Human, mouse, rat, and chicken have two copies of this enzyme: one that acts in the cytosol (HMGCS1) and one that acts in the mitochondria (HMGCS2) [31]. In contrast, fish, frogs, and sharks have a single copy of the enzyme. Based on this phylogenetic distribution, gain-loss parsimony infers a recent gain on the branch leading to amniotes. The HMGCS gene tree tells a different story: the two HMGCS subfamilies arose via an early duplication at the base of the vertebrate lineage followed by three parallel losses in shark, fish and amphibians. The branching order of the gene tree, with branch support values > 0.9, strongly supports these conclusions and rejects the seemingly more parsimonious history with a single, more recent gain. Gene tree inferred using PhyML [52] from sequences aligned with MAFFT [53] and rooted using three invertebrate outgroup sequences. Branch support was assessed using aLRT scores [52]. Abbreviations: Human = Homo sapiens; Rat = Rattus norvegicus; Mouse = Mus musculus; Chicken = Gallus gallus; Frog = Xenopus tropicalis; Pufferfish = Takifugu rubripes; Zebrafish = Danio rerio; Shark = Callorhinchus milii; Fly = Drosophila melanogaster; Yeast=Saccharomyces cerevisiae; Arabidopsis = Arabidopsis thaliana.