Institutional Animal Care and Use Committee (A-3009-01, IACUC protocol #08-13). Phylogenetic Analysis Alignments of ALDH1A proteins from vertebrates and cephalochordates were generated with clustalX [99] and corrected by eye. Only the unambiguous part of the alignment was considered for phylogenetic tree reconstructions (Figure S1 provides sequence alignments). The ProtTest tool was used to choose the best-fit protein evolutionary model [100], resulting in the LG+I+G [101] and the JTT+I+G [102] as the top two selected, with a relatively low value of deltaAIC = 92.92 (AIC = 18797.45 and 18890.37, respectively). Because different phylogenetic methods have different limitations [103], we compared results from four phylogenetic approaches: i) Bayesian phylogenetic inferences were calculated with MrBayes [104], using the JTT model as well as a gamma distribution for rate variation (divided into four categories) and a proportion of invariant sites. We ran two chains for 5 million generations, sampling every 100 iterations with a 25% burn-in. ii) Maximum-likelihood (ML) analysis was conducted using PHYML [105], with an LG+I+G and JTT+I+G model. The alpha parameter of the gamma distribution (1.41) and the proportion of invariable sites (0.19) were estimated from the sample, considering four categories of substitution rates. The topology, branch lengths, and rate parameters of the tree were required to be optimized. iii) Maximum-parsimony (MP) analysis (MEGA package, [106] used the close-neighbor-interchange approach with one level of search, and added 10 replicas of random trees, and 100 replications to calculate the bootstrap value that supports each node of the tree. iv) Neighbor-joining phylogenetic (NJ) tree (MEGA package, [106] was inferred taking into account among-site rate heterogeneity with four gamma-distributed categories. This approach has been previously shown to provide equivalent results to those obtained by ML under conditions of low sequence divergence, with the advantage of a low computing-time cost [107]. The alpha parameter 1.41 was estimated from the sample using PHYML under a JTT substitution model. Concordance of trees from each of the different methods, bootstrap proportions and posterior probability estimates were used to examine the robustness of nodes. Aldh1a1/2/3 proteins predicted from gene sequence data from the cephalochordate Branchiostoma floridae were used to root the phylogenetic tree of the vertebrate Aldh1a family. Tunicate Aldh1a1/2/3 proteins were not included to avoid possible artifacts arising from long branches shown previously for Aldh genes [59]. Comparative Genomic Tools: Dotplots, Circleplots, and Orthologous and Paralogous Syntenic Gene Cluster Database The automatic tools developed by Catchen et al. [48] to detect synteny conservation allowed us to perform comprehensive genomic comparisons between the human genome and other fully or partially assembled genomes from a wide variety of model organisms. These automatic tools use a reciprocal best hit BLAST analysis pipeline to define groups of paralogy between a primary genome and an outgroup genome. For instance, when the human genome is compared with outgroup genomes that diverged prior the two rounds of genome duplication R1 and R2 (i.e. the urochordate Ciona intestinallis or the cephalochordate Branchiostoma floridae in Figure 2A), each human gene will belong to a group of paralogy that is anchored to a gene from the outgroup genome. Use of multiple outgroup genomes and merging clusters anchored by outgroup paralogs help to minimize errors derived from the Gene Loss and Evolution of Surviving Paralogs automatic reciprocal best hit BLAST pipeline due to the effect of losses, duplications or sequence divergence of outgroup genes (for details on best hit BLAST pipeline analysis, see [48]). Dotplots graphically represent the distribution of paralogous genes (crosses) within the primary genome (e.g. Figure 2A), or the distribution of orthologous genes between the primary and outgroup genomes (e.g. Figure 3A), using the results generated with the automatic BLAST analysis pipeline. In the case of an orthology dotplot, genes belonging to a selected chromosome in the outgroup are displayed along the x-axis of the plot in the order they appear in that genome. Orthologs of those genes are displayed on their respective chromosomes in the primary genome directly above the location of the gene on the selected chromosome in the outgroup, not in their order in the second genome. Scaled dotplots represent a variant in which the paralogs (or orthologs) of genes on the selected chromosome are displayed according to their natural chromosomal positions in the genome (e.g. Figure 2A). For instance, given an orthologous dotplot with Danio rerio as the primary genome and human as the outgroup (Figure 4A), each two paralog genes originated by R3 in Danio will be aligned above their human ortholog on the x-axis. Composite dotplots overlap multiple dotplots from the analyses of various regions of interest (crosses labeled with different colors) and different outgroup genomes (e.g. Figure 2A). Circleplots represent user-selected chromosomes as arcs along the circumference of a circle. The origins of lines connecting positions along the arcs represent pairs of paralogous genes within the same species (Figure 2D–F) or orthologous genes between two different species (Figure 3B). Gene loci that are close to each other may appear overlapped as single crosses in the dotplot or a single connecting line in circle-plots due to the selected graph resolution. Clusters in the Synteny Database were created by coupling results from the reciprocal best hit BLAST pipeline with the use of a sliding window analysis that links chromosome segments with conserved synteny (for details see [48]). Clusters that link chromosomal segments within the same species represent para- logous syntenic conservation (e.g. Figure 2B–C), and clusters that link chromosomal segments between different species represent orthologous syntenic conservation (e.g. Figure 4E–G). The Synteny Database provides clusters produced using several different sliding window sizes measured in terms of contiguous gene number. Smaller window sizes identify tightly-conserved syntenic regions in which gene order and orientation are well preserved while larger window sizes can accommodate chromo- somal rearrangements (inversions, transpositions, translocations, and small duplications). The Synteny Database is especially useful to provide evidence of ohnologs gone missing (ogm) by uncovering the putative chromosomal region that still preserves paralogous syntenic conservation, but lacks a certain ohnolog of interest (e.g. Figures 5 and 6). Gene Cloning and Expression Analysis Full coding sequence of aldh1a2 cDNA from Medaka Oryzias latipes (Cab strain) and the aldh1a3 cDNA from zebrafish Danio rerio were cloned after being amplified from cDNA by PCR with specific primers designed from genomic scaffold sequence data (medaka: 200506- scaffold21 and zebrafish: Zv5Scaffold1492 and NA2068) (Ola1a2F: 59ATGACTTCCAGTAAGATCGAGATCCC39 and Ola1a2R: 59CATTAACGTTTCATCCATTACTGTCC39; Dre1a3F: 59GT- CCACACAATAATCTACTCTACAGC39; Dre1a3R 59CAT- ATGTTTGCGCTTAGCTGCCATG39). Full length cDNA se- quences were submitted to GenBank (medaka aldh1a2: FJ516380, and zebrafish aldh1a3: DQ300198). A zebrafish adh1a2 clone [86], a clone containing a zebrafish aldh1a3 800 nt-fragment from exon 7 to PLoS Genetics | www.plosgenetics.org 16 May 2009 | Volume 5 | Issue 5 | e1000496