Gene Loss and Evolution of Surviving Paralogs Results Phylogenetic Analysis of the Vertebrate ALDH1A Gene Family To understand the history of gene gain and loss in the Aldh1a family, it is important to first understand the phylogeny of family members. Unfortunately, evolutionary relationships among verte- brate Aldh1a paralogs are currently unclear. In one analysis, the three vertebrate Aldh1a clades collapsed to an unresolved trichotomy [59], and in another, Aldh1a2 and Aldh1a3 appeared as sister groups (Aldh1a1, (Aldh1a2, Aldh1a3)), supported by low bootstrap values [65]. These problems may stem from sequence similarities among the Aldh1a1, Aldh1a2 and Aldh1a3 proteins and the use of the evolutionarily distant mitochondrial Aldh2 family to root the tree. To overcome this uncertainty, we turned to a chordate outgroup, the cephalochordate amphioxus, whose lineage diverged from that of the vertebrates before the R1 and R2 events [66,67]. Amphioxus has both Aldh1a and Aldh2 gene families [59], and hence its Aldh1a genes are much more closely related to vertebrate Aldh1a1 genes than is the Aldh2 gene family. We found that several different phylogenetic methodologies, including Bayesian inference, Maximum-likelihood, gamma-cor- rected Neighbor-Joining and Maximum-Parsimony all agreed on the same tree topology ((Aldh1a1, Aldh1a2), Aldh1a3)), with Aldh1a1 and Aldh1a2 as sister groups (Figures 1 and S1). This phylogeny differs from both published results: the trichotomy result and the view of Aldh1a2 and Aldh1a3 as sister clades [59,65]. Our results still provided only a moderately high probability of 0.76 supporting the Aldh1a1/2 clade under the Bayesian phylogenetic inference (Figure 1); thus, phylogenetic Figure 1. Phylogenetic tree of the vertebrate Aldh1A gene family. All phylogenetic methodologies (Bayesian, Maximum-likelihood, Neighbor-joining and Maximum-parsimony; included in Figure S1) agreed on a unique gene topology in which Aldh1a1 (green background) and Aldh1a2 (tan background) are the closest sister clades, while Aldh1a3 (blue background) diverged basally: ((Aldh1a1, Aldh1a2), Aldh1a3). Values at nodes correspond to the posterior probabilities inferred from the Bayesian method and generally show a highly supported tree topology. The only exception is a moderately high value of 0.76 for the Aldh1a1-Aldh1a2 node (for this node, the ML, NJ and MP supporting values are also shown). While Aldh1a2 and Aldh1a3 are present in both tetrapods (red lines) and teleosts (blue lines), Aldh1a1 is absent from teleost genomes. Scale bar indicates amino-acid substitutions. Tetrapods: Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus novergicus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Teleosts: Dr, Danio rerio; Ga, Gasterosteus aculeatus; Ol, Oryzias latipes; Tn, Tetraodon nigroviridis; Tr, Takifugu rubripes; Cephalochordates: Bf, Branchiostoma floridae. doi:10.1371/journal.pgen.1000496.g001 PLoS Genetics | www.plosgenetics.org 3 May 2009 | Volume 5 | Issue 5 | e1000496