Aldh1a3-ogm were syntenic in the ancestral chromosome A0 (Figure 7 step 1), which broke apart in tetrapods (Figure 7 step 4) but remained intact in the teleost lineage (Figure 7 step 3). Consistent with our finding that Hsa1 and Hsa9 are related to the ALDH1A genomic neighborhoods in the human genome despite their lack of ALDH1A genes (Figure 2A yellow boxes), Nakatani’s reconstruction also shows that most of Hsa1 and Hsa19 derive from ‘‘A2–A5’’ and ‘‘A1–A3’’, respectively, which are the other post-R2 homeologs derived from the ancestral chromosome ‘‘A’’ present in the genome of the last common pre-R1 vertebrate ancestor. Therefore, we conclude that the conserved synteny related to ALDH1A we detected in Hsa1, Hsa5, Hsa9, Hsa15 and Hsa19 originated by R1 and R2 from the ancestral chromosome ‘‘A’’ in the genome of the last common ancestor of vertebrates. In Figure 7, we propose two hypotheses to explain how a single gene located in pre-duplication chromosome ‘‘A’’ generated Aldh1a2 and Aldh1a3 in ancestral chromosome ‘‘A4’’, and Aldh1a1 and Aldh1a3-ogm in ancestral chromosome ‘‘A0’’ inferred after R2 (Figure 7 step 1). The first hypothesis suggests Aldh1a duplication before R1 (pink box in Figure 7), and the second hypothesis invokes a translocation either before or after R2 (tan box in Figure 7) (see legend in Figure 7 for details). Independently of the order of events, however, both scenarios agree that the first duplication generated Aldh1a1/2 and Aldh3/3-ogm ancestral genes from the precursor Aldh1a1/2/3/3-ogm gene in the ancestral chromosome ‘‘A’’. Because the available genomic databases of basally divergent vertebrates such as cartilaginous fishes (e.g. dogfish, little skate or elephant shark), or from basally divergent craniates (e.g. lampreys or hagfish), are still too fragmented to perform a comprehensive analysis of conserved synteny, testing the hypothetical ‘‘pre-R1 duplication’’ or ‘‘translocation’’ scenarios must be delayed. Asymmetric Chromosomal Distribution of Fish Surviving Co-Orthologs after R3 Supporting the postulated R3 teleost-specific genome duplica- tion, our analysis of conserved synteny between the ALDH1A genomic neighborhoods and teleost genomes (Figure 4) revealed that fish orthologs of human ALDH1A neighbors are mostly confined to two main chromosomes in each fish species, and no extra R3-generated aldh1a ohnologs have been preserved in duplicated copies (Figure 7 step 5). Analysis of conserved synteny (Figure 4) supports the conclusion that each preserved duplicated Aldh1a gene is an actual ortholog of its partners within teleosts, and no evidence supports the complementary loss of aldh1a paralogs after R3 in different teleost lineages. The distribution of conserved co-orthologs in teleost paralogons, however, was asymmetric. In each of four genomic regions for three teleost species, one homeolog (the primary chromosome) conserved substantially more genes in the observed region than the other chromosome (the secondary chromosome) (Figure 4D). This asymmetric distribution of syntenic gene conservation appears to be a common characteristic for R3-generated genomic neighbor- hoods, in agreement with previous observations of the analysis of the hox and parahox genomic neighborhoods in teleosts [17,89–91] and the analysis of syntenic blocs formed following tetraploidy in Arabidopsis [92]. Evolutionary sequence divergence among paralogs also often display asymmetry, with one paralog evolving at a rate similar to its tetrapod ortholog and the other paralog evolving at an accelerated rate, suggesting neofunctionalization [39,93–95]. During the analysis of the hox cluster it was noted that the fastest evolving hox genes belong to clusters that tend to lose their hox genes faster [89,96]. Furthermore, the asymmetric distribution of synteny conservation between parahox cluster paralogons in teleosts, was accompanied by asymmetric accumulation of introns Gene Loss and Evolution of Surviving Paralogs and repetitive DNA elements in type III RTK genes, and asymmetric conservation of potential regulatory elements [91]. Thus, our observation of asymmetric chromosomal distribution of surviving co-orthologs in the aldh1a genomic neighborhoods extend previous observations in the hox and parahox genomic regions, to genomic neighborhoods with a great variety of gene types, suggesting that the probability of duplicate gene preserva- tion depends not only on inherent evolutionary forces depending on gene function (i.e. subfunctionalization and neofunctionaliza- tion), but also on properties pertaining to the architecture of the local genomic neighborhood. The R3 Aldh1a-ogm genes appear to represent cases in which, once gene organization had become altered in one of the duplicated regions, constraints that preserve genes became more relaxed, and therefore the chances of additional gene losses and further chromosomal rearrangements in the secondary chromosome were increased. At least two possible mechanisms could explain asymmetric co- ortholog retention: first, enhancers shared or embedded in genes at distant sites [70,91], or second, epigenetic regulatory mecha- nisms based on chromatin architecture [71,72]. In principle, shared or distant enhancers or epigenetic regulatory signals must be retained in one homeolog, thus facilitating neighborhood gene retention, but can be lost from the other, allowing more gene loss and more rapid gene evolution due to greater relaxation of evolutionary constraints. Effects of Lineage-Specific Gene Loss on the Functional Fate of the Surviving Paralogs In addition to the loss of aldh1a1 in stem teleosts (Figure 7 step 3), our genomic surveys revealed that aldh1a3 is absent from the genomic database of medaka fish (Figure 7 step 6). Identification of a genomic neighborhood in medaka that shows conserved ortholo- gous synteny with the stickleback and human Aldh1a3 genomic neighborhoods (Figure 6) provides evidence that aldh1a3 was lost in the medaka lineage after it diverged from the stickleback lineage (Figure 7 step 6). This finding illustrates again the power of comparative analysis of conserved synteny to provide evidence of gene loss. The finding of an apparent LTR-retrotransposon in the orthologous position occupied by aldh1a3 in stickleback and human suggests that the insertion of this mobile element may have caused the loss of aldh1a3 in medaka. Genomic data from medaka species phylogenetically close to Oryzias latipes is not yet available to more narrowly define the timing of this insertion event. The finding of the loss of aldh1a3 in medaka makes this organism the first known vertebrate with a single surviving Aldh1a paralog (i.e. aldh1a2), and made us wonder about the functional implications of gene loss. As a measure of gene function, consider expression patterns of Aldh1a genes. In the developing retina of mouse, frog, zebrafish and medaka, Aldh1a genes are expressed in a dorsal sector and in a ventral sector at the completion of optic cup invagination (about E11.5 in mouse, stage 35 in frog, and 1.5 days post fertilization in zebrafish and medaka; Figure 8A). Different vertebrates express different Aldh1a genes in different dorso-ventral sectors of the eye. The right column of Figure 8 summarizes the main expression patterns of the Aldh1a family in the retina of different animals (Aldh1a1 in red, Aldh1a2 in blue, and Aldh1a3 in green). Aldh1a paralogs expressed in the dorsal sector of the retina include Aldh1a1 (but not Aldh1a2) in mouse; both Aldh1a1 and Aldh1a2 in frogs and birds (e.g. chicken and quail, not included in Figure 8A for simplicity); and Aldh1a2 (but not Aldh1a1) in teleosts (e.g. zebrafish and medaka). The main Aldh1a paralog expressed in the ventral sector of the retina is Aldh1a3 both in tetrapods (e.g. mouse, frog and birds) and in at least one teleost (e.g. zebrafish). In contrast, in medaka, which lacks an aldh1a3 paralog, we found strong expression of aldh1a2 ventrally (Figure 6). Dotted regions PLoS Genetics | www.plosgenetics.org 13 May 2009 | Volume 5 | Issue 5 | e1000496