Functional constraint underlies 60 million year stasis of Dipteran testis-specific beta-tubulin.

How do proteins evolve while maintaining their function? Previous studies find a highly stringent structure/function relationship between the Drosophila melanogaster testis-specific tubulin beta2 and the spermtail axoneme, such that small changes in the beta2 protein render it unable to generate a motile axoneme. This raises the question, how does beta2 evolve while maintaining its function? To address this question we cloned full- and partial-length beta2 sequences from 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. Not a single amino acid difference is coded among them-beta2 maintains its function by not evolving. We also performed gene genealogical analyses to determine ortholog/paralog relationships among insect tubulins. We find that the Lepidopteran and Dipteran testis-specific beta-tubulins are likely orthologs, and surprisingly, despite functioning in the same structure, the Lepidopteran orthologs are evolving rapidly. We argue that differences in tubulin isoform use in the testes cause the Dipteran axoneme to be less evolvable than the Lepidopteran axoneme, which has facilitated the evolution of a unique amino acid synergism in Drosophila and Hirtodrosophilabeta2 that is resistant to change, contributing to its evolutionary stasis.