Protein sequence evolution underlies interspecies incompatibility of a cell fate determinant

Novel and rapidly evolving genes can integrate into conserved gene networks and play critical roles in development. Understanding how sequence variation in the orthologs of such genes influences functional interactions with the molecular products of older, more conserved genes requires investigation at the level of protein function. Here, we ask this question using the oskar gene as a case study. oskar is essential for primordial germ cell specification and embryonic patterning in fruit flies. Previous work showed functional incompatibility between oskar orthologs from the congeneric fruit fly species Drosophila melanogaster and Drosophila virilis. However, the region(s) of Oskar in which protein-coding sequence evolution led to this incompatibility, and the molecular mechanisms underlying it, remained unknown. To address these questions, we generated chimeric versions of oskar comprising different combinations of Oskar protein domains from each species, expressed these chimeric oskar sequences in D. melanogaster, and quantified their ability to assemble functional germ line and abdominal patterning determinants (germ plasm). We found that a specific portion of D. virilis Oskar, namely the OSK domain, was primarily responsible for the cross-species incompatibility of Oskar. In the absence of endogenous D. melanogaster Oskar, chimeras containing the D. virilis OSK domain could not localize posterior germ plasm well enough to generate primordial germ cells, but were sufficient to specify the anteroposterior axis. We also found that the D. virilis OSK domain had dominant-negative effects on D. melanogaster Oskar's ability to localize germ plasm mRNAs, resulting in severe axial patterning defects. Both in vivo and in silico analyses support the hypothesis that evolved changes in the ability of the OSK domain to interact with conserved germ plasm mRNAs played a significant role in the evolution of interspecies incompatibility of Oskar. Under this hypothesis, an essential germ line determinant evolved to be incompatible across species of the same genus in less than 50 million years, while retaining functional within-species molecular interactions. Thus, by investigating in vivo protein function, we link genomic and molecular evolution with phenotypic variation and fitness at higher scales.