Gene Loss and Evolution of Surviving Paralogs Figure 4. Syntenic conservation between human and fish ALDH1A genomic neighborhoods and asymmetric distribution of surviving fish co-orthologs. (A–C) Dotplots display the distribution of zebrafish (A), stickleback (B) and medaka (C) orthologs (red dots for Aldh1a paralogs and black crosses for neighbor genes on teleost chromosomes in y-axis) of human ALDH1A genes, and their neighbor genes (red dots and black dots on x-axis, respectively) within a 10 Mb-window. The dotplot reveals that all genomic neighborhoods (GN) related to the Aldh1a family were duplicated during R3 in teleosts, but no additional Aldh1a-ohnologs from R3 (e.g. Aldh1a29) currently survive. Gene loci that are close to each other may appear overlapped as single crosses in the plot due to the selected graph resolution. Table S3 provides gene accession numbers and genomic information for all genes and each orthology group shown in the dotplot. (D) Bar-graph representing the asymmetric distribution of conserved co-orthologs in different chromosomes resulting from the analysis of twelve genomic neighborhoods related to the ALDH1A family (a1: ALDH1A1; a2: Aldh1a2; a3: Aldh1a3; a3-ogm: Aldh1a3-ogm). Values represent the percentage of conserved ALDH1A gene neighbor co-orthologs in each chromosome. (E–G) Co-orthologous gene clusters of zebrafish (E), stickleback (F), and medaka (G) related to the human ALDH1A2 genomic neighborhood exemplify the asymmetric distribution of conserved co-orthologs after R3. Fish co-orthologs conserved between both fish clusters and the human cluster are colored gold, fish co-orthologs preserved only in the primary chromosome are in green, and fish co-orthologs preserved only in the secondary chromosome are in blue. ALDH1A2 orthologs are highlighted in red, and co-orthologs present in both fish chromosomes, but absent in the human ALDH1A2 genomic neighborhood are in purple. doi:10.1371/journal.pgen.1000496.g004 and teleosts (Figure 5) supports the hypothesis that Aldh1a1 and Aldh1a2 originated by a large-scale genomic duplication event before the divergence of tetrapods and teleosts, and therefore that Aldh1a1 was lost during teleost evolution. Thus, we can conclude that Aldh1a1 is not a tetrapod innovation, but its absence from teleost genomes is due to an evolutionary simplification in this lineage (Figure 5). Evidence for loss of aldh1a3 in medaka. Just as aldh1a1 is missing in the teleost lineage, dotplot analysis (Figure 4C) showed that aldh1a3 appeared to be missing from medaka but not from other percomorphs (Figure 4A–B). RT-PCR experiments with degenerate primers and in silico genomic surveys failed to identify aldh1a3 in the medaka Oryzia latipes. BLAST searches of the medaka genome database, which has 9-fold coverage (http:// dolphin.lab.nig.ac.jp/medaka), and EST databases, which have a total 584,144 sequences deposited in NCBI at http://www.ncbi. nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=8090, (including the sequences from University of Tokyo at http://medaka.lab.nig.ac. jp/est_index.html [73] and the sequences from the National Bioresource Project at http://www.shigen.nig.ac.jp/medaka) did not identify any Aldh1a3 hits, while we found 28 EST sequences of the Aldh1a2, which appeared therefore as the only member of the Aldh1a family in medaka. Important evidence for the loss of aldh1a3 from medaka comes from BLAST analysis that identified an unassembled medaka scaffold (scaffold572) containing genes that immediately flank aldh1a3 in other species (i.e. lins1, asb7, lrrk1, chsy1, sels and snrpa1; Figure 6A and Table S3). We did not find evidence of a segment duplication of the genomic region in the scaffold572 that could be hiding a putative aldh1a3 as an artifact of whole genome shotgun sequence assemblies [44]. No aldh1a3-like sequences could be identified to suggest the presence of an aldh1a3 pseudogene in the 20-kilobase (Kb) long intergenic region between the asb7 and lrrk1 genes, where the aldh1a3 should have been located according to syntenic conservation data from other species (Figure 6). Significantly, however, this intergenic region contains a 17 Kb genomic segment that is flanked by 140 bp long terminal repeats (LTR) (scaffold572 positions 43,994–44,136 and 60,837– 60,972) and contains a retrotranscriptase sequence (ENSORLG00000019477) similar to the ORF2 of retrotransposons related to LINE elements of the CR1 family in PLoS Genetics | www.plosgenetics.org 8 May 2009 | Volume 5 | Issue 5 | e1000496