Author Summary Gene duplication may facilitate the acquisition of genetic diversity. Little is known, however, about the impact of gene loss on the functions of surviving genes. When a gene is lost, can other closely related genes evolve to perform the functions of the lost gene? Answering this question can be difficult because the proof for gene loss is based on negative evidence and thus can easily pass unnoticed. Here, we illustrate how the comparison of genomic neighborhoods in different species can help reconstruct the chromosomal history of a gene family and provide robust evidence for gene loss, even without an appropriate early-diverging comparator group. Identifying gene loss is important because it helps distinguish between gene gain as a lineage-specific innovation and gene loss as a lineage-specific simplification. As a case study, we investigated the expression of the Aldh1a family, which is crucial for retinoic acid signaling in development of eyes, limbs, the brain, and in cancer. Results showed that gene loss is indeed associated with the evolution of functional change in surviving gene family members. Our results highlight the relevance of comparative genomics for identifying gene loss and improving the functional connectivity among human and model organism ge- nomes. for human origins [35]. Furthermore, reciprocal loss of even fully redundant gene duplicates in two populations may contribute to speciation [33,36]. Global estimations of gene loss in fully sequenced vertebrate genomes have been inferred by massive phylogenetic reconstruc- tions of gene families [37–39]. Large-scale analyses, however, are sensitive to uncertainties of phylogenetic analysis, for example, asymmetric rates of evolution among paralogs can affect tree topologies and generate gene phylogenies that are not congruent with the species phylogenies of which they are a part [40,41]. Furthermore, published genome-wide studies have not addressed gene function. In principle, gene functions that are associated exclusively with a certain gene may disappear if the gene is lost. It is possible, however, that exclusive gene functions might not disappear in situations in which a surviving paralog might acquire or maintain the expression domain of the lost paralog, and thereby the ancestral developmental or physiological program can remain unaltered [42,43]. Because the evidence for gene loss is negative and can pass unnoticed and is subject to uncertainties in the completion or assembly of sequenced genomes and in copy number polymorphisms [44,45], the impact of gene loss in the evolution of function of surviving paralogs is under-investigated. Identification of gene loss is especially important to avoid misinterpretations when human gene functions are inferred from the study of model organisms that might have suffered lineage- specific paralog loss, so that the model has no true ortholog of the phylogenetically most closely related human gene, or vice versa. To evaluate the evolutionary relevance of gene loss on the functions of surviving paralogs, it is first important to understand gene phylogeny. For genes lost following large-scale genome duplications, conserved syntenies can identify duplicated genomic regions and provide evidence for gene loss, often even in situations lacking a proper outgroup [46]. Genes lost after genome duplication events have been called ‘‘ohnologs gone missing’’ (ogm), and their identification is important to properly distinguish orthologs from other types of paralogs [32,47,48]. We propose that identification of gene loss by automated comparative genomic analysis of conserved syntenies can: (1) help resolve uncertain gene Gene Loss and Evolution of Surviving Paralogs phylogenies; (2) help discriminate cases of evolutionary innovations from evolutionary simplifications; (3) facilitate understanding of the diversification of gene functions among species; and, importantly, (4) improve functional connectivity of human and model organism genomes. To explore the roles of gene loss in a functional context, work reported here focuses on the vertebrate Aldh1a retinaldehyde dehydrogenase gene family (formerly known as Raldh) as a case study. Understanding the evolution of Aldh1a genes is important because this family encodes enzymes responsible for the synthesis of retinoic acid (RA), the active derivative of vitamin A (retinol). In humans, as in other vertebrates, RA plays important roles during embryogenesis, for example, in axial patterning, limb develop- ment, and differentiation of eyes and nervous system, as well as functioning in adult organ homeostasis (recently reviewed in [49,50]). Alterations of RA metabolism can lead to human pathologies including breast and prostate cancers, osteoporosis, rheumatoid arthritis, dermatologic diseases, developmental anom- alies and premature births. The evolutionary origin of the Aldh1a family probably predates the origin of stem bilaterians [51,52], but the ability of the Aldh1a enzyme of basally diverging bilaterians to synthesize RA remains unknown. Aldh1a likely arose by duplication of an ancestral gene related to the Aldh2 gene family, which encodes a mitochondrial Aldh that plays a major role in acetaldehyde oxidation and is broadly represented in most extant organisms from bacteria to humans [53]. Humans and many other vertebrates have three genes that encode Aldh1a family enzymes: ALDH1A1, ALDH1A2 and ALDH1A3 [54]. Studies of model organisms such as mouse, chicken, frog and zebrafish have provided insights into the roles of each Aldh1a gene in the synthesis of RA (reviewed in [49,50,55– 58]). Variation in Aldh1a gene number in different animal lineages has been hypothesized to be relevant to animal evolution due to potential effects of RA metabolism on the mechanisms of development [59–61]; reviewed in [62]. Rodents have a fourth Aldh1a paralog that is mostly expressed in kidney (termed, Aldh1a4 in rat [63], and its ortholog Aldh1a7 in mouse [64]); these genes originated by a tandem gene duplication in the rodent lineage after it diverged from the human lineage. Experiments using a heterologous Xenopus system to express mouse Aldh1a7 suggested that Aldh1a7 might not be involved in RA synthesis [64]. In contrast to rodents with four Aldh1 genes, most teleost fish have just two, aldh1a2 and aldh1a3, but they lack aldh1a1 [59,65]. Phylogenetic relationships of vertebrate Aldh1a1 genes are still controversial, and whether Aldh1a1 is a tetrapod innovation or its absence from teleosts is due to gene loss is still unknown. Furthermore, the functional consequences of these gene copy number variations have not yet been investigated. Here, we show how comprehensive comparative genomic analyses of syntenic conservation provides a framework necessary for the examination of the general mechanisms by which lineage- specific gene loss can impact the functions of surviving paralogs. This work reveals multiple losses of Aldh1a ohnologs and proposes an evolutionary genomic model that reconstructs the history of Aldh1a-related vertebrate chromosomes and the evolution of Aldh1a gene functions during and subsequent to the R1, R2, and R3 genome duplications. Results show that acquisition or modification of expression domains by surviving paralogs may lead to lineage-specific innovations that preserve unaltered ancestral developmental programs in the face of gene loss. This work highlights the importance of comparative genomics for understanding the historical basis of gene loss, and to improve functional connectivity between model organism and human genomes. PLoS Genetics | www.plosgenetics.org 2 May 2009 | Volume 5 | Issue 5 | e1000496