Abstract
BACKGROUND: Testes vary widely in mass relative to body mass across species, but we know very little about which genes underlie and contribute to such variation. This is partly because evidence for which genes are implicated in testis size variation tends to come from investigations involving just one or a few species. Contemporary comparative phylogenetic methods provide an opportunity to test candidate genes for their role in phenotypic change at a macro-evolutionary scale-across species and over millions of years. Previous attempts to detect genotype-phenotype associations across species have been limited in that they can only detect where genes have driven directional selection (e.g. brain size increase). RESULTS: Here, we introduce an approach that uses rates of evolutionary change to overcome this limitation to test whether any of twelve candidate genes have driven testis size evolution across tetrapod vertebrates-regardless of directionality. We do this by seeking a relationship between the rates of genetic and phenotypic evolution. Our results reveal five genes (Alkbh5, Dmrtb1, Pld6, Nlrp3, Sp4) that each have played unique and complex roles in tetrapod testis size diversity. In all five genes, we find strong significant associations between the rate of protein-coding substitutions and the rate of testis size evolution. Such an association has never, to our knowledge, been tested before for any gene or phenotype. CONCLUSIONS: We describe a new approach to tackle one of the most fundamental questions in biology: how do individual genes give rise to biological diversity? The ability to detect genotype-phenotype associations that have acted across species has the potential to build a picture of how natural selection has sculpted phenotypic change over millions of years.