fem-3 plays a central role in both somatic and germline sex determination in C. elegans , and its careful translational regulation is thought to be a key feature of hermaphrodite development (reviewed by Puoti et al., 1997). FEM-3 functions via a direct antagonistic physical interaction with a cytoplasmic domain of TRA-2 (Mehra et al., 1999), but little else is known about this novel protein. We are studying sex determination genes in non- elegans species of Caenorhabditis for both structure-function and regulatory purposes, and also to gain insight into the mechanisms by which hermaphrodites evolved from gonochoristic ancestors. Using a synteny approach, we have cloned
fem-3 homologues from both C. briggsae and C. remanei , which together form a clade that is the sister taxon to C. elegans (Fitch et al., 1995; Rudel and Kimble, 2001). The FEM-3-binding domain of TRA-2 is hypervariable in evolution relative to the rest of the TRA-2 (Mehra et al., 1999; Haag and Kimble, 2000), and we find that FEM-3 itself is also extremely divergent. Cr-FEM-3 consists of 439 aa and Cb-FEM-3 412 aa, compared with 388 aa for Ce-FEM-3, the differences due primarily to variation in the start codon location. The pairwise identity across the domain common to all three is 33% (Ce-FEM-3 vs. Cr-FEM-3), and 39% (Ce-FEM-3 vs. Cb-FEM-3), making FEM-3 the most rapidly evolving sex determination gene yet discovered, and therefore one of the most quickly evolving genes in the entire genome. Such rapid evolution of FEM-3 and the TRA-2 domain with which it interacts suggests three possibilities. First, many sequences may be able accomplish the same function (lack of constraint). Second, high mutation rates in the sequence of one binding partner may have been offset by selection for compensatory changes in the other to produce a constant affinity (compensatory coevolution). Third, there may have been selection for a distinct phenotype that led to a net change in the affinity of interaction (directional selection). We are attempting to distinguish between these scenarios by yeast two-hybrid and in vitro assays. Unexpectedly, the role of
fem-3 in sex determination appears to have diverged among these species, as tested by
fem-3(RNAi) . Although
fem-3 promotes male development in somatic tissues in C. elegans and C. remanei , it is apparently not required for spermatogenesis in C. remanei . RNAi analysis of
Cb-fem-3 is underway, as are experiments to test for conservation of the translational control of
fem-3 by FBF in both briggsae and remanei . Taken together, our results lead us to two major conclusions. First, FEM-3 and the FEM-3/TRA-2 interaction are "hotspots" of especially rapid sequence change during the evolution of sex determination, for reasons that are not yet clear. Second, the essential role in germline sex determination played by
fem-3 in C. elegans is not conserved in its sister species. Whether control of germline sex by
fem-3 is an ancestral or derived feature remains to be determined. *These authors made equal contributions to this work. Fitch, D.H.A., et al. (1995) Mol. Biol. Evol. 12: 346-358. Haag, E.S. and Kimble, J. (2000). Genetics 155: 105-116. Rudel, D. and Kimble, J. (2001). Genetics 157: 639-654. Mehra, A., et al. (1999) Genes Dev . 13: 1453-1463. Puoti, A., et al. (1997). Cold Spring Harbor Symp. Quant. Biol. 62: 19-24.