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ARTICLE OPEN doi:10.1038/nature12027 The African coelacanth genome provides insights into tetrapod evolution Chris T. Amemiya1,2*, Jessica Alföldi3*, Alison P. Lee4, Shaohua Fan5, Hervé Philippe6, Iain MacCallum3, Ingo Braasch7, Tereza Manousaki5,8, Igor Schneider9, Nicolas Rohner10, Chris Organ11, Domitille Chalopin12, Jeramiah J. Smith13, Mark Robinson1, Rosemary A. Dorrington14, Marco Gerdol15, Bronwen Aken16, Maria Assunta Biscotti17, Marco Barucca17, Denis Baurain18, Aaron M. Berlin3, Gregory L. Blatch14,19, Francesco Buonocore20, Thorsten Burmester21, Michael S. Campbell22, Adriana Canapa17, John P. Cannon23, Alan Christoffels24, Gianluca De Moro15, Adrienne L. Edkins14, Lin Fan3, Anna Maria Fausto20, Nathalie Feiner5,25, Mariko Forconi17, Junaid Gamieldien24, Sante Gnerre3, Andreas Gnirke3, Jared V. Goldstone26, Wilfried Haerty27, Mark E. Hahn26, Uljana Hesse24, Steve Hoffmann28, Jeremy Johnson3, Sibel I. Karchner26, Shigehiro Kuraku5{, Marcia Lara3, Joshua Z. Levin3, Gary W. Litman23, Evan Mauceli3{, Tsutomu Miyake29, M. Gail Mueller30, David R. Nelson31, Anne Nitsche32, Ettore Olmo17, Tatsuya Ota33, Alberto Pallavicini15, Sumir Panji24{, Barbara Picone24, Chris P. Ponting27, Sonja J. Prohaska34, Dariusz Przybylski3, Nil Ratan Saha1, Vydianathan Ravi4, Filipe J. Ribeiro3{, Tatjana Sauka-Spengler35, Giuseppe Scapigliati20, Stephen M. J. Searle16, Ted Sharpe3, Oleg Simakov5,36, Peter F. Stadler32, John J. Stegeman26, Kenta Sumiyama37, Diana Tabbaa3, Hakim Tafer32, Jason Turner-Maier3, Peter van Heusden24, Simon White16, Louise Williams3, Mark Yandell22, Henner Brinkmann6, Jean-Nicolas Volff12, Clifford J. Tabin10, Neil Shubin38, Manfred Schartl39, David B. Jaffe3, John H. Postlethwait7, Byrappa Venkatesh4, Federica Di Palma3, Eric S. Lander3, Axel Meyer5,8,25 & Kerstin Lindblad-Toh3,40 The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution. In 1938 Marjorie Courtenay-Latimer, the curator of a small natural history museum in East London, South Africa, discovered a large, unusual-looking fish among the many specimens delivered to her by a local fish trawler. Latimeria chalumnae, named after its discoverer1, was over 1 m long, bluish in colour and had conspicuously fleshy fins that resembled the limbs of terrestrial vertebrates. This discovery is considered to be one of the most notable zoological finds of the twen- tieth century. Latimeria is the only living member of an ancient group of lobe-finned fishes that was known previously only from fossils and believed to have been extinct since the Late Cretaceous period, approximately 70 million years ago (Myr ago)1. It was almost 15 years before a second specimen of this elusive species was discovered in the *These authors contributed equally to this work. 1Molecular Genetics Program, Benaroya Research Institute, Seattle, Washington 98101, USA. 2Department of Biology, University of Washington, Seattle, Washington 98105, USA. 3Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 4Comparative Genomics Laboratory, Institute of Molecular and Cell Biology, A*STAR, Biopolis, Singapore 138673, Singapore. 5Department of Biology, University of Konstanz, Konstanz 78464, Germany. 6Département de Biochimie, Université de Montréal, Centre Robert Cedergren, Montréal H3T 1J4, Canada. 7Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403, USA. 8Konstanz Research School of Chemical Biology, University of Konstanz, Konstanz 78464, Germany. 9Instituto de Ciencias Biologicas, Universidade Federal do Para, Belem 66075-110, Brazil. 10Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 11Department of Anthropology, University of Utah, Salt Lake City, Utah 84112, USA. 12Institut de Genomique Fonctionnelle de Lyon, Ecole Normale Superieure de Lyon, Lyon 69007, France. 13Department of Biology, University of Kentucky, Lexington, Kentucky 40506, USA. 14Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry, Microbiology & Biotechnology, Rhodes University, Grahamstown 6139, South Africa. 15Department of Life Sciences, University of Trieste, Trieste 34128, Italy. 16Department of Informatics, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. 17Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona 60131, Italy. 18Department of Life Sciences, University of Liege, Liege 4000, Belgium. 19College of Health and Biomedicine, Victoria University, Melbourne VIC 8001, Australia. 20Department for Innovation in Biological, Agro-food and Forest Systems, University of Tuscia, Viterbo 01100, Italy. 21Department of Biology, University of Hamburg, Hamburg 20146, Germany. 22Eccles Institute of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA. 23Department of Pediatrics, University of South Florida Morsani College of Medicine, Children’s Research Institute, St. Petersburg, Florida 33701, USA. 24South African National Bioinformatics Institute, University of the Western Cape, Bellville 7535, South Africa. 25International Max-Planck Research School for Organismal Biology, University of Konstanz, Konstanz 78464, Germany. 26Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA. 27MRC Functional Genomics Unit, Oxford University, Oxford OX1 3PT, UK. 28Transcriptome Bioinformatics Group, LIFE Research Center for Civilization Diseases, Universität Leipzig, Leipzig 04109, Germany. 29Graduate School of Science and Technology, Keio University, Yokohama 223-8522, Japan. 30Department of Molecular Genetics, All Children’s Hospital, St. Petersburg, Florida 33701, USA. 31Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. 32Bioinformatics Group, Department of Computer Science, Universität Leipzig, Leipzig 04109, Germany. 33Department of Evolutionary Studies of Biosystems, The Graduate University for Advanced Studies, Hayama 240-0193, Japan. 34Computational EvoDevo Group, Department of Computer Science, Universität Leipzig, Leipzig 04109, Germany. 35Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX1 2JD, UK. 36European Molecular Biology Laboratory, Heidelberg 69117, Germany. 37Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japan. 38University of Chicago, Chicago, Illinois 60637, USA. 39Department Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg 97070, Germany. 40Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala 751 23, Sweden. {Present addresses: Genome Resource and Analysis Unit, Center for Developmental Biology, RIKEN, Kobe, Japan (S.K.); Boston Children’s Hospital, Boston, Massachusetts, USA (E.M.); Computational Biology Unit, Institute of Infectious Disease and Molecular Medicine, University of Cape Town Health Sciences Campus, Anzio Road, Observatory 7925, South Africa (S.P.); New York Genome Center, New York, New York, USA (F.J.R.). 1 8 A P R I L 2 0 1 3 | V O L 4 9 6 | N A T U R E | 3 1 1 Macmillan Publishers Limited. All rights reserved©2013 www.nature.com/doifinder/10.1038/nature12027 Comoros Islands in the Indian Ocean, and only 309 individuals have been recorded in the past 75 years (R. Nulens, personal communication)2. The discovery in 1997 of a second coelacanth species in Indonesia, Latimeria menadoensis, was equally surprising, as it had been assumed that living coelacanths were confined to small populations off the East African coast3,4. Fascination with these fish is partly due to their pre- historic appearance—remarkably, their morphology is similar to that of fossils that date back at least 300 Myr, leading to the supposition that, among vertebrates, this lineage is markedly slow to evolve1,5. Latimeria has also been of particular interest to evolutionary biologists, owing to its hotly debated relationship to our last fish ancestor, the fish that first crawled onto land6. In the past 15 years, targeted sequencing efforts have produced the sequences of the coelacanth mitochondrial genomes7, HOX clusters8 and a few gene families9,10. Nevertheless, coelacanth research has felt the lack of large-scale sequencing data. Here we describe the sequencing and comparative analysis of the genome of L. chalumnae, the African coelacanth. Genome assembly and annotation The African coelacanth genome was sequenced and assembled using DNA from a Comoros Islands Latimeria chalumnae specimen (Sup- plementary Fig. 1). It was sequenced by Illumina sequencing tech- nology and assembled using the short read genome assembler ALLPATHS-LG11. The L. chalumnae genome has been reported previ- ously to have a karyotype of 48 chromosomes12. The draft assembly is 2.86 gigabases (Gb) in size and is composed of 2.18 Gb of sequence plus gaps between contigs. The coelacanth genome assembly has a contig N50 size (the contig size above which 50% of the total length of the sequence assembly can be found) of 12.7 kilobases (kb) and a scaffold N50 size of 924 kb, and quality metrics comparable to other Illumina genomes (Supplementary Note 1, and Supplementary Tables 1 and 2). The genome assembly was annotated separately by both the Ensembl gene annotation pipeline (Ensembl release 66, February 2012) and by MAKER13. The Ensembl gene annotation pipeline created gene models using protein alignments from the Universal Protein Resource (Uni- prot) database, limited coelacanth complementary DNA data, RNA-seq data generated from L. chalumnae muscle (18 Gb of paired-end reads were assembled using Trinity software14, Supplementary Fig. 2) as well as orthology with other vertebrates. This pipeline produced 19,033 protein-coding genes containing 21,817 transcripts. The MAKER pipeline used the L. chalumnae Ensembl gene set, Uniprot protein alignments, and L. chalumnae (muscle) and L. menadoensis (liver and testis)15 RNA-seq data to create gene models, and this produced 29,237 protein-coding gene annotations. In addition, 2,894 short non- coding RNAs, 1,214 long non-coding RNAs, and more than 24,000 conserved RNA secondary structures were identified (Supplementary Note 2, Supplementary Tables 3 and 4, Supplementary Data 1–3 and Supplementary Fig. 3). It was inferred that 336 genes underwent spe- cific duplications in the coelacanth lineage (Supplementary Note 3, Supplementary Tables 5 and 6, and Supplementary Data 4). The closest living fish relative of tetrapods The question of which living fish is the closest relative to ‘the fish that first crawled on to land’ has long captured our imagination: among scientists the odds have been placed on either the lungfish or the coelacanth16. Analyses of small to moderate amounts of sequence data for this important phylogenetic question (ranging from 1 to 43 genes) has tended to favour the lungfishes as the extant sister group to the land vertebrates17. However, the alternative hypothesis that the lung- fish and the coelacanth are equally closely related to the tetrapods could not be rejected with previous data sets18. To seek a comprehensive answer we generated RNA-seq data from three samples (brain, gonad and kidney, and gut and liver) from the West African lungfish, Protopterus annectens, and compared it to gene sets from 21 strategically chosen jawed vertebrate species. To perform a reliable analysis we selected 251 genes in which a 1:1 orthology ratio was clear and used CAT-GTR, a complex site-heterogeneous model of sequence evolution that is known to reduce tree-reconstruction arte- facts19 (see Supplementary Methods). The resulting phylogeny, based on 100,583 concatenated amino acid positions (Fig. 1, posterior prob- ability 5 1.0 for the lungfish–tetrapod node) is maximally supported except for the relative positions of the armadillo and the elephant. It corroborates known vertebrate phylogenetic relationships and strongly supports the conclusion that tetrapods are more closely related to lungfish than to the coelacanth (Supplementary Note 4 and Supplementary Fig. 4). The slowly evolving coelacanth The morphological resemblance of the modern coelacanth to its fossil ancestors has resulted in it being nicknamed ‘the living fossil’1. This invites the question of whether the genome of the coelacanth is as slowly evolving as its outward appearance suggests. Earlier work showed that a few gene families, such as Hox and protocadherins, have comparatively slower protein-coding evolution in coelacanth than in other vertebrate lineages8,10. To address the question, we compared several features of the coelacanth genome to those of other vertebrate genomes. Protein-coding gene evolution was examined using the phyloge- nomics data set described above (251 concatenated proteins) (Fig. 1). Pair-wise distances between taxa were calculated from the branch lengths of the tree using the two-cluster test proposed previously20 to test for equality of average substitution rates. Then, for each of the following species and species clusters (coelacanth, lungfish, chicken and mammals), we ascertained their respective mean distance to an outgroup consisting of three cartilaginous fishes (elephant shark, little skate and spotted catshark). Finally, we tested whether there was any significant difference in the distance to the outgroup of cartilaginous fish for every pair of species and species clusters, using a Dog 0.1 substitutions per site Human Mouse Elephant Armadillo Opossum Platypus Chicken Turkey Zebra finch Lizard Western clawed frog Chinese brown frog Lungfish Coelacanth Tilapia Pufferfish Zebrafish Elephant shark Little skate Spotted catshark Tetrapods Lobe-finned fish Cartilaginous fish Ray-finned fish Tammar wallaby Figure 1 | A phylogenetic tree of a broad selection of jawed vertebrates shows that lungfish, not coelacanth, is the closest relative of tetrapods. Multiple sequence alignments of 251 genes with a 1:1 ratio of orthologues in 22 vertebrates and with a full sequence coverage for both lungfish and coelacanth were used to generate a concatenated matrix of 100,583 unambiguously aligned amino acid positions. The Bayesian tree was inferred using PhyloBayes under the CAT 1 GTR 1 C4 model with confidence estimates derived from 100 gene jack-knife replicates (support is 100% for all clades but armadillo 1 elephant with 45%)48. The tree was rooted on cartilaginous fish, and shows that the lungfish is more closely related to tetrapods than the coelacanth, and that the protein sequence of coelacanth is evolving slowly. Pink lines (tetrapods) are slightly offset from purple lines (lobe-finned fish), to indicate that these species are both tetrapods and lobe-finned fish. RESEARCH ARTICLE 3 1 2 | N A T U R E | V O L 4 9 6 | 1 8 A P R I L 2 0 1 3 Macmillan Publishers Limited. All rights reserved©2013 Z statistic. When these distances to the outgroup of cartilaginous fish were compared, we found that the coelacanth proteins that were tested were significantly more slowly evolving (0.890 substitutions per site) than the lungfish (1.05 substitutions per site), chicken (1.09 substitutions per site) and mammalian (1.21 substitutions per site) orthologues (P , 1026 in all cases) (Supplementary Data 5). In addition, as can be seen in Fig. 1, the substitution rate in coelacanth is approxi- mately half that in tetrapods since the two lineages diverged. A Tajima’s relative rate test21 confirmed the coelacanth’s significantly slower rate of protein evolution (P , 10220) (Supplementary Data 6). We next examined the abundance of transposable elements in the coelacanth genome. Theoretically, transposable elements may make their greatest contribution to the evolution of a species by generating templates for exaptation to form novel regulatory elements and exons, and by acting as substrates for genomic rearrangement22. We found that the coelacanth genome contains a wide variety of transposable- element superfamilies and has a relatively high transposable-element content (25%); this number is probably an underestimate as this is a draft assembly (Supplementary Note 5 and Supplementary Tables 7–10). Analysis of RNA-seq data and of the divergence of individual transposable-element copies from consensus sequences show that 14 coelacanth transposable-element superfamilies are currently active (Supplementary Note 6, Supplementary Table 10 and Supplementary Fig. 5). We conclude that the current coelacanth genome shows both an abundance and activity of transposable elements similar to many other genomes. This contrasts with the slow protein evolution observed. Analyses of chromosomal breakpoints in the coelacanth genome and tetrapod genomes reveal extensive conservation of synteny and indicate that large-scale rearrangements have occurred at a generally low rate in the coelacanth lineage. Analyses of these rearrangement classes detected several fission events published previously23 that are known to have occurred in tetrapod lineages, and at least 31 inter- chromosomal rearrangements that occurred in the coelacanth lineage or the early tetrapod lineage (0.063 fusions per 1 Myr), compared to 20 events (0.054 fusions per 1 Myr) in the salamander lineage and 21 events (0.057 fusions per 1 Myr) in the Xenopus lineage23 (Sup- plementary Note 7 and Supplementary Fig. 6). Overall, these analyses indicate that karyotypic evolution in the coelacanth lineage has occurred at a relatively slow rate, similar to that of non-mammalian tetrapods24. In a separate analysis we also examined the evolutionary divergence between the two species of coelacanth, L. chalumnae and L. menadoensis, found in African and Indonesian waters, respectively. Previous ana- lysis of mitochondrial DNA showed a sequence identity of 96%, but estimated divergence times range widely from 6 to 40 Myr25,26. When we compared the liver and testis transcriptomes of L. menadoensis27 to the L. chalumnae genome, we found an identity of 99.73% (Sup- plementary Note 8 and Supplementary Fig. 7), whereas alignments between 20 sequenced L. menadoensis bacterial artificial chromosomes (BACs) and the L. chalumnae genome showed an identity of 98.7% (Supplementary Table 11 and Supplementary Fig. 8). Both the genic and genomic divergence rates are similar to those seen between the human and chimpanzee genomes (99.5% and 98.8%, respectively; divergence time of 6 to 8 Myr ago)28, whereas the rates of molecular evolution in Latimeria are probably affected by several factors, includ- ing the slower substitution rate seen in coelacanth. This suggests a slightly longer divergence time for the two coelacanth species. The adaptation of vertebrates to land As the species with a sequenced genome closest to our most recent aquatic ancestor, the coelacanth provides a unique opportunity to identify genomic changes that were associated with the successful adaptation of vertebrates to the land environment. Over the 400 Myr that vertebrates have lived on land, some genes that are unnecessary for existence in their new environment have been eliminated. To understand this aspect of the water-to-land transition, we surveyed the Latimeria genome annotations to identify genes that were present in the last common ancestor of all bony fish (including the coelacanth) but that are missing from tetrapod genomes. More than 50 such genes, including components of fibroblast growth factor (FGF) signalling, TGF-b and bone morphogenic protein (BMP) sig- nalling, and WNT signalling pathways, as well as many transcription factor genes, were inferred to be lost based on the coelacanth data (Supplementary Data 7 and Supplementary Fig. 9). Previous studies of genes that were lost in this transition could only compare teleost fish to tetrapods, meaning that differences in gene content could have been due to loss in the tetrapod or in the lobe-finned fish lineages. We were able to confirm that four genes that were shown previously to be absent in tetrapods (And1 and And2 (ref. 29), Fgf24 (ref. 30) and Asip2 (ref. 31)), were indeed present and intact in Latimeria, support- ing the idea that they were lost in the tetrapod lineage. We functionally annotated more than 50 genes lost in tetrapods using zebrafish data (gene expression, knock-downs and knockouts). Many genes were classified in important developmental categories (Supplementary Data 7): fin development (13 genes); otolith and ear development (8 genes); kidney development (7 genes); trunk, somite and tail development (11 genes); eye (13 genes); and brain development (23 genes). This implies that critical characters in the morphological transition from water to land (for example, fin-to-limb transition and remodelling of the ear) are reflected in the loss of specific genes along the phylogenetic branch leading to tetrapods. However, homeobox genes, which are responsible for the develop- ment of an organism’s basic body plan, show only slight differences between Latimeria, ray-finned fish and tetrapods; it would seem that the protein-coding portion of this gene family, along with several others (Supplementary Note 9, Supplementary Tables 12–16 and Sup- plementary Fig. 10), have remained largely conserved during the vertebrate land transition (Supplementary Fig. 11). As vertebrates transitioned to a new land environment, changes occurred not only in gene content but also in the regulation of existing genes. Conserved non-coding elements (CNEs) are strong candidates for gene regulatory elements. They can act as promoters, enhancers, repressors and insulators32,33, and have been implicated as major faci- litators of evolutionary change34. To identify CNEs that originated in the most recent common ancestor of tetrapods, we predicted CNEs that evolved in various bony vertebrate (that is, ray-finned fish, coela- canth and tetrapod) lineages and assigned them to their likely branch points of origin. To detect CNEs, conserved sequences in the human genome were identified using MULTIZ alignments of bony vertebrate genomes, and then known protein-coding sequences, untranslated regions (UTRs) and known RNA genes were excluded. Our ana- lysis identified 44,200 ancestral tetrapod CNEs that originated after the divergence of the coelacanth lineage. They represent 6% of the 739,597 CNEs that are under constraint in the bony vertebrate lin- eage. We compared the ancestral tetrapod CNEs to mouse embryo ChIP-seq (chromatin immunoprecipitation followed by sequencing) data obtained using antibodies against p300, a transcriptional coacti- vator. This resulted in a sevenfold enrichment in the p300 binding sites for our candidate CNEs and confirmed that these CNEs are indeed enriched for gene regulatory elements. Each tetrapod CNE was assigned to the gene whose transcription start site was closest, and gene-ontology category enrichment was cal- culated for those genes. The most enriched categories were involved with smell perception (for example, sensory perception of smell, detection of chemical stimulus and olfactory receptor activity). This is consistent with the notable expansion of olfactory receptor family genes in tetrapods compared with teleosts, and may reflect the neces- sity of a more tightly regulated, larger and more diverse repertoire of olfactory receptors for detecting airborne odorants as part of the terrestrial lifestyle. Other significant categories include morphoge- nesis (radial pattern formation, hind limb morphogenesis, kidney mor- phogenesis) and cell differentiation (endothelial cell fate commitment, ARTICLE RESEARCH 1 8 A P R I L 2 0 1 3 | V O L 4 9 6 | N A T U R E | 3 1 3 Macmillan Publishers Limited. All rights reserved©2013 epithelial cell fate commitment), which is consistent with the body- plan changes required for land transition, as well as immunoglobulin VDJ recombination, which reflects the presumed response differences required to address the novel pathogens that vertebrates would encoun- ter on land (Supplementary Note 10 and Supplementary Tables 17–24). A major innovation of tetrapods is the evolution of limbs charac- terized by digits. The limb skeleton consists of a stylopod (humerus or femur), the zeugopod (radius and ulna, or tibia and fibula), and an autopod (wrist or ankle, and digits). There are two major hypotheses about the origins of the autopod; that it was a novel feature of tetra- pods, and that it has antecedents in the fins of fish35 (Supplementary Note 11 and Supplementary Fig. 12). We examine here the Hox regulation of limb development in ray-finned fish, coelacanth and tetrapods to address these hypotheses. In mouse, late-phase digit enhancers are located in a gene desert that is proximal to the HOX-D cluster36. Here we provide an align- ment of the HOX-D centromeric gene desert of coelacanth with those of tetrapods and ray-finned fishes (Fig. 2a). Among the six cis-regulatory sequences previously identified in this gene desert36, three sequences show sequence conservation restricted to tetrapods (Supplementary Fig. 13). However, one regulatory sequence (island 1) is shared by tetra- pods and coelacanth, but not by ray-finned fish (Fig. 2b and Supplemen- tary Fig. 14). When tested in a transient transgenic assay in mouse, the coelacanth sequence of island 1 was able to drive reporter expression in a limb-specific pattern (Fig. 2c). This suggests that island 1 was a lobe- fin developmental enhancer in the fish ancestor of tetrapods that was then coopted into the autopod enhancer of modern tetrapods. In this case, the autopod developmental regulation was derived from an ances- tral lobe-finned fish regulatory element. Changes in the urea cycle provide an illuminating example of the adaptations associated with transition to land. Excretion of nitrogen is a major physiological challenge for terrestrial vertebrates. In aquatic environments, the primary nitrogenous waste product is ammonia, which is readily diluted by surrounding water before it reaches toxic levels, but on land, less toxic substances such as urea or uric acid must be produced instead (Supplementary Fig. 15). The widespread and almost exclusive occurrence of urea excretion in amphibians, some turtles and mammals has led to the hypothesis that the use of urea as the main nitrogenous waste product was a key innovation in the vertebrate transition from water to land37. With the availability of gene sequences from coelacanth and lungfish, it became possible to test this hypothesis. We used a branch-site model in the HYPHY package38, which estimates the ratio of synonymous (dS) to non-synonymous (dN) substitutions (v values) among different branches and among different sites (codons) across a multiple-species sequence alignment. For the rate-limiting enzyme of the hepatic urea cycle, carbamoyl phosphate synthase I (CPS1), only one branch of the tree shows a strong signature of selection (P 5 0.02), namely the branch leading to tetrapods and the branch leading to amniotes (Fig. 3); no other enzymes in this cycle showed a signature of selection. Conversely, mitochondrial arginase (ARG2), which produces extrahepatic urea as a byproduct of arginine metabolism but is not involved in the production of urea for nitrogenous waste disposal, did not show any evidence of selection in vertebrates (Supplementary Fig. 16). This leads us to con- clude that adaptive evolution occurred in the hepatic urea cycle during the vertebrate land transition. In addition, it is interesting to note that of the five amino acids of CPS1 that changed between coelacanth and tetrapods, three are in important domains (the two ATP-binding sites and the subunit interaction domain) and a fourth is known to cause a malfunctioning enzyme in human patients if mutated39. The adaptation to a terrestrial lifestyle necessitated major changes in the physiological environment of the developing embryo and fetus, resulting in the evolution and specialization of extra-embryonic mem- branes of the amniote mammals40. In particular, the placenta is a com- plex structure that is critical for providing gas and nutrient exchange between mother and fetus, and is also a major site of haematopoiesis41. We have identified a region of the coelacanth HOX-A cluster that may have been involved in the evolution of extra-embryonic struc- tures in tetrapods, including the eutherian placenta. Global alignment of the coelacanth Hoxa14–Hoxa13 region with the homologous regions of the horn shark, chicken, human and mouse revealed a CNE just upstream of the coelacanth Hoxa14 gene (Supplementary Fig. 17a). This conserved stretch is not found … ARTICLE OPEN doi:10.1038/nature12027 The African coelacanth genome provides insights into tetrapod evolution Chris T. Amemiya1,2*, Jessica Alföldi3*, Alison P. Lee4, Shaohua Fan5, Hervé Philippe6, Iain MacCallum3, Ingo Braasch7, Tereza Manousaki5,8, Igor Schneider9, Nicolas Rohner10, Chris Organ11, Domitille Chalopin12, Jeramiah J. Smith13, Mark Robinson1, Rosemary A. Dorrington14, Marco Gerdol15, Bronwen Aken16, Maria Assunta Biscotti17, Marco Barucca17, Denis Baurain18, Aaron M. Berlin3, Gregory L. Blatch14,19, Francesco Buonocore20, Thorsten Burmester21, Michael S. Campbell22, Adriana Canapa17, John P. Cannon23, Alan Christoffels24, Gianluca De Moro15, Adrienne L. Edkins14, Lin Fan3, Anna Maria Fausto20, Nathalie Feiner5,25, Mariko Forconi17, Junaid Gamieldien24, Sante Gnerre3, Andreas Gnirke3, Jared V. Goldstone26, Wilfried Haerty27, Mark E. Hahn26, Uljana Hesse24, Steve Hoffmann28, Jeremy Johnson3, Sibel I. Karchner26, Shigehiro Kuraku5{, Marcia Lara3, Joshua Z. Levin3, Gary W. Litman23, Evan Mauceli3{, Tsutomu Miyake29, M. Gail Mueller30, David R. Nelson31, Anne Nitsche32, Ettore Olmo17, Tatsuya Ota33, Alberto Pallavicini15, Sumir Panji24{, Barbara Picone24, Chris P. Ponting27, Sonja J. Prohaska34, Dariusz Przybylski3, Nil Ratan Saha1, Vydianathan Ravi4, Filipe J. Ribeiro3{, Tatjana Sauka-Spengler35, Giuseppe Scapigliati20, Stephen M. J. Searle16, Ted Sharpe3, Oleg Simakov5,36, Peter F. Stadler32, John J. Stegeman26, Kenta Sumiyama37, Diana Tabbaa3, Hakim Tafer32, Jason Turner-Maier3, Peter van Heusden24, Simon White16, Louise Williams3, Mark Yandell22, Henner Brinkmann6, Jean-Nicolas Volff12, Clifford J. Tabin10, Neil Shubin38, Manfred Schartl39, David B. Jaffe3, John H. Postlethwait7, Byrappa Venkatesh4, Federica Di Palma3, Eric S. Lander3, Axel Meyer5,8,25 & Kerstin Lindblad-Toh3,40 The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution. In 1938 Marjorie Courtenay-Latimer, the curator of a small natural history museum in East London, South Africa, discovered a large, unusual-looking fish among the many specimens delivered to her by a local fish trawler. Latimeria chalumnae, named after its discoverer1, was over 1 m long, bluish in colour and had conspicuously fleshy fins that resembled the limbs of terrestrial vertebrates. This discovery is considered to be one of the most notable zoological finds of the twen- tieth century. Latimeria is the only living member of an ancient group of lobe-finned fishes that was known previously only from fossils and believed to have been extinct since the Late Cretaceous period, approximately 70 million years ago (Myr ago)1. It was almost 15 years before a second specimen of this elusive species was discovered in the *These authors contributed equally to this work. 1Molecular Genetics Program, Benaroya Research Institute, Seattle, Washington 98101, USA. 2Department of Biology, University of Washington, Seattle, Washington 98105, USA. 3Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 4Comparative Genomics Laboratory, Institute of Molecular and Cell Biology, A*STAR, Biopolis, Singapore 138673, Singapore. 5Department of Biology, University of Konstanz, Konstanz 78464, Germany. 6Département de Biochimie, Université de Montréal, Centre Robert Cedergren, Montréal H3T 1J4, Canada. 7Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403, USA. 8Konstanz Research School of Chemical Biology, University of Konstanz, Konstanz 78464, Germany. 9Instituto de Ciencias Biologicas, Universidade Federal do Para, Belem 66075-110, Brazil. 10Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 11Department of Anthropology, University of Utah, Salt Lake City, Utah 84112, USA. 12Institut de Genomique Fonctionnelle de Lyon, Ecole Normale Superieure de Lyon, Lyon 69007, France. 13Department of Biology, University of Kentucky, Lexington, Kentucky 40506, USA. 14Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry, Microbiology & Biotechnology, Rhodes University, Grahamstown 6139, South Africa. 15Department of Life Sciences, University of Trieste, Trieste 34128, Italy. 16Department of Informatics, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. 17Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona 60131, Italy. 18Department of Life Sciences, University of Liege, Liege 4000, Belgium. 19College of Health and Biomedicine, Victoria University, Melbourne VIC 8001, Australia. 20Department for Innovation in Biological, Agro-food and Forest Systems, University of Tuscia, Viterbo 01100, Italy. 21Department of Biology, University of Hamburg, Hamburg 20146, Germany. 22Eccles Institute of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA. 23Department of Pediatrics, University of South Florida Morsani College of Medicine, Children’s Research Institute, St. Petersburg, Florida 33701, USA. 24South African National Bioinformatics Institute, University of the Western Cape, Bellville 7535, South Africa. 25International Max-Planck Research School for Organismal Biology, University of Konstanz, Konstanz 78464, Germany. 26Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA. 27MRC Functional Genomics Unit, Oxford University, Oxford OX1 3PT, UK. 28Transcriptome Bioinformatics Group, LIFE Research Center for Civilization Diseases, Universität Leipzig, Leipzig 04109, Germany. 29Graduate School of Science and Technology, Keio University, Yokohama 223-8522, Japan. 30Department of Molecular Genetics, All Children’s Hospital, St. Petersburg, Florida 33701, USA. 31Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. 32Bioinformatics Group, Department of Computer Science, Universität Leipzig, Leipzig 04109, Germany. 33Department of Evolutionary Studies of Biosystems, The Graduate University for Advanced Studies, Hayama 240-0193, Japan. 34Computational EvoDevo Group, Department of Computer Science, Universität Leipzig, Leipzig 04109, Germany. 35Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX1 2JD, UK. 36European Molecular Biology Laboratory, Heidelberg 69117, Germany. 37Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japan. 38University of Chicago, Chicago, Illinois 60637, USA. 39Department Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg 97070, Germany. 40Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala 751 23, Sweden. {Present addresses: Genome Resource and Analysis Unit, Center for Developmental Biology, RIKEN, Kobe, Japan (S.K.); Boston Children’s Hospital, Boston, Massachusetts, USA (E.M.); Computational Biology Unit, Institute of Infectious Disease and Molecular Medicine, University of Cape Town Health Sciences Campus, Anzio Road, Observatory 7925, South Africa (S.P.); New York Genome Center, New York, New York, USA (F.J.R.). 1 8 A P R I L 2 0 1 3 | V O L 4 9 6 | N A T U R E | 3 1 1 Macmillan Publishers Limited. All rights reserved©2013 www.nature.com/doifinder/10.1038/nature12027 Comoros Islands in the Indian Ocean, and only 309 individuals have been recorded in the past 75 years (R. Nulens, personal communication)2. The discovery in 1997 of a second coelacanth species in Indonesia, Latimeria menadoensis, was equally surprising, as it had been assumed that living coelacanths were confined to small populations off the East African coast3,4. Fascination with these fish is partly due to their pre- historic appearance—remarkably, their morphology is similar to that of fossils that date back at least 300 Myr, leading to the supposition that, among vertebrates, this lineage is markedly slow to evolve1,5. Latimeria has also been of particular interest to evolutionary biologists, owing to its hotly debated relationship to our last fish ancestor, the fish that first crawled onto land6. In the past 15 years, targeted sequencing efforts have produced the sequences of the coelacanth mitochondrial genomes7, HOX clusters8 and a few gene families9,10. Nevertheless, coelacanth research has felt the lack of large-scale sequencing data. Here we describe the sequencing and comparative analysis of the genome of L. chalumnae, the African coelacanth. Genome assembly and annotation The African coelacanth genome was sequenced and assembled using DNA from a Comoros Islands Latimeria chalumnae specimen (Sup- plementary Fig. 1). It was sequenced by Illumina sequencing tech- nology and assembled using the short read genome assembler ALLPATHS-LG11. The L. chalumnae genome has been reported previ- ously to have a karyotype of 48 chromosomes12. The draft assembly is 2.86 gigabases (Gb) in size and is composed of 2.18 Gb of sequence plus gaps between contigs. The coelacanth genome assembly has a contig N50 size (the contig size above which 50% of the total length of the sequence assembly can be found) of 12.7 kilobases (kb) and a scaffold N50 size of 924 kb, and quality metrics comparable to other Illumina genomes (Supplementary Note 1, and Supplementary Tables 1 and 2). The genome assembly was annotated separately by both the Ensembl gene annotation pipeline (Ensembl release 66, February 2012) and by MAKER13. The Ensembl gene annotation pipeline created gene models using protein alignments from the Universal Protein Resource (Uni- prot) database, limited coelacanth complementary DNA data, RNA-seq data generated from L. chalumnae muscle (18 Gb of paired-end reads were assembled using Trinity software14, Supplementary Fig. 2) as well as orthology with other vertebrates. This pipeline produced 19,033 protein-coding genes containing 21,817 transcripts. The MAKER pipeline used the L. chalumnae Ensembl gene set, Uniprot protein alignments, and L. chalumnae (muscle) and L. menadoensis (liver and testis)15 RNA-seq data to create gene models, and this produced 29,237 protein-coding gene annotations. In addition, 2,894 short non- coding RNAs, 1,214 long non-coding RNAs, and more than 24,000 conserved RNA secondary structures were identified (Supplementary Note 2, Supplementary Tables 3 and 4, Supplementary Data 1–3 and Supplementary Fig. 3). It was inferred that 336 genes underwent spe- cific duplications in the coelacanth lineage (Supplementary Note 3, Supplementary Tables 5 and 6, and Supplementary Data 4). The closest living fish relative of tetrapods The question of which living fish is the closest relative to ‘the fish that first crawled on to land’ has long captured our imagination: among scientists the odds have been placed on either the lungfish or the coelacanth16. Analyses of small to moderate amounts of sequence data for this important phylogenetic question (ranging from 1 to 43 genes) has tended to favour the lungfishes as the extant sister group to the land vertebrates17. However, the alternative hypothesis that the lung- fish and the coelacanth are equally closely related to the tetrapods could not be rejected with previous data sets18. To seek a comprehensive answer we generated RNA-seq data from three samples (brain, gonad and kidney, and gut and liver) from the West African lungfish, Protopterus annectens, and compared it to gene sets from 21 strategically chosen jawed vertebrate species. To perform a reliable analysis we selected 251 genes in which a 1:1 orthology ratio was clear and used CAT-GTR, a complex site-heterogeneous model of sequence evolution that is known to reduce tree-reconstruction arte- facts19 (see Supplementary Methods). The resulting phylogeny, based on 100,583 concatenated amino acid positions (Fig. 1, posterior prob- ability 5 1.0 for the lungfish–tetrapod node) is maximally supported except for the relative positions of the armadillo and the elephant. It corroborates known vertebrate phylogenetic relationships and strongly supports the conclusion that tetrapods are more closely related to lungfish than to the coelacanth (Supplementary Note 4 and Supplementary Fig. 4). The slowly evolving coelacanth The morphological resemblance of the modern coelacanth to its fossil ancestors has resulted in it being nicknamed ‘the living fossil’1. This invites the question of whether the genome of the coelacanth is as slowly evolving as its outward appearance suggests. Earlier work showed that a few gene families, such as Hox and protocadherins, have comparatively slower protein-coding evolution in coelacanth than in other vertebrate lineages8,10. To address the question, we compared several features of the coelacanth genome to those of other vertebrate genomes. Protein-coding gene evolution was examined using the phyloge- nomics data set described above (251 concatenated proteins) (Fig. 1). Pair-wise distances between taxa were calculated from the branch lengths of the tree using the two-cluster test proposed previously20 to test for equality of average substitution rates. Then, for each of the following species and species clusters (coelacanth, lungfish, chicken and mammals), we ascertained their respective mean distance to an outgroup consisting of three cartilaginous fishes (elephant shark, little skate and spotted catshark). Finally, we tested whether there was any significant difference in the distance to the outgroup of cartilaginous fish for every pair of species and species clusters, using a Dog 0.1 substitutions per site Human Mouse Elephant Armadillo Opossum Platypus Chicken Turkey Zebra finch Lizard Western clawed frog Chinese brown frog Lungfish Coelacanth Tilapia Pufferfish Zebrafish Elephant shark Little skate Spotted catshark Tetrapods Lobe-finned fish Cartilaginous fish Ray-finned fish Tammar wallaby Figure 1 | A phylogenetic tree of a broad selection of jawed vertebrates shows that lungfish, not coelacanth, is the closest relative of tetrapods. Multiple sequence alignments of 251 genes with a 1:1 ratio of orthologues in 22 vertebrates and with a full sequence coverage for both lungfish and coelacanth were used to generate a concatenated matrix of 100,583 unambiguously aligned amino acid positions. The Bayesian tree was inferred using PhyloBayes under the CAT 1 GTR 1 C4 model with confidence estimates derived from 100 gene jack-knife replicates (support is 100% for all clades but armadillo 1 elephant with 45%)48. The tree was rooted on cartilaginous fish, and shows that the lungfish is more closely related to tetrapods than the coelacanth, and that the protein sequence of coelacanth is evolving slowly. Pink lines (tetrapods) are slightly offset from purple lines (lobe-finned fish), to indicate that these species are both tetrapods and lobe-finned fish. RESEARCH ARTICLE 3 1 2 | N A T U R E | V O L 4 9 6 | 1 8 A P R I L 2 0 1 3 Macmillan Publishers Limited. All rights reserved©2013 Z statistic. When these distances to the outgroup of cartilaginous fish were compared, we found that the coelacanth proteins that were tested were significantly more slowly evolving (0.890 substitutions per site) than the lungfish (1.05 substitutions per site), chicken (1.09 substitutions per site) and mammalian (1.21 substitutions per site) orthologues (P , 1026 in all cases) (Supplementary Data 5). In addition, as can be seen in Fig. 1, the substitution rate in coelacanth is approxi- mately half that in tetrapods since the two lineages diverged. A Tajima’s relative rate test21 confirmed the coelacanth’s significantly slower rate of protein evolution (P , 10220) (Supplementary Data 6). We next examined the abundance of transposable elements in the coelacanth genome. Theoretically, transposable elements may make their greatest contribution to the evolution of a species by generating templates for exaptation to form novel regulatory elements and exons, and by acting as substrates for genomic rearrangement22. We found that the coelacanth genome contains a wide variety of transposable- element superfamilies and has a relatively high transposable-element content (25%); this number is probably an underestimate as this is a draft assembly (Supplementary Note 5 and Supplementary Tables 7–10). Analysis of RNA-seq data and of the divergence of individual transposable-element copies from consensus sequences show that 14 coelacanth transposable-element superfamilies are currently active (Supplementary Note 6, Supplementary Table 10 and Supplementary Fig. 5). We conclude that the current coelacanth genome shows both an abundance and activity of transposable elements similar to many other genomes. This contrasts with the slow protein evolution observed. Analyses of chromosomal breakpoints in the coelacanth genome and tetrapod genomes reveal extensive conservation of synteny and indicate that large-scale rearrangements have occurred at a generally low rate in the coelacanth lineage. Analyses of these rearrangement classes detected several fission events published previously23 that are known to have occurred in tetrapod lineages, and at least 31 inter- chromosomal rearrangements that occurred in the coelacanth lineage or the early tetrapod lineage (0.063 fusions per 1 Myr), compared to 20 events (0.054 fusions per 1 Myr) in the salamander lineage and 21 events (0.057 fusions per 1 Myr) in the Xenopus lineage23 (Sup- plementary Note 7 and Supplementary Fig. 6). Overall, these analyses indicate that karyotypic evolution in the coelacanth lineage has occurred at a relatively slow rate, similar to that of non-mammalian tetrapods24. In a separate analysis we also examined the evolutionary divergence between the two species of coelacanth, L. chalumnae and L. menadoensis, found in African and Indonesian waters, respectively. Previous ana- lysis of mitochondrial DNA showed a sequence identity of 96%, but estimated divergence times range widely from 6 to 40 Myr25,26. When we compared the liver and testis transcriptomes of L. menadoensis27 to the L. chalumnae genome, we found an identity of 99.73% (Sup- plementary Note 8 and Supplementary Fig. 7), whereas alignments between 20 sequenced L. menadoensis bacterial artificial chromosomes (BACs) and the L. chalumnae genome showed an identity of 98.7% (Supplementary Table 11 and Supplementary Fig. 8). Both the genic and genomic divergence rates are similar to those seen between the human and chimpanzee genomes (99.5% and 98.8%, respectively; divergence time of 6 to 8 Myr ago)28, whereas the rates of molecular evolution in Latimeria are probably affected by several factors, includ- ing the slower substitution rate seen in coelacanth. This suggests a slightly longer divergence time for the two coelacanth species. The adaptation of vertebrates to land As the species with a sequenced genome closest to our most recent aquatic ancestor, the coelacanth provides a unique opportunity to identify genomic changes that were associated with the successful adaptation of vertebrates to the land environment. Over the 400 Myr that vertebrates have lived on land, some genes that are unnecessary for existence in their new environment have been eliminated. To understand this aspect of the water-to-land transition, we surveyed the Latimeria genome annotations to identify genes that were present in the last common ancestor of all bony fish (including the coelacanth) but that are missing from tetrapod genomes. More than 50 such genes, including components of fibroblast growth factor (FGF) signalling, TGF-b and bone morphogenic protein (BMP) sig- nalling, and WNT signalling pathways, as well as many transcription factor genes, were inferred to be lost based on the coelacanth data (Supplementary Data 7 and Supplementary Fig. 9). Previous studies of genes that were lost in this transition could only compare teleost fish to tetrapods, meaning that differences in gene content could have been due to loss in the tetrapod or in the lobe-finned fish lineages. We were able to confirm that four genes that were shown previously to be absent in tetrapods (And1 and And2 (ref. 29), Fgf24 (ref. 30) and Asip2 (ref. 31)), were indeed present and intact in Latimeria, support- ing the idea that they were lost in the tetrapod lineage. We functionally annotated more than 50 genes lost in tetrapods using zebrafish data (gene expression, knock-downs and knockouts). Many genes were classified in important developmental categories (Supplementary Data 7): fin development (13 genes); otolith and ear development (8 genes); kidney development (7 genes); trunk, somite and tail development (11 genes); eye (13 genes); and brain development (23 genes). This implies that critical characters in the morphological transition from water to land (for example, fin-to-limb transition and remodelling of the ear) are reflected in the loss of specific genes along the phylogenetic branch leading to tetrapods. However, homeobox genes, which are responsible for the develop- ment of an organism’s basic body plan, show only slight differences between Latimeria, ray-finned fish and tetrapods; it would seem that the protein-coding portion of this gene family, along with several others (Supplementary Note 9, Supplementary Tables 12–16 and Sup- plementary Fig. 10), have remained largely conserved during the vertebrate land transition (Supplementary Fig. 11). As vertebrates transitioned to a new land environment, changes occurred not only in gene content but also in the regulation of existing genes. Conserved non-coding elements (CNEs) are strong candidates for gene regulatory elements. They can act as promoters, enhancers, repressors and insulators32,33, and have been implicated as major faci- litators of evolutionary change34. To identify CNEs that originated in the most recent common ancestor of tetrapods, we predicted CNEs that evolved in various bony vertebrate (that is, ray-finned fish, coela- canth and tetrapod) lineages and assigned them to their likely branch points of origin. To detect CNEs, conserved sequences in the human genome were identified using MULTIZ alignments of bony vertebrate genomes, and then known protein-coding sequences, untranslated regions (UTRs) and known RNA genes were excluded. Our ana- lysis identified 44,200 ancestral tetrapod CNEs that originated after the divergence of the coelacanth lineage. They represent 6% of the 739,597 CNEs that are under constraint in the bony vertebrate lin- eage. We compared the ancestral tetrapod CNEs to mouse embryo ChIP-seq (chromatin immunoprecipitation followed by sequencing) data obtained using antibodies against p300, a transcriptional coacti- vator. This resulted in a sevenfold enrichment in the p300 binding sites for our candidate CNEs and confirmed that these CNEs are indeed enriched for gene regulatory elements. Each tetrapod CNE was assigned to the gene whose transcription start site was closest, and gene-ontology category enrichment was cal- culated for those genes. The most enriched categories were involved with smell perception (for example, sensory perception of smell, detection of chemical stimulus and olfactory receptor activity). This is consistent with the notable expansion of olfactory receptor family genes in tetrapods compared with teleosts, and may reflect the neces- sity of a more tightly regulated, larger and more diverse repertoire of olfactory receptors for detecting airborne odorants as part of the terrestrial lifestyle. Other significant categories include morphoge- nesis (radial pattern formation, hind limb morphogenesis, kidney mor- phogenesis) and cell differentiation (endothelial cell fate commitment, ARTICLE RESEARCH 1 8 A P R I L 2 0 1 3 | V O L 4 9 6 | N A T U R E | 3 1 3 Macmillan Publishers Limited. All rights reserved©2013 epithelial cell fate commitment), which is consistent with the body- plan changes required for land transition, as well as immunoglobulin VDJ recombination, which reflects the presumed response differences required to address the novel pathogens that vertebrates would encoun- ter on land (Supplementary Note 10 and Supplementary Tables 17–24). A major innovation of tetrapods is the evolution of limbs charac- terized by digits. The limb skeleton consists of a stylopod (humerus or femur), the zeugopod (radius and ulna, or tibia and fibula), and an autopod (wrist or ankle, and digits). There are two major hypotheses about the origins of the autopod; that it was a novel feature of tetra- pods, and that it has antecedents in the fins of fish35 (Supplementary Note 11 and Supplementary Fig. 12). We examine here the Hox regulation of limb development in ray-finned fish, coelacanth and tetrapods to address these hypotheses. In mouse, late-phase digit enhancers are located in a gene desert that is proximal to the HOX-D cluster36. Here we provide an align- ment of the HOX-D centromeric gene desert of coelacanth with those of tetrapods and ray-finned fishes (Fig. 2a). Among the six cis-regulatory sequences previously identified in this gene desert36, three sequences show sequence conservation restricted to tetrapods (Supplementary Fig. 13). However, one regulatory sequence (island 1) is shared by tetra- pods and coelacanth, but not by ray-finned fish (Fig. 2b and Supplemen- tary Fig. 14). When tested in a transient transgenic assay in mouse, the coelacanth sequence of island 1 was able to drive reporter expression in a limb-specific pattern (Fig. 2c). This suggests that island 1 was a lobe- fin developmental enhancer in the fish ancestor of tetrapods that was then coopted into the autopod enhancer of modern tetrapods. In this case, the autopod developmental regulation was derived from an ances- tral lobe-finned fish regulatory element. Changes in the urea cycle provide an illuminating example of the adaptations associated with transition to land. Excretion of nitrogen is a major physiological challenge for terrestrial vertebrates. In aquatic environments, the primary nitrogenous waste product is ammonia, which is readily diluted by surrounding water before it reaches toxic levels, but on land, less toxic substances such as urea or uric acid must be produced instead (Supplementary Fig. 15). The widespread and almost exclusive occurrence of urea excretion in amphibians, some turtles and mammals has led to the hypothesis that the use of urea as the main nitrogenous waste product was a key innovation in the vertebrate transition from water to land37. With the availability of gene sequences from coelacanth and lungfish, it became possible to test this hypothesis. We used a branch-site model in the HYPHY package38, which estimates the ratio of synonymous (dS) to non-synonymous (dN) substitutions (v values) among different branches and among different sites (codons) across a multiple-species sequence alignment. For the rate-limiting enzyme of the hepatic urea cycle, carbamoyl phosphate synthase I (CPS1), only one branch of the tree shows a strong signature of selection (P 5 0.02), namely the branch leading to tetrapods and the branch leading to amniotes (Fig. 3); no other enzymes in this cycle showed a signature of selection. Conversely, mitochondrial arginase (ARG2), which produces extrahepatic urea as a byproduct of arginine metabolism but is not involved in the production of urea for nitrogenous waste disposal, did not show any evidence of selection in vertebrates (Supplementary Fig. 16). This leads us to con- clude that adaptive evolution occurred in the hepatic urea cycle during the vertebrate land transition. In addition, it is interesting to note that of the five amino acids of CPS1 that changed between coelacanth and tetrapods, three are in important domains (the two ATP-binding sites and the subunit interaction domain) and a fourth is known to cause a malfunctioning enzyme in human patients if mutated39. The adaptation to a terrestrial lifestyle necessitated major changes in the physiological environment of the developing embryo and fetus, resulting in the evolution and specialization of extra-embryonic mem- branes of the amniote mammals40. In particular, the placenta is a com- plex structure that is critical for providing gas and nutrient exchange between mother and fetus, and is also a major site of haematopoiesis41. We have identified a region of the coelacanth HOX-A cluster that may have been involved in the evolution of extra-embryonic struc- tures in tetrapods, including the eutherian placenta. Global alignment of the coelacanth Hoxa14–Hoxa13 region with the homologous regions of the horn shark, chicken, human and mouse revealed a CNE just upstream of the coelacanth Hoxa14 gene (Supplementary Fig. 17a). This conserved stretch is not found …