Gene Loss and Evolution of Surviving Paralogs in the dotplot (golden crosses: Hsa1 ALDH1A-related GN; black crosses: Hsa5 ALDH1A-related GN; pink crosses: Hsa9 ALDH1A-related GN; brown crosses: Hsa15 ALDH1A-related GN). (B) Two clusters of genes in Hsa15 and Hsa9 display a substantial number of conserved syntenies between the ALDH1A1 and ALDH1A2 gene neighborhoods (red lines), but fewer conserved syntenies with the ALDH1A3 GN (green lines), supporting the idea that ALDH1A1 and ALDH1A2 are the closest sister paralogs, consistent with the phylogenetic tree ((ALDH1A1, ALDH1A2), ALDH1A3) in Figure 1. Golden lines show conserved synteny between Hsa15 and parts of Hsa9 probably due to a local transposition that moved material between ALDH1A2 and ALDH1A3 from its original location to the right of ALDH1A1 to the left of ALDH1A1 (or vice versa). Colored boxes correspond to regions shown in A. Figure S2 provides high-resolution images including the name of conserved syntenic genes. (C) Representation of a pair of paralogous gene clusters in Hsa15 and Hsa5 displaying high amounts of conserved synteny between the ALDH1A3 and ALDH1A3-ogm GNs (green lines). (D–F) Circleplots display the patterns of conserved synteny between the ALDH1A GN (labeled with black arcs outside of each chromosome) revealed by the dotplot in A for Hsa1, Hsa5, Hsa9, Hsa15 and Hsa19 (see main text for explanations). While the patterns of conserved synteny between ALDH1A1 and ALDH1A2 GNs (red lines) and between ALDH1A3 and ALDH1A3-ogm GNs (green lines) are restricted to defined dense bundles (D), lines originating from Hsa1 (E) and Hsa19 (F) are not restricted to any particular ALDH1A GN (the different colors of the lines in E and F label various chromosomes). Circles represent chromosome centromeres, and dotted arcs label the approximate ALDH1A GN positions in the chromosomes. doi:10.1371/journal.pgen.1000496.g002 Figure 2D); these lines may connect conserved paralogs (e.g. GCNT, CCNB, FAM81 and PCSK paralogs) derived from genes already present in the genomic neighborhood of the original ALDH1A1/2/3 gene before the expansion of the ALDH1A gene family. Besides the genomic neighborhood of the ALDH1A3-ogm on Hsa5, the dotplot revealed two additional chromosome regions, one on Hsa1 and one on Hsa19, that also lacked an ALDH1A paralog but displayed syntenic conservation with ALDH1A genomic neighborhoods (Figure 2A, yellow-shaded boxes in Hsa1 and Hsa19). Circleplots, however, showed that while the neighbor-paralogs of the ADH1A1, ALDH1A2, ALDH1A3 and ALDH1A3-ogm genomic neighborhoods formed tight clusters (red and green bundles in Figure 2D), the pattern of conserved syntenies between the paralogous regions of Hsa1 (Figure 2E) and Hsa19 (Figure 2F) was not specifically restricted to ALDH1A- related genomic neighborhoods, but were broadly distributed over most of each chromosome (Hsa1, Hsa5, Hsa9, Hsa15 and Hsa19, colored arcs in Figure 2E, F). Thus, our data, in agreement with previous work [26,68], suggest that portions of Hsa1, Hsa5, Hsa9, Hsa15 and Hsa19 form part of a paralogy group with high syntenic conservation that evolved during the R1 and R2 genome duplications. Our results suggest that ALDH1A genes may have been duplicated but not preserved in ancestral chromosomes leading to parts of today’s Hsa1 and Hsa19. In summary, syntenic analysis of the genomic neighborhoods of the human ALDH1A gene family supported the phylogenetic results of Figure 1 in showing that ALDH1A1 and ALDH1A2 are sisters, and furthermore, suggested the location of an ALDH1A ohnolog gone missing on Hsa5 (ALDH1A3-ogm). All evidence thus supports the historical relationship: ((ALDH1A1, ALDH2), (ALDH1A3, ALDH1A3-ogm)). Are Murine and Human ALDH1A3 Genes Orthologs or Paralogs? When gene functions are compared among different organisms, it is important to distinguish whether the compared genes are orthologs or paralogs. In some cases, reciprocal loss of paralogs in different organisms can lead to the misinterpretation of paralogs as orthologs. Intriguingly, while in most vertebrates Aldh1a2 and Aldh1a3 are on the same chromosome separated by an intervening region of few tens of megabases, in rodents Aldh1a2 and Aldh1a3 are on different chromosomes [63,64]. In rats, for instance, Aldh1a3 is on the same chromosome as Aldh1a1 rather than being on the same chromosome as Aldh1a2 as in human. This arrangement would be expected if the rat Aldh1a3 gene were a paralog rather than an ortholog of human ALDH1A3. Phyloge- netic analysis provided strong support for the conclusion that all vertebrate Aldh1a3 genes are orthologs [59,65,69], but evidence for an ALDH1A3 ohnolog gone missing from the human genome raises the possibility of reciprocal paralog loss that would have caused human and rodent Aldh1a3 genes to be paralogs rather than orthologs. To see whether the mouse Aldh1a3 gene is orthologous to human ALDH1A3 or to ALDH1A3-ogm, we first constructed a dotplot that displayed the distribution of the mouse orthologs of human genes within 10 Mb of ALDH1A1, ALDH1A2, ALDH1A3 and ALDH1A3-ogm (Figure 3A and Table S2). The dotplot revealed that most mouse orthologs of the human ALDH1A- neighbor genes tightly clustered on four mouse chromosomes (Mmu7, Mmu9, Mmu13, and Mmu19) (Figure 3A). Next, we compared these four mouse chromosomes to their orthologons on human chromosomes Hsa5, Hsa9 and Hsa15 in a circleplot (Figure 3B). These analyses identified four clusters of orthology in the Synteny database [48] that unequivocally related mouse orthologs of human ALDH1A1, ALDH1A2, ALDH1A3, and ALDH1A3-ogm genome neighborhoods (Figure S2). The identifi- cation of a genomic region on Mmu13 that lacks any Aldh1a gene but that nevertheless possesses orthologous syntenic conservation to ALDH1A3-ogm genomic neighborhood on Hsa5 (golden bundle in Figure 3B) provides strong evidence that the loss of Aldh1a3-ogm predated the split between the lineages leading to humans and rodents, and discards the hypothesis of reciprocal paralog loss. These results conclusively rule out the hypothesis that the rodent Aldh1a3 is an ortholog of human ALDH1A3-ogm, and indepen- dently supports orthologous relationships between human and mouse Aldh1a3 genes inferred by phylogenetic methods (Figure 1). Evolution of the Aldh1a Family in Teleosts Because the number of Aldh1a paralogs detected in genome databases is lower in teleosts than in tetrapods [59,65], we performed a comparative genomic analysis of conserved synteny between Aldh1a genomic neighborhoods in the genomes of three teleosts and human to learn the historical basis of different numbers of gene family members (Figure 4). Teleost aldh1a-ohnologs gone missing. Dotplot representations comparing the 10 Mb genomic neighborhood surrounding each human ALDH1A gene to the genomes of three teleosts (zebrafish (Danio rerio), stickleback (Gasterosteus aculeatus), and medaka (Oryzias latipes)) revealed that most fish orthologs of human ALDH1A neighbor genes were not randomly scattered throughout the genome, but were mostly confined to two main chromosomes in each fish species for each human ALDH1A genomic neighborhood (Figure 4A–C). This finding is consistent with an extra round of genome duplication (R3) that occurred in the teleost lineage after diverging from the tetrapod lineage [17–26]. The distribution of conserved co-orthologs between the pair of chromosomes in each teleost, however, was asymmetric. In general, one chromosome (the primary chromosome) showed considerably more syntenic conservation than the other PLoS Genetics | www.plosgenetics.org 6 May 2009 | Volume 5 | Issue 5 | e1000496