Evolutionary divergence of the sex-determining gene MID uncoupled from the transition to anisogamy in volvocine algae

Volvocine algae comprise a unique comparative model for investigating the evolution of distinct male and female sexes (oogamy) from an isogamous ancestral state with mating types. The mating-type or sex-determining gene MID encodes a putative RWP-RK family transcription factor, and orthologs of MID are present throughout the volvocine algal lineage in either the MT- or male mating locus of dioecious species. It was previously found that ectopic expression of isogamous Chlamydomonas reinhardtii MID (CrMID) in a C. reinhardtii MT+ strain caused differentiation of gametes with a minus phenotype, while ectopic expression of oogamous Volvox carteri MID (VcMID) in V. carteri females induced spermatogenesis, but the CrMID gene could not induce ectopic spermatogenesis when expressed heterologously in V. carteri females, suggesting coevolution of Mid function with gamete dimorphism. Here we used heterologous expression experiments to test whether Mid function was coupled to the emergence of anisogamy or oogamy in the volvocine lineage. We found that PsMID from the anisogamous multicellular species, Pleodorina starrii, could efficiently induce spermatogenesis when expressed in V. carteri females and, unexpectedly, GpMID from the isogamous multicellular species Gonium pectorale was also able to induce V. carteri spermatogenesis. MID genes from multicellular volvocine species (VcMID and GpMID) could not complement CrMID function when expressed in C. reinhardtii, at least partly due to instability of non-native Mid proteins. Our data show that Mid divergence was not a major contributor to the transition between isogamy and anisogamy/oogamy in volvocine algae, and instead implicate changes in cis-regulatory interactions and/or trans-acting factors of the Mid network in the evolution of sexual dimorphism.