analysis alone is insufficient to definitively resolve Aldh1a relationships. To further test historical relationships among Aldh1a paralogs, we examined a data set independent of Aldh1a gene sequence by conducting comparative genomic analyses of the entire genomic neighborhoods (GN) surrounding Aldh1a genes in the genomes of humans and other vertebrates. Analysis of Conserved Syntenies for the Human ALDH1A Gene Family The results of our phylogenetic analysis ((Aldh1a1, Aldh1a2), Aldh1a3) (Figure 1) implies that the duplication event that gave rise to Aldh1a1 and Aldh1a2 was more recent than the duplication event that gave rise to Aldh1a3 and the ancestral Aldh1a1/2 gene. If the duplication events that produced the Aldh1a family involved whole genomes or large chromosomal segments, then the phylogenic hypothesis of relationships (Figure 1) predicts more syntenic conservation between the genomic neighborhoods (GN) surrounding Aldh1a1 and Aldh1a2 than between the genomic neighborhood of Aldh1a3 and either Aldh1a1 or Aldh1a2. To test this hypothesis, we conducted a comparative genomic analysis of conserved synteny among the genomic neighborhoods of each ALDH1A paralog in the human genome. The three human ALDH1A genes are located on two chromosomes: ALDH1A1 is on Hsa9 (human chromosome 9), while ALDH1A2 and ALDH1A3 are on Hsa15 separated by 43 megabases (Mb). We first made a composite dotplot to represent the genome-wide distribution of the paralogs of all genes within a 10 Mb-window surrounding each human ALDH1A gene through- out the 23 human chromosomes (y-axis) (we refer to this set of genes as ALDH1A-neighbor paralogs (red, blue and green crosses in Figure 2A)). Table S1 lists gene names, reference numbers, genomic positions and outgroup (i.e. Branchiostoma floridae and Ciona intestinalis) gene information used to construct each paralogy group in the dotplot. This plot showed that while some ALDH1A- neighbor paralogs appeared randomly scattered throughout the genome, some chromosomal regions contained a concentration of ALDH1A-neighbor paralogs (yellow and pink boxes in Figure 2A). These chromosome regions with syntenic conservation to ALDH1A-neighbor paralogs likely represent chromosome frag- ments that were duplicated during the whole genome duplication events R1 and R2 and are historically related to the expansion of the Aldh1a family. The presence of ALDH1A-neighbor genes conserved among ALDH1A genomic neighborhoods (pink-shaded dotted boxes) suggests that the ALDH1A family expanded by large- scale genome duplications rather than by local tandem gene duplications. The dotplot analysis also identified four genomic regions that share syntenic conservation with ALDH1A genomic neighborhoods, but do not contain ALDH1A genes (yellow-shaded boxes on Hsa1, Hsa5, Hsa9 and Hsa19). The paralogs of each gene contained in these four yellow boxes were also included in the dotplot (Figure 2A: golden, black, pink and brown crosses). In principle, the existence of the yellow-boxed regions that lack ALDH1A paralogs but show syntenic conservation with the ALDH1A genomic neighborhood could be explained by genome duplications followed by a loss of the ALDH1A paralog (i.e. ALDH1A ohnologs gone missing), or alternatively by the translocation of a portion of the genomic neighborhood away from the ALDH1A gene itself. Syntenic analysis supports the hypothesis that ALDH1A1 and ALDH1A2 are the closest sister paralogs, and suggests an ALDH1A3 ohnolog gone missing. The dotplot (Figure 2A) revealed a region about 36 Mb from ALDH1A1 on Hsa9 that contained ALDH1A2- and ALDH1A1-neighbor paralogs but had no ALDH1A gene (yellow in Figure 2A). The proximity of this Gene Loss and Evolution of Surviving Paralogs region to ALDH1A1 mirrored the proximity of ALDH1A2 and ALDH1A3, suggesting it as a candidate region paralogous to the ALDH1A3 genomic neighborhood. To decrease possible effects of local chromosome rearrangements (i.e. inversions, translocations and local duplications), we looked for ALDH1A-neighbor paralogs shared by Hsa9 and Hsa15 using the Synteny Database and a 100- gene sliding window [48] (Figure 2B). Analysis identified a cluster that clearly showed that the ALDH1A2 and ALDH1A1 genomic neighborhoods share more paralog conservation to each other (red lines (n = 15) in Figure 2B), than either does to ALDH1A3 genomic neighborhood (green lines (n = 3) in Figure 2B). Paralogs shared between the ALDH1A1 and ALDH1A2 genomic neighborhoods include members of the FOXB, ROR, VPS13, GCNT and TRPM gene families. Figure S2 provides high-resolution images including the name of each conserved syntenic ALDH1A-neighbor in the gene clusters shown in Figure 2. This analysis provides robust evidence independent of ALDH1A sequence that ALDH1A1 and ALDH1A2 are sister clades, and supports the tree topology obtained in our phylogenetic analysis ((ALDH1A1, ALDH1A2), ALDH1A3) (Figure 1). The small number of paralogs shared by the ALDH1A3 genomic neighborhood and the region 36 Mb upstream of ALDH1A1 made it unlikely that they are evolutionarily related by the R1 and R2 genomic duplication events. The dotplot in Figure 2A, however, also revealed a region on Hsa5 that lacks an ALDH1A3 paralog but was enriched in ALDH1A3-neighbor paralogs (Figure 2A, green crosses in yellow-shaded box on Hsa5). This region is a candidate for a chromosome segment that is the sister of the ALDH1A3- containing region (pink-shaded box on Hsa15) but that subsequent to the duplication event, lost the ALDH1A gene copy. We refer to the hypothesized absent ALDH1A3 paralog on Hsa5 as an ALDH1A3 ohnolog gone missing (ALDH1A3-ogm) from the human genome. Neighboring genes in the Hsa5 and Hsa15 regions showed clear syntenic conservation with gene order also preserved (note that the arbitrary convention of chromosome orientation displays them as inverted) (Figure 2C). This arrangement is consistent with these regions being paralogons that both contained ALDH1A3 paralogs before ALDH1A3-ogm was lost (Figure 2C). Genes including MEF2C, ARRDC3, NR2F1, MCTP1 and PCSK1 are neighbors of the ALDH1A3-ogm that have been preserved in Hsa5 after the genomic duplication, with their paralogs MEF2A, ARRDC4, NR2F2, MCTP2, and PCSK3 and PCSK6 on Hsa15 near ALDH1A3 (Figure 2C). To further investigate spatial relationships along chromosomes, we used circleplots connecting genes on a chromosome to their paralogs positioned on other chromosomes [23]. The circleplot of Figure 2D shows connections found among paralogs from the ALDH1A genomic neighborhoods on Hsa5, Hsa9 and Hsa15. This representation highlighted two well-defined dense bundles: one mostly restricted to ALDH1A3 and ALDH1A3-ogm genomic neighborhoods (green lines between Hsa15 and Hsa5 in Figure 2D), and one mostly restricted to ALDH1A2 and ALDH1A1 genomic neighborhoods (red lines between Hsa15 and Hsa9 in Figure 2D). The presence of red lines linking the ALDH1A1 genomic neighborhood and the 36 Mb upstream region of ALDH1A1 in Hsa9 are likely due to local gene duplications followed by inversions within Hsa9, including duplicates of the DNAJ, ZFAND and TLE families. The fact that most of the Hsa15 paralogs are located between ALDH1A2 and ALDH1A3 supports this argument (gold lines in Figure 2B). The circleplot of Figure 2D also showed a few scattered lines that likely represent inversions of ALDH1A-neighbor genes to other regions (grey lines in Figure 2D). Interestingly some lines also connect the ALDH1A1/ALDH1A2 and the ALDH1A3/ALDH1A3-ogm groups, (black lines in PLoS Genetics | www.plosgenetics.org 4 May 2009 | Volume 5 | Issue 5 | e1000496