Genetic changes affecting gene expression contribute to phenotypic divergence; thus, understanding how regulatory networks controlling gene expression change over time is critical for understanding evolution. Prior studies of expression differences within and between species have identified properties of regulatory divergence, but technical and biological differences among these studies make it difficult to assess the generality of these properties or to understand how regulatory changes accumulate with divergence time. Here, we address these issues by comparing gene expression among strains and species of Drosophila with a range of divergence times and use F1 hybrids to examine inheritance patterns and disentangle cis- and trans-regulatory changes. We find that the fixation of compensatory changes has caused the regulation of gene expression to diverge more rapidly than gene expression itself. Specifically, we observed that the proportion of genes with evidence of cis-regulatory divergence has increased more rapidly with divergence time than the proportion of genes with evidence of expression differences. Surprisingly, the amount of expression divergence explained by cis-regulatory changes did not increase steadily with divergence time, as was previously proposed. Rather, one species (Drosophila sechellia) showed an excess of cis-regulatory divergence that we argue most likely resulted from positive selection in this lineage. Taken together, this work reveals not only the rate at which gene expression evolves, but also the molecular and evolutionary mechanisms responsible for this evolution.

BackgroundRNA-seq can be used to measure allele-specific expression (ASE) by assigning sequence reads to individual alleles; however, relative ASE is systematically biased when sequence reads are aligned to a single reference genome. Aligning sequence reads to both parental genomes can eliminate this bias, but this approach is not always practical, especially for non-model organisms. To improve accuracy of ASE measured using a single reference genome, we identified properties of differentiating sites responsible for biased measures of relative ASE.ResultsWe found that clusters of differentiating sites prevented sequence reads from an alternate allele from aligning to the reference genome, causing a bias in relative ASE favoring the reference allele. This bias increased with greater sequence divergence between alleles. Increasing the number of mismatches allowed when aligning sequence reads to the reference genome and restricting analysis to genomic regions with fewer differentiating sites than the number of mismatches allowed almost completely eliminated this systematic bias. Accuracy of allelic abundance was increased further by excluding differentiating sites within sequence reads that could not be aligned uniquely within the genome (imperfect mappability) and reads that overlapped one or more insertions or deletions (indels) between alleles.ConclusionsAfter aligning sequence reads to a single reference genome, excluding differentiating sites with at least as many neighboring differentiating sites as the number of mismatches allowed, imperfect mappability, and/or an indel(s) nearby resulted in measures of allelic abundance comparable to those derived from aligning sequence reads to both parental genomes.

Summary Genomic imprinting occurs when expression of an allele differs based on the sex of the parent that transmitted the allele. In D. melanogaster, imprinting can occur, but its impact on allelic expression genome-wide is unclear. Here, we search for imprinted genes in D. melanogaster using RNA-seq to compare allele-specific expression between pools of 7–10 day old adult female progeny from reciprocal crosses. 119 genes with allelic expression patterns consistent with imprinting were identified and showed significant clustering within the genome. Surprisingly, additional analysis of several of these genes showed that either genomic heterogeneity or high levels of intrinsic noise caused imprinting-like allelic expression. Consequently, our data provide no convincing evidence of imprinting for D. melanogaster genes in their native genomic context. Elucidating sources of false positive signals for imprinting in allele-specific RNA-seq data, as done here, is critical given the growing popularity of this method for identifying imprinted genes.

In species with a heterogametic sex, population genetics theory predicts that DNA sequences on the X chromosome can evolve faster than comparable sequences on autosomes. Both neutral and nonneutral evolutionary processes can generate this pattern. Complex traits like gene expression are not predicted to have accelerated evolution by these theories, yet a "faster-X" pattern of gene expression divergence has recently been reported for both Drosophila and mammals. Here, we test the hypothesis that accelerated adaptive evolution of cis-regulatory sequences on the X chromosome is responsible for this pattern by comparing the relative contributions of cis- and trans-regulatory changes to patterns of faster-X expression divergence observed between strains and species of Drosophila with a range of divergence times. We find support for this hypothesis, especially among male-biased genes, when comparing different species. However, we also find evidence that trans-regulatory differences contribute to a faster-X pattern of expression divergence both within and between species. This contribution is surprising because trans-acting regulators of X-linked genes are generally assumed to be randomly distributed throughout the genome. We found, however, that X-linked transcription factors appear to preferentially regulate expression of X-linked genes, providing a potential mechanistic explanation for this result. The contribution of trans-regulatory variation to faster-X expression divergence was larger within than between species, suggesting that it is more likely to result from neutral processes than positive selection. These data show how accelerated evolution of both coding and noncoding sequences on the X chromosome can lead to accelerated expression divergence on the X chromosome relative to autosomes.

The authors would like to correct an error in Supplemental Data Set 2, which inadvertently included 40 genes that were excluded prior to the allele-specific expression analyses presented in the publication. The error does not affect the conclusions of the paper. The revised supplemental file, containing 4851 genes, is now available online.