The Drosophila Y chromosome is a degenerated, heterochromatic chromosome with few functional genes. Nonetheless, natural variation on the Y chromosome in Drosophila melanogaster has substantial trans-acting effects on the regulation of X-linked and autosomal genes. However, the contribution of Y chromosome divergence to gene expression divergence between species is unknown. In this study, we constructed a series of Y chromosome introgression lines, in which Y chromosomes from either Drosophila sechellia or Drosophila simulans are introgressed into a common D. simulans genetic background. Using these lines, we compared genome-wide gene expression and male reproductive phenotypes between heterospecific and conspecific Y chromosomes. We find significant differences in expression for 122 genes, or 2.84% of all genes analyzed. Genes down-regulated in males with heterospecific Y chromosomes are significantly biased toward testis-specific expression patterns. These same lines show reduced fecundity and sperm competitive ability. Taken together, these results imply a significant role for Y/X and Y/autosome interactions in maintaining proper expression of male-specific genes, either directly or via indirect effects on male reproductive tissue development or function.evolution | regulatory divergence | male fitness T he Y chromosome in Drosophila melanogaster comprises ∼40 MB of sequence (∼20% of the male genome), but contains fewer than 20 protein-coding genes, most of which are specialized male reproductive genes (1-5). The vast bulk of the chromosome is comprised of large blocks of repetitive sequences, including large microsatellite repeats, transposable element-derived sequence, and bobbed, the Y-linked rDNA array (6). The heterochromatic and gene-poor nature of the Y chromosome is consistent with theoretical models that predict rapid degeneration and specialization for male-specific functions of Y chromosomes in the absence of recombination (7-9). Furthermore, the hemizygous nature of the Y chromosome, combined with the lack of recombination, makes it uniquely susceptible to population genetic processes that reduce genetic variation and limit adaptation (10-13).As predicted by theory, little or no SNP polymorphism exists in single-copy protein-coding sequences on the Y chromosome (7, 14-16). However, several cytological forms of the Y chromosome segregate in at least some species of Drosophila (17), and molecular evidence also suggests that the Y chromosome is not monomorphic. Variation exists for the size of repetitive DNA blocks (18,19), and Y-linked genetic variation associates with variation in organismal phenotypes, including thermal tolerance of spermatogenesis (20, 21) and male mating success (22). Nonetheless, direct tests of association between variation at the rDNA locus and phenotypes reveal no significant effect (18) and theoretical predictions suggest that nonneutral variation on the Y chromosome can only be maintained under very restrictive conditions (23,24). We have previously shown that Y-linked gen...