Because distant species often share similar macromolecules, regulatory mutations are often considered responsible for much of their biological differences. Recently, a large portion of regulatory changes has been attributed to cis-regulatory mutations. Here, we examined an alternative possibility that the putative contribution of cis-regulatory changes was, in fact, caused by compensatory action of cis-and trans-regulatory elements. First, we show by stochastic simulations that compensatory cis-trans evolution maintains the binding affinity of a transcription factor at a constant level, thereby spuriously exaggerating the contribution of cis-regulatory mutations to gene expression divergence. This exaggeration was not observed when changes in the binding affinity were compensated by variable transcription factor concentration. Second, using reciprocal introgressions of Drosophila, we show that relative expression of heterozygous alleles from two distinct species often varied significantly between different species backgrounds, indicating the possible action of cis-trans compensation. Taken together, we propose that cis-trans hybrid incompatibilities are accumulating much faster than generally considered. compensatory evolution | trans-regulatory mutation | epistasis | cis-trans interaction | reproductive isolation A first step to understanding the evolution of gene expression is to decompose expression variation into cis-and transregulatory components (1-4). This decomposition can be done by combining an allele-specific expression assay using F 1 hybrids with expression assays in pure species backgrounds (1, 4). From these assays, two kinds of expression differences are determined for a gene: (i) difference between two parental strains and (ii) difference between two alleles in an F 1 hybrid. When the difference between parental strains is entirely due to trans-regulatory changes, we would not expect any difference between the two alleles in the hybrid condition. However, when the difference between parental strains is entirely due to cis-regulatory changes, this difference should be reproduced as an allelic difference in the hybrid. A recent application of this method identified a greater contribution of cis-regulatory changes in inter-than intraspecific comparisons, suggesting that cis-regulatory mutations are fixed more preferentially during evolution than transregulatory mutations (5).In the above decomposition, allelic differences in F 1 hybrids are entirely attributed to cis-regulatory variation under the assumption that trans-regulatory factors should affect both alleles equally. However, some types of cis-trans interactions can violate this assumption by differently affecting the two alleles. For example, by adapting to an evolving cis-regulatory element of the same species, a transcription factor may bind less efficiently with the cis-regulatory element from the distinct species. In the extreme case where the binding between species does not occur at all, an allelic difference in a hybrid would be simil...

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Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.