Gene Loss and Evolution of Surviving Paralogs Figure 3. Orthologous syntenic conservation between human and mouse ALDH1A genomic neighborhoods. (A) Dotplot displays of the genomic distribution of the mouse orthologs (crosses) of the human ALDH1A-neighbor genes within a 10 Mb-window of the human ALDH1A gene (red: ALDH1A1-neighbor genes; blue: ALDH1A2-neighbor genes; green: ALDH13-neighbor genes; and yellow: ALDH1A3-ogm-neighbor genes), and reveals the presence of four regions (colored boxes) in Mmu7, Mmu9, Mmu13 and Mmu19 orthologous to the human ALDH1A genomic neighborhoods (GN). Mouse chromosomes are represented in the y-axis, and chromosome position is in the x-axis. Table S2 provides gene accession numbers and genomic information for each orthology group represented in the dotplot. (B) A circle-plot shows the pattern of syntenic correspondence between the human ALDH1A GNs (black arcs outside chromosomes) and the candidate mouse orthologous ALDH1A-related chromosomes revealed in A. Figure S2 provide high resolution images of pair-wise gene clusters identified in the orthologous syntenic database [48] showing orthologous regions of high syntenic conservation between the human and mouse Aldh1a GNs shown in A and B. The correspondence of ALDH1A3 GNs in Hsa 15 and Mmu7 (green lines), and ALDH1A3-ogm GNs in Hsa5 and Mmu13 (golden) strongly supports the conclusion that ALDH1A3 in human is an ortholog, not a paralog, of Aldh1a3 in mouse. These results show that the loss of ALDH1A3-ogm predated the divergence of mouse and human. Circles represent chromosome centromeres. doi:10.1371/journal.pgen.1000496.g003 chromosome (the secondary chromosome) (red and blue bars, respectively, in Figure 4D). Among a total of twelve chromosomal distributions of conserved orthologs related to the four human ALDH1A genomic neighborhoods analyzed in three teleost species (zebrafish, stickleback and medaka), on average 55%67 of orthologs were in a primary chromosome, 25%610 in a secondary chromosome, 8%64 in a tertiary chromosome, 4%62 in a fourth chromosome, and 8%68 distributed throughout the rest of the genome (Figure 4D and Table S3). Fish orthologs of conserved neighbor genes surrounding human ALDH1A2, for instance, were mostly on a primary chromosome that also contained aldh1a2: in zebrafish, 46% of neighbor genes were located on chromosome Dre7; in stickleback 58% were in groupII; and in medaka 55% were on chromosome Ola3. Most of the remaining conserved synteny appeared in secondary chromosomes in which no aldh1a gene was present: in zebrafish 17% of genes were located on Dre18 and 17% on Dre25; in stickleback 33% were in groupXIX; and in medaka 29% were in Ola6 (Figure 4D). This asymmetric pattern of co-ortholog distribution is exemplified in the comparative genomic analysis of the human and teleost co- orthologous gene clusters with high conserved synteny related to the ALDH1A2 genomic neighborhood (Figure 4E–G). In addition to the co-orthologs conserved in both gene clusters in fishes (shaded gold), we found that a majority of fish orthologs (shaded green) was preserved only on the primary chromosome, and just a few were preserved only on the secondary chromosome (shaded blue, Figure 4E–G). The results of Figure 4 provide strong evidence that all ALDH1A-related genomic regions were duplicated in R3 during the evolution of teleosts but that each aldh1a gene reverted to single copy status. The fact that pairs of aldh1a genes in each teleost are in orthologous genomic neighborhoods, as revealed by the high syntenic conservation shared among them (e.g. aldh1a2 orthologs are in zebrafish Dre7, stickleback groupII and medaka Ola3), suggests that the loss of the aldh1a-ogm probably occurred in stem teleosts prior to the teleost radiation. Because asymmetric distribution of preserved duplicates appeared as a common feature of most of aldh1a genomic neighborhoods analyzed, the preserva- tion of duplicated genes might be subject to functional constraints that depend on the local architecture of the genomic neighbor- hood. For instance, enhancers shared by several genes or embedded in distant genes [70] or coordinated epigenomic regulation based on chromatin architecture of the genomic neighborhood, as in the Hox clusters [71,72], could bias paralog retention – loss from the first paralogon might be without penalty due to redundancy, but then the second paralogon would have to maintain certain aspects of its gene content. Aldh1a1 was lost during teleost evolution. Despite the absence of Aldh1a1 orthologs in teleost genomes, dotplots in Figure 4A–C revealed candidate primary chromosome regions that conserved synteny with the human ALDH1A1 genomic neighborhood on zebrafish Dre5, stickleback groupXIII and medaka Ola9. Screening of these candidate orthologous chromosomal regions identified pair-wise clusters of human and teleost genes in the Synteny Database [48], and pointed to the putative chromosome location where aldh1a1 would have been before it was lost (Figure 5). The lower clade of Figure 5 shows that in the human genome, ALDH1A1 lies between TMC1 and ANXA1 (purple in Figure 5). In zebrafish, stickleback, and medaka, tmc1 and anxa1 are near neighbors, transcribed in the same direction as in human, but with no aldh1a gene between them. Instead, teleost-specific tmc tandemly duplicated genes occupy this gap, suggesting that local genomic reorganizations, including tandem tmc duplications, might be functionally related to the loss of the teleost aldh1a1 gene. The identification of conserved paralogous synteny between the ALDH1A1 and the ALDH1A2 genomic neighborhoods in human PLoS Genetics | www.plosgenetics.org 7 May 2009 | Volume 5 | Issue 5 | e1000496