of the Y h chromosome and V. monoica have all gained and lost genes, including a surprising amount of changes in the X.Carica papaya | gene gains and losses | sex chromosome evolution | suppression of recombination | centromere of X chromosome P apaya (Carica papaya L.) is a trioecious tropical fruit crop that has a nascent XY sex chromosome system, in which the sex determining region occupies a small fraction of the X/Y chromosome pair (1-3). Papaya has two slightly different Y chromosomes that diverged about 73,000 y ago, Y in males and Y h in hermaphrodites (4). All genotypes without X chromosomes (YY, YY h , and Y h Y h ) are lethal in early development, resulting in 25% aborted seeds in selfed hermaphrodites and in crosses between hermaphrodites and males (5), indicating that both Y types have lost at least one gene essential for development.Papaya belongs to the family Caricaceae, with six genera and 35 species, 32 of which are dioecious, two are trioecious (with male, female, and hermaphrodite individuals) and one, Vasconcellea monoica, is monoecious (with male and female flowers on a single plant). The predominance of dioecious species suggests that dioecy is ancestral in this family and that the trioecious and monoecious species evolved recently. V. monoica has no sex chromosomes, because there is no sexual dimorphism among individuals, and there is a single sequence corresponding to each of the several papaya X/Y h gene pairs tested, whereas distinct X and Y alleles were detected in several dioecious Vasconcellea species (6), which indicates that the sex chromosomes in these species are homologous with those in papaya.V. monoica therefore provides an opportunity to compare the recentl...