Dramatic Expansion and Developmental Expression Diversification of the M ethuselah Gene Family During Recent D rosophila Evolution

Functional studies of the methuselah/methuselah‐like ( mth / mthl ) gene family have focused on the founding member mth , but little is known regarding the developmental functions of this receptor or any of its paralogs. We undertook a comprehensive analysis of developmental expression and sequence divergence in the mth / mthl gene family. Using in situ hybridization techniques, we detect expression of six genes ( mthl1 , 5 , 9 , 11 , 13 , and 14 ) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors ( mthl3 , 4 , 6 , and 8 ) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors ( mthl10 and mth ) are expressed in both embryos and larvae. Phylogenetic analysis of all mth / mthl genes in five D rosophila species, mosquito and flour beetle structured the mth / mthl family into several subclades. mthl1 , 5 , and 14 are present in most species, each forming a separate clade. A newly identified D rosophila mthl gene ( CG 31720; herein mthl15 ) formed another ancient clade. The remaining D rosophila receptors, including mth , are members of a large “superclade” that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth / mthl “superclade” paralogs to the embryonic expression of the singleton ortholog in T ribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, our findings shed novel light on mth as a young member of an adaptively evolving developmental gene family. J. Exp. Zool. (Mol. Dev. Evol.) 318B:368‐387, 2012. © 2012 Wiley Periodicals, Inc.