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The dynamic genome of Hydra

The dynamic genome of Hydra Vol 464 |25 March 2010 |doi:10.1038/nature08830 LETTERS 1 2 3,4 5 4 Jarrod A. Chapman *, Ewen F. Kirkness *, Oleg Simakov *, Steven E. Hampson {, Therese Mitros , 6 6 3 2 2 Thomas Weinmaier , Thomas Rattei , Prakash G. Balasubramanian , Jon Borman , Dana Busam , 2 2 2 2 2 Kathryn Disbennett , Cynthia Pfannkoch , Nadezhda Sumin , Granger G. Sutton , Lakshmi Devi Viswanathan , 2 1 1 4 1 Brian Walenz , David M. Goodstein , Uffe Hellsten , Takeshi Kawashima , Simon E. Prochnik , 1,4 1 7,8 8,9 7,8 5 Nicholas H. Putnam {, Shengquiang Shu , Bruce Blumberg , Catherine E. Dana , Lydia Gee , Dennis F. Kibler , 7,8 7,8 10 7,8 11 3 Lee Law , Dirk Lindgens , Daniel E. Martinez , Jisong Peng , Philip A. Wigge {, Bianca Bertulat , 3 3 3 3 12 12 Corina Guder , Yukio Nakamura , Suat Ozbek , Hiroshi Watanabe , Konstantin Khalturin , Georg Hemmrich , 12 12 12 13 13 13 ´ ´ Andre Franke , Rene Augustin , Sebastian Fraune , Eisuke Hayakawa , Shiho Hayakawa , Mamiko Hirose , 13 13 13 13 14 Jung Shan Hwang , Kazuho Ikeo , Chiemi Nishimiya-Fujisawa , Atshushi Ogura {, Toshio Takahashi , 15 16 17 17 17 Patrick R. H. Steinmetz , Xiaoming Zhang , Roland Aufschnaiter , Marie-Kristin Eder , Anne-Kathrin Gorny {, 17 18 19 18 20 Willi Salvenmoser , Alysha M. Heimberg , Benjamin M. Wheeler , Kevin J. Peterson , Angelika Bo¨ttger , 6 20 13 2 2 Patrick Tischler , Alexander Wolf , Takashi Gojobori , Karin A. Remington {, Robert L. Strausberg , 2 15 17 12 3 J. Craig Venter , Ulrich Technau , Bert Hobmayer , Thomas C. G. Bosch , Thomas W. Holstein , 13 7,8 20 1,4 8,9 Toshitaka Fujisawa , Hans R. Bode , Charles N. David , Daniel S. Rokhsar & Robert E. Steele The freshwater cnidarian Hydra was first described in 1702 and has (71% A1T), and includes ,57% transposable elements (see below). been the object of study for 300 years. Experimental studies of Hydra Although the sequenced strain reproduces clonally in the laboratory between 1736 and 1744 culminated in the discovery of asexual repro- by asexual budding, it is diploid with substantial heterozygosity duction of an animal by budding, the first description of regeneration (,0.7% single nucleotide polymorphism between alleles), which in an animal, and successful transplantation of tissue between ani- we find is distributed along the genome as expected if it were drawn mals .Today, Hydra is an important model for studies of axial pat- from a randomly mating population (Supplementary Information 3 4 5 terning , stem cell biology and regeneration .Herewereportthe section 3). These features complicate shotgun sequencing and assembly. genome of Hydra magnipapillata and compare it to the genomes of Two complementary assemblies (CA and RP) were generated (Sup- the anthozoan Nematostella vectensis and other animals. The Hydra plementary Information section 3) and deposited in GenBank. The genome has been shaped by bursts of transposable element expan- CA assembly (1.5 gigabases (Gb)) has contig and scaffold N50 values sion, horizontal gene transfer, trans-splicing, and simplification of of 12.8 kilobases (kb) and 63.4 kb, respectively. The RP assembly (1.0 gene structure and gene content that parallel simplification of the Gb) has a contig N50 length of 9.7 kb and a scaffold N50 length of Hydra life cycle. We also report the sequence of the genome of a novel 92.5 kb. The CA assembly gives an estimated non-redundant genome bacterium stably associated with H. magnipapillata. Comparisons of size of 1.05 Gb. The RP assembly gives an estimated non-redundant genome size of 0.9 Gb (see Supplementary Information section 3 for a the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated discussion of genome size calculations). For analysis, we chose the transcription factors, the Spemann–Mangold organizer, pluripotency assembly that minimized sequence redundancy owing to the separate genes and the neuromuscular junction. assembly of haplotypes (see Supplementary Information section 3 for The genomic basis of cnidarian evolution has so far been viewed further discussion). Approximately 99% of known Hydra genes are found in both assemblies, attesting to their completeness with respect from the perspective of an anthozoan, the sea anemone Nematostella vectensis . Hydra is a medusozoan that diverged from anthozoans to protein-coding genes. at least 540 millions year ago. Features of Hydra and Nematostella Although the present Hydra assembly is too fragmented for a are compared in Supplementary Table 1. We generated draft chromosome-scale analysis, we found evidence for synteny with other assemblies of the Hydra magnipapillata genome using a whole- metazoans. Of the 33 longest gene-rich Hydra scaffolds (that is, those genome shotgun approach (Supplementary Information sections containing genes from at least 10 Hydra/Nematostella orthologue 1–3 and Supplementary Figs 1–3). The Hydra genome is (A1T)-rich groups), 15 (45%) were significantly enriched (P, 0.01) for genes from 1 2 3 US Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA. The J. Craig Venter Institute, Rockville, Maryland 20850, USA. Institute of Zoology, Department of Molecular Evolution and Genomics, University of Heidelberg, D-69120 Heidelberg, Germany. Center for Integrative Genomics, Department of Molecular and Cell 5 6 Biology, University of California, Berkeley, California 94720, USA. Department of Computer Science, University of California, Irvine, California 92697-3435, USA. Department of 7 8 Genome-Oriented Bioinformatics, Technische Universita¨tMu¨nchen, D-85354 Freising, Germany. Department of Developmental and Cell Biology, Developmental Biology Center, 9 10 University of California, Irvine, California 92697-2275, USA. Department of Biological Chemistry, University of California, Irvine, California 92697-1700, USA. Department of 11 12 Biology, Pomona College, Claremont, California 91711, USA. The Salk Institute, La Jolla, California 92037, USA. Zoologisches Institu¨t, Christian-Albrechts-University, D-24098 Kiel, 13 14 15 Germany. National Institute of Genetics, Yata 1, 111, Mishima 411-8540, Japan. Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan. Department of Molecular Evolution and Development, University of Vienna, A-1090 Vienna, Austria. Department of Anatomy and Cell Biology, The University of Kansas Medical Center, Kansas City, Kansas 17 18 66160, USA. Institute of Zoology and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria. Department of Biological Sciences, Dartmouth College, 19 20 Hanover, New Hampshire 03755, USA. Department of Computer Science, North Carolina State University, Raleigh, North Carolina 27695, USA. Department of Biology II, Ludwig- Maximilians-University, D-82152 Planegg-Martinsried, Germany. {Present addresses: Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK (P.A.W.); Institute of Human Genetics, University of Heidelberg, D-69120 Heidelberg, Germany (A.-K.G.); Center for Bioinformatics and Computational Biology, National Institute of General Medical Sciences, Bethesda, Maryland 20892-6200, USA (K.A.R.); Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas 77251-1892, USA (N.H.P.); Ochadai Academic Production, Ochanomizu University, Ohtsuka, Bunkyo, 1128610 Tokyo, Japan (A.O.). *These authors contributed equally to this work. {Deceased. ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 |25 March 2010 LETTERS CR_Y a b H. viridissima (380 Mb) H. oligactis (1,450 Mb) H. vulgaris (1,300 Mb) III H. magnipapillata (1,300 Mb) 8 II Inferred ‘bursts’ of TE activity Transib 0.0 0.1 0.2 0.3 0.4 0.5 1.5 Chapaev Jukes–Cantor distance CR1 hAT Penelope 1.0 Mariner Gypsy Other III 0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 I II Jukes–Cantor distance from ReAS consensus Figure 1 | Dynamics of transposable element expansion in Hydra reveals is per cent dissimilarity on the nucleotide level from the repeat consensus. several periods of transposon activity. a, The top panel shows phylogenetic This substitution level for transposons is equivalent to Nei-Gojobori relationships between four Hydra species based on ESTs (using Nei- synonymous substitution rates in the ESTs. Three element expansions are Gojobori synonymous substitution rates; see Supplementary Fig. 8). The inferred, the most distinct are the most ancient at ,0.4 and the most recent bottom panel shows the fraction of the genome that is occupied by a specific at 0.05 divergence levels. The middle expansion at about ,0.2 is not well repeat class at a given divergence from the repeat consensus generated by the synchronized and is more clearly seen for individual element classes in ReAS (recovery of ancestral sequences) algorithm (see Supplementary Supplementary Figs 5 and 6. b, c, Example of periods of activity of a single Information section 9). Substitution levels are corrected for multiple Hydra CR1 retrotransposon family (b) and the maximum likelihood substitutions using the Jukes–Cantor formula K523/4ln(12i4/3), where i phylogeny of the family (c). specificeumetazoanlinkagegroups ,indicatingthatvestigesoftheancestral Most individual Hydra transposable element families show discrete eumetazoan genome organization persist in Hydra.Thisisincontrastto bursts of expansion (Fig. 1b, c) that are possibly associated with popu- the highly diverged genomes of Drosophila and Caenorhabditis elegans, lation bottlenecks . The correspondence between speciation times in which show no synteny with other metazoans by these methods. the genus Hydra and the timing of transposon activity may have been We estimate that the Hydra genome contains ,20,000 bona fide associated with the approximately threefold increase in genome size protein-coding genes (excluding transposable elements), based on (Fig. 1a) in H. magnipapillata, H. vulgaris and H. oligactis relative to H. expressed sequence tags (ESTs), homology and ab initio gene prediction viridissima (380 megabases (Mb)) . (Supplementary Information section 6). The amino acid substitution Addition of short RNA leader sequences to the 59 ends of messenger rate in the Hydra lineage is enhanced relative to the Nematostella RNAs by trans-splicing occurs in a subset of metazoans and unicellular lineage; the sequence divergence between a Hydra peptide and its human eukaryotes . Transcripts from at least one-third of EST-supported orthologue is typically greater than the sequence divergence between genes in Hydra undergo trans-spliced leader addition (Supplemen- Nematostella and human (Supplementary Information section 8 and tary Information section 10). Hydra has multiple spliced leader genes Supplementary Fig. 4) as expected based on the longer branch leading to (Supplementary Table 9), and a given transcript may be trans-spliced Hydra in peptide-based phylogenies . Similarly, the rate of intron loss with several different spliced leaders. Notably, trans-splicing is has been higher in the Hydra lineage; we find that 22% (126 out of 575) absent from Nematostella (Supplementary Information section 10). of the introns shared by Nematostella and human in well-aligned coding It now seems likely that trans-splicing has evolved multiple times regions have been lost in Hydra. Conversely, only 6% (28 out of 476) of independently . the introns shared by Hydra and human are absent in Nematostella. Trans-splicing occurs in Hydra viridissima (N. A. Stover and R.E.S., Transposable elements make up ,57% of the Hydra genome and unpublished data; GenBank accession number DQ092354) and in represent over 500 different families (Supplementary Information several other hydrozoans (Supplementary Table 10), and may be an section 9). The most abundant element, comprising ,15% of the ancestral feature of the class. Spliced leader addition gives a eukaryotic genome (Fig. 1 and Supplementary Table 3), is a non-long-terminal- cell the opportunity to combine genes into operons, the multi-cistronic repeat (non-LTR) retroelement of the chicken repeat 1 (CR1) family. To transcripts of which can be resolved into individual mRNAs by trans- our knowledge, elements of this family are more abundant in the Hydra splicing. We found 32 potential Hydra operons (Supplementary genome than in any other sequenced animal genome (in comparison, Information section 10, Supplementary Table 11 and Supplementary the CR1 family occupies only,1% of the Nematostella assembly and 3% Fig. 9), but no obvious evidence for functional relationships between of the chicken assembly). This retrotransposon is still active in Hydra,as genes in these operons. indicated by its representation in 105 ESTs. We also found 789 cases of Bacteria are stably associated with Hydra . Electron micrographs intronless genes that were derived recently from multi-exon genes, most reveal bacterial cells underneath the glycocalyx, the coat that overlies probably through retrotransposition. DNAtransposons (predominantly the apical surface of the ectodermal epithelial layer of Hydra (Sup- ‘cut-and-paste’ elements of the mariner, Transib and hAT (hobo- plementary Fig. 10). Our assembly yielded eight large putative bacterial Ac-Tam3) types) occupy ,20% of both the Hydra and Nematostella scaffolds as evidenced by: (1) high G1C content (in contrast to the low genomes, and are also active in Hydra based on the presence of ESTs. G1C content of the Hydra genome); (2) no high-copy repeat Timing of transposable element activity using sequence divergence sequences typical of Hydra scaffolds; and (3) closely spaced single-exon of extant copies reveals at least three periods of element expansion (at open reading frames with best hits to bacterial genes (Supplemen- ,5%, ,20% and ,40% nucleotide substitutions; Fig. 1 and Sup- tary Information section 11, Supplementary Fig. 11 and Supplemen- plementary Figs 5 and 6). In marked contrast, comparable expansions tary Table 12). These scaffolds span a total of 4 Mb encoding 3,782 are absent from the Nematostella genome (Supplementary Fig. 7). single-exon genes and represent an estimated 98% of the bacterial ©2010 Macmillan Publishers Limited. All rights reserved Percentage of the current genome Counts LETTERS NATURE |Vol 464 | 25 March 2010 (Supplementary Fig. 13). This pathway could modify endogenous glycoproteins or proteoglycans in Hydra. We also identified 90 transposable elements that were potentially hori- zontally transferred into the Hydra genome. These elements have expanded recently (less than 10% nucleotide divergence from their con- sensus) and have no older copies in the genome. The most frequent nv nv nv element class consists of hAT transposons with 34 different families, although all major classes of transposable element (DNA transposon, LTR and non-LTR elements) are represented. Transposable elements have been shown previously to be horizontally transferred in metazoans . emc emc emc We identified 51 unique non-tRNA/non-rRNA transcripts that correspond to putative non-coding RNA genes based on 454 sequencing of short transcripts from Hydra (Supplementary Information section 13 Ac-CoA + Ch Nerve and Supplementary Table 16). At least 17 of these are microRNAs cell (miRNAs), compared to 40 identified miRNAs in Nematostella . Surprisingly, only a single miRNA gene in the available data sets, miR- 2022, is common to both cnidarian species. ChT Hox and ParaHox gene families arose from a megacluster that AChE Ch included a number of other homeobox genes (for example, NK genes) . Agrin With the exception of engrailed, descendants of all of the classes of 16,17 homeobox genes in the megacluster are found in Nematostella . Muscle cell Hydra is missing a substantial fraction of megacluster descendants , Rapsyn Rapsyn indicating secondary loss. For example, the eve and emx genes are absent from Hydra, although they are present in Nematostella and several Figure 2 | The neuromuscular junction in Hydra.a, Electron micrograph of hydrozoans (Supplementary Table 17). The loss of these genes from a nerve synapsing on a Hydra epitheliomuscular cell. emc, epitheliomuscular Hydra is therefore recent in relation to the diversification of hydro- cell; nv, nerve cell. Three vesicles are located in the nerve cell at the site of contact with the epitheliomuscular cell. Scale bar, 200 nm. b, Schematic zoans. These genes are expressed in a cell-type-specific manner in larvae 17 18 diagram of a canonical neuromuscular junction. Yellow indicates presence and adults of Nematostella and Hydractinia ;itisintriguingthatthe in Hydra. Choline acetyltransferase (ChAT) is shown in red because it is not loss of these genes correlates with the absence of a larval stage in Hydra clear whether Hydra has an enzyme that prefers choline (Ch) as a substrate. (Supplementary Table 17). The absence of these genes in Hydra indi- Acetylcholine (ACh) molecules are shown as blue circles. The nicotinic cates that despite their near-universal presence in animals, it is possible acetylcholine receptor (nAChR) is shown in the open state with to construct a metazoan without either of them. In addition to the loss acetylcholine bound (left), and in the closed state in the absence of bound of emx and eve genes, Hydra has undergone several other marked gene acetylcholine (right). AChE, acetylcholinesterase; ChT, choline transporter; losses; for example, it lacks fluorescent protein genes and key circadian MuSK, muscle-specific kinase; VAChT, vesicular acetylcholine transporter. rhythm genes (Supplementary Information section 14). All major bilaterian signalling pathways, including Wnt, transform- chromosome. Phylogenetic analysis of 16S rRNA (Supplementary ing growth factor-b, Hedgehog, receptor tyrosine kinase and Notch, Fig. 12) and conserved clusters of orthologous groups of proteins are present in Hydra and Nematostella. An important signalling centre (COGs) indicate that this bacterium is a novel Curvibacter species in Hydra is the head organizer, which uses the Wnt signalling pathway belonging to the family Comamonadaceae (order Burkholderiales) . 19,20 to establish positional values along the body column . The head About 60% of annotated Curvibacter sp. genes have an orthologue in organizer, which is located at the apical tip of the adult polyp, is another species of Comamonadaceae (Supplementary Table 13). derived from the gastrula blastopore in cnidarians. A transplanted Notably, the Curvibacter sp. genome encodes nine different ABC sugar head organizer has the capacity to induce axis formation , similar transporters, compared to only one or two in other species of to the Spemann–Mangold organizer in Xenopus. Orthologues of a Comamonadaceae (Supplementary Table 14), possibly reflecting an number of genes known to act in the Spemann–Mangold organizer adaptation to life in association with Hydra. in Xenopus are present in the Hydra and Nematostella genomes. Non-metazoan genes among cnidarian ESTs have been reported Moreover, several of the secreted signalling molecules and trans- previously , and we have now found further examples of such genes cription factors encoded by these genes are expressed specifically in the Hydra genome assembly. These genes are candidates for hori- in the Hydra head organizer and the blastopore organizer in the zontal gene transfer (HGT) (Supplementary Information section 12). Nematostella gastrula (Supplementary Information section 15 and Seventy-one Hydra gene models showed closer relationships to bac- Supplementary Table 18). Thus, the Hydra head organizer and the terial genes than to metazoan genes based on sequence similarity and Xenopus Spemann–Mangold organizer may share common descent phylogenetic analysis (Supplementary Table 15). Of these, 51 have no from an organizer in the ancestor of cnidarians and bilaterians. 22,23 blast hits to other metazoans, except in a few cases to Nematostella. The extracellular portions of two Hydra receptor tyrosine kinases Potential donors of these HGT candidates are widely distributed contain a novel protein domain, sweet tooth (SWT). The SWT domain among different bacterial phyla (Supplementary Table 15) and show is also present in ESTs from the hydrozoan Clytia, but is absent from all no enrichment for close relatives of Curvibacter. Approximately 70% other sequenced genomes, including that of Nematostella (Supplemen- of the HGT candidates have EST support, and transcripts from 30% tary Fig. 15). SWT is among the most abundant protein domains of the genes have spliced leaders, indicating unambiguously that they encoded in the Hydra genome. The SWT domain is present in one are derived from Hydra and not from associated bacteria (Sup- or more copies in predicted secreted proteins. Given its presence in plementary Table 15). The HGT candidates generally have fewer receptors and secreted proteins, we deduce that the SWT domain introns than Hydra genes and nearly one-half are single-exon genes defines a large, diverse and novel set of signalling proteins. (Supplementary Fig. 14), as expected if they were relatively recently Hydra contains a pluripotent stem cell type that gives rise to germ acquired by Hydra. A number of the HGT candidates encode sugar- cells, nerve cells, nematocytes and secretory cells . Of the five genes that modifying enzymes. Three genes encode enzymes in the branch of the have been shown to induce pluripotency in differentiated somatic cells bacterial lipopolysaccharide synthesis pathway that leads to formation of mammals (Myc, Nanog, Klf4, Oct4 and Sox2) , homologues of three of the activated heptose precursor of the lipopolysaccharide inner core (Nanog, Klf4 and Oct4) are clearly not present in the Hydra genome. ©2010 Macmillan Publishers Limited. All rights reserved ChAT VAChT nAChR nAChR MuSK MuSK nAChR nAChR NATURE | Vol 464 |25 March 2010 LETTERS Hydra has four Myc homologues. There are two members of the Sox B ac b group in Hydra. The Sox B group includes Sox2, but the evolutionary relationship between vertebrate Sox2 genes and Hydra Sox B genes is not clear . We conclude that the stem cell genetic network in Hydra probably has an evolutionary origin independent from the network Ecto used in mammalian stem cells. Studies of diverse cnidarians support this scenario (see Supplementary Information section 14 for details). Hydra’s shape is formed by epitheliomuscular cells, a cell type unique to cnidarians. A survey of genes that encode muscle structural and d e regulatory proteins in Hydra and Nematostella reveals a conserved eumetazoan core actin-myosin contractile machinery shared with bilaterians (Supplementary Table 19). Both cnidarians, however, lack Endo crucial, specific regulators associated with vertebrate striated (troponin complex) or smooth muscles (caldesmon), indicating that these spe- cializations arose after the cnidarian–bilaterian split. Hydra also shows secondary simplifications relative to Nematostella, which has a greater degree of muscle-cell-type specialization, including specialized retrac- tor muscle cells. Hydra lacks several components of the dystroglycan complex (a/e-sarcoglycan and b-sarcoglycan, a/b-dystroglycan and c-syntrophin), which may lead to a less robust tethering of actin to the cell membrane than in Nematostella. Similarly, the absence of a bona fide myosin light chain kinase and phosphatase in Hydra indicates adivergenceorlossofregulationbymyosinregulatorylight chain phosphorylation. The greater degree of muscle-cell-type specialization in Nematostella is also mirrored in the higher number of myosin light Adherens junction/ Structural Classic cadherin ? desmosome Associated β-Catenin chain genes in this species. Thus, even among cnidarians, we see sub- p120/δ-catenin stantial variation in muscle-associated components superimposed on α-Catenin the eumetazoan core, with the Hydra muscular system representing a Vinculin α-Actinin secondary simplification from a more complex cnidarian ancestor. Afadin Ultrastructural studies show that nerve cells in Hydra form synapses Nonclassic cadherin on contractile epitheliomuscular cells (Fig. 2a), and that these synapses Tight junction Structural Occludin contain dense core vesicles, paramembranous densities and cleft fila- Claudin Associated ZO-1 ments similar to canonical neuromuscular junctions in bilaterians. Stardust/PALS1 Several components of the bilaterian neuromuscular junction (choline PATJ transporter, nicotinic acetylcholine receptor) are encoded in the Hydra Par3 Par6 genome (Supplementary Information section 16 and Supplementary Septate junction Structural Neurexin Table 20) and their expression is consistent with a role in neuromus- Neuroglian cular signalling (Supplementary Figs 16 and 17). Other components, Contactin Gliotactin however, are found only in a possibly primitive form (putative carni- Associated Discs large tine acetyltransferases that lack the diagnostic residues for choline Scribble selectivity), and some components are absent (the vesicular acetylcholine Coracle Gap junction Structural Innexin/Pannexin transporter; Fig. 2b). Together, these data indicate that a canonical bila- Connexin terian neuromuscular junction was probably not present in the last Hemidesmosome Structural Integrin-α common ancestor of cnidarians and bilaterians. Hydra is known to Integrin-β Associated Talin use neuropeptides for the control of behaviour , and these may be Paxillin contained in the dense-core vesicles seen at Hydra synapses. Tensin In Hydra and Nematostella, epitheliomuscular cells have an apical Integrin-linked kinase Focal adhesion kinase junctional belt in the form of a septate junction, clear apical–basal polar- ity, and hemidesmosome-like contact sites with the extracellular matrix Figure 3 | Hydra cell junctions. a, Schematic diagram of the positions of (mesoglea) on their basal surface (Fig. 3a). The Hydra and Nematostella cell–cell and cell–matrix contacts in Hydra epitheliomuscular cells. Septate genomes encode almost all of the proteins known from bilaterians to be junction, red; gap junctions, green; spot desmosomes, blue; hemidesmosome- involved in the establishment of cell–cell and cell–substrate contacts like cell–matrixcontact,yellow.Ecto,ectodermal cell; Endo, endodermal cell; (Fig. 3b and Supplementary Fig. 18). This indicates that the common M, mesoglea. For simplicity the nervous system has been omitted. b–e,Electron micrographs of cell–cell and cell–matrix contacts in Hydra. b,Apical septate cnidarian–bilaterian ancestor possessed a genetic inventory for the junction. c, Spot desmosome between basal muscle processes. d, Gap junction formation of all types of eumetazoan cell–cell and cell–substrate junc- 28 in the lateral cell membrane. e, Hemidesmosome-like cell–mesoglea contact tions. The presence of innexin genes in the Hydra and Nematostella site. Scale bars in b–e indicate 100 nm. f, Phylogenetic distribution of cell–cell genomes (Fig. 3b and Supplementary Fig. 19) combined with the lack of and cell–substrate contact proteins. A filled box indicates the presence of an connexin genes in non-chordate genomes clearly support the view that orthologue from the corresponding protein family as identified by SMART/ innexin-based gap junctions are an ancestral eumetazoan feature, and Pfam analysis or conserved cysteine patterns. See Supplementary Information that gap junctions formed by connexins arose later in animal evolution. section 17 and Supplementary Table 21 for details. Similarly, the lack of occludin genes in cnidarians and other non- chordates (Fig. 4) indicates that occludins and their function in tight bilaterians. For example, Hydra and Nematostella have classic cadherins junction formation first arose in the deuterostome lineage. exhibiting a highly conserved, bilaterian-type cytoplasmic (CCD) Although some gene families associated with cell–cell and cell– domain (Fig.3band SupplementaryFig.18)thatisabletointeractwith substrate interactions are also found in placozoans, demosponges b-and p120/d-catenin (Supplementary Information section 17). So far, and choanoflagellates, it is important to note that there are cell- only one sponge cadherin gene that encodes a cytoplasmic domain with adhesion-associated protein domains specific to cnidarians and weak similarity to the eumetazoan CCD domain has been detected. ©2010 Macmillan Publishers Limited. All rights reserved Monosiga Amphimedon Trichoplax Nematostella Hydra Drosophila Human LETTERS NATURE |Vol 464 | 25 March 2010 20. Broun, M., Gee, L., Reinhardt, B. & Bode, H. R. Formation of the head organizer in The sequencing of the Hydra genome has revealed unexpected rela- hydra involves the canonical Wnt pathway. Development 132, 2907–2916 (2005). tionships between the genetic makeup of the animal and its biology. 21. Browne, E. The production of new hydranths in Hydra by the insertion of small The genes encoding the proteins that form epithelial junctions in grafts. J. Exp. Zool. 7, 1–23 (1909). bilaterians are present in Hydra yet there are obvious differences in 22. Bridge, D. M., Stover, N. A. & Steele, R. E. Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in structures of the junctional complexes. Despite the morphological patterning at the oral and aboral ends of hydra. Dev. Biol. 220, 253–262 (2000). similarity of neuromuscular junctions in bilaterians and Hydra,several 23. Reidling, J. C., Miller, M. A. & Steele, R. E. Sweet Tooth, a novel receptor protein- of the key genes required to make this junction in bilaterians are absent tyrosine kinase with C-type lectin-like extracellular domains. J. Biol. Chem. 275, from Hydra. Hydra has a complete set of muscle genes but lacks meso- 10323–10330 (2000). 24. Feng, B., Ng, J. H., Heng, J. C. & Ng, H. H. Molecules that promote or enhance derm and forms muscles only in epithelial cells. Most of the genes reprogramming of somatic cells to induced pluripotent stem cells. Cell Stem Cell 4, required for stem cell pluripotency in mammals are absent from 301–312 (2009). Hydra,yet Hydra has a multipotent stem cell system that functions 25. Jager, M., Queinnec, E., Houliston, E. & Manuel, M. Expansion of the SOX gene family similarly to stem cell systems in bilaterians. The availability of the predated the emergence of the Bilateria. Mol. Phylogenet. Evol. 39, 468–477 (2006). provide an 26. Westfall, J. A., Yamataka, S. & Enos, P. D. Ultrastructural evidence of polarized Hydra genome sequence and methods to manipulate it synapses in the nerve net of Hydra. J. Cell Biol. 51, 318–323 (1971). opportunity to understand how this remarkable animal evolved. 27. Takahashi, T. et al. Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families. Proc. Natl Acad. Sci. USA 94, METHODS SUMMARY 1241–1246 (1997). 28. Alexopoulos, H. et al. Evolution of gap junctions: the missing link? Curr. Biol. 14, The genome of Hydra magnipapillata strain 105 was sequenced at the J. Craig Venter R879–R880 (2004). Institute using the whole genome shotgun approach. Two different assemblies were 29. Yen, M. R. & Saier, M. H. Jr. Gap junctional proteins of animals: the innexin/ generated and deposited in GenBank (accession numbers ABRM00000000 and pannexin superfamily. Prog. Biophys. Mol. Biol. 94, 5–14 (2007). ACZU00000000). Complementary DNA libraries were prepared using standard 30. Wittlieb, J., Khalturin, K., Lohmann, J. U., Anton-Erxleben, F. & Bosch, T. C. G. methods and ESTs were generated at the National Institute of Genetics (Mishima, Transgenic Hydra allow in vivo tracking of individual stem cells during Japan) and the Genome Sequencing Center (Washington University, St Louis). ESTs morphogenesis. Proc. Natl Acad. Sci. USA 103, 6208–6211 (2006). have been deposited in the dbEST database at the National Center for Biotechnology Supplementary Information is linked to the online version of the paper at Information. The Curvibacter sp. genome sequence has been deposited in GenBank www.nature.com/nature. (accession numbers FN543101, FN543102, FN543103, FN543104, FN543105, FN543106, FN543107 and FN543108). Acknowledgements We aregratefultoS.Clifton,R.Wilsonand theEST sequencing group at the Genome Sequencing Center at the Washington University School of Received 9 April 2009; accepted 11 January 2010. Medicine for their efforts in generating the Hydra ESTs and to the National Science Published online 14 March 2010. Foundation for its support of the Hydra EST project (grant number IBN-0120591). Funding for the sequencing of the Hydra genome was provided by the National 1. van Leeuwenhoek, A. in The Collected Letters of Antoni van Leeuwenhoek Vol. XIV Human Genome Research Institute. We thank J. Gerhart who, as co-chair of the (ed. Palm, L. C.) 169–173 (Swets and Zeitlinger, 1996). National Human Genome Research Institute Working Group on Comparative 2. Trembley, A. Me´moires, pour servir a ` l’historie d’un genre de polypes d’eau douce, a` Genome Evolution, advocated sequencing of the Hydra genome. The septate junction bras en forme de cornes (Jean and Herman Verbeek, 1744). electron micrograph in Fig. 3 was provided by G. Rieger.K.J.P.thanks N.Margulisfor 3. Bode, P. M. & Bode, H. R. in Pattern Formation, A Primer in Developmental Biology technical assistance and the NSF for support (grant number DEB-0716960). U.T. was (eds Malacinski, G. M. & Bryant, S. V.) 213–241 (Macmillan, 1984). supported by the Austrian Science Fund and the Norwegian Research Council. B.H. 4. David, C. N. & Murphy, S. Characterization of interstitial stem cells in Hydra by was supported by Austrian Science Fund grants FWF P16685 and FWF P20734. cloning. Dev. Biol. 58, 372–383 (1977). D.S.R. was supported by R. Melmon and the Gordon and Betty Moore Foundation. 5. Holstein, T. W., Hobmayer, E. & Technau, U. Cnidarians: an evolutionarily Work at the US Department of Energy Joint Genome Institute was supported by the conserved model system for regeneration? Dev. Dyn. 226, 257–267 (2003). Office of Science of the US Department of Energy under Contract No. 6. Putnam, N. H. et al. Sea anemone genome reveals ancestral eumetazoan gene DE-AC02-05CH11231. T.F. and T.G. were supported by Grants-In-Aid for Scientific repertoire and genomic organization. Science 317, 86–94 (2007). Research from the Ministry of Education, Culture, Sports, Science, and Technology of 7. Lynch, M. The evolution of genetic networks by non-adaptive processes. Nature Japan. T.C.G.B. was supported by grants from the Deutsche Forschungsgemeinschaft Genet. 8, 803–813 (2007). (DFG SFB617-A1) and from the DFG Cluster of Excellence programs ‘The Future 8. Zacharias, H., Anokhin, B., Khalturin, K. & Bosch, T. C. G. Genome sizes and Ocean’ and ‘Inflammation at Interfaces’. T.W.H. was supported by grants from the chromosomes in the basal metazoan Hydra. Zoology 107, 219–227 (2004). Deutsche Forschungsgemeinschaft including SFB488-A12 and the DFG Cluster of 9. Douris, V., Telford, M. J. & Averof, M. Evidence for multiple independent origins of Excellence program ‘CellNetworks’. Support for H.W. was provided by the TOYOBO trans-splicing in Metazoa. Mol. Biol. Evol. doi:10.1093/molbev/msp286 (25 Biotechnology Foundation and the Alexander von Humboldt Foundation. Support for November 2009). A.B., B.B., C.G., C.N.D., H.W., P.G.B., S.O., T.F. (Mercator Professor) and Y.N. was 10. Fraune, S. & Bosch, T. C. Long-term maintenance of species-specific bacterial microbiota provided by the Deutsche Forschungsgemeinschaft. in the basal metazoan Hydra. Proc. Natl Acad. Sci. USA 104, 13146–13151 (2007). Author Contributions J.A.C., E.F.K., O.S., H.R.B., C.N.D., D.S.R. and R.E.S. directed 11. Ding,L.& Yokota, A.Proposals of Curvibacter gracilis gen. nov., sp. nov. and the project and wrote the manuscript. E.F.K., K.A.R., R.L.S. and J.C.V. directed Herbaspirillum putei sp. nov. for bacterial strains isolated from well water and genome sequencing and assembly at JCVI. J.B., D.B., K.D., C.P., N.S., G.G.S., L.D.V. reclassification of [Pseudomonas] huttiensis,[Pseudomonas] lanceolata,[Aquaspirillum] and B.W. were responsible for library construction, sequence production and delicatum and [Aquaspirillum] autotrophicum as Herbaspirillum huttiense comb. nov., genome assembly at JCVI. D.S.R., J.A.C., O.S., T.M., D.M.G., U.H., T.K., S.E.P., S.S. Curvibacter lanceolatus comb. nov., Curvibacter delicatus comb. nov. and Herbaspirillum and N.H.P. carried out genome assembly and gene annotation at UC Berkeley. autotrophicum comb. nov. Int. J. Syst. Evol. Microbiol. 54, 2223–2230 (2004). Construction of cDNA libraries and analysis of ESTs was carried out by H.R.B., 12. Technau, U. et al. Maintenance of ancestral complexity and non-metazoan genes R.E.S., D.F.K., S.E.H., L.G., D.L., L.L., J.P., B.B., P.A.W., T.F., C.N.-F., T.G., J.S.H., E.H., in two basal cnidarians. Trends Genet. 21, 633–639 (2005). S.H., M.H., K.I., A.O., T.T., T.C.G.B., K.K., G.H., A.F., R.A., S.F., T.W.H., C.G., P.G.B., 13. Pace, J. K. II, Gilbert, C., Clark, M. S. & Feschotte, C. Repeated horizontal transfer of B.B., Y.N., S.O., H.W. and D.E.M. T.W., T.R., P.G.B., C.E.D., P.T. and C.N.D. carried a DNA transposon in mammals and other tetrapods. Proc. Natl Acad. Sci. USA 105, out analysis of the Curvibacter genome and HGT candidate genes. A.M.H., B.M.W., 17023–17028 (2008). O.S. and K.J.P. carried out the microRNA analyses. U.T., B.H., A.W., P.R.H.S., X.Z., 14. Grimson, A. et al. Early origins and evolution of microRNAs and Piwi-interacting R.A., M.-K.E., A.-K.G., W.S., T.F. and A.B. carried out analyses of genes involved in RNAs in animals. Nature 455, 1193–1197 (2008). various biological processes. J.A.C., E.F.K. and O.S. are joint first authors. 15. Butts, T., Holland, P. W. & Ferrier, D. E. The urbilaterian Super-Hox cluster. Trends Genet. 24, 259–262 (2008). Author Information Two different assemblies of the Hydra magnipapillata strain 16. Chourrout, D. et al. Minimal ProtoHox cluster inferred from bilaterian and 105 genome were generated and deposited in GenBank under accession numbers cnidarian Hox complements. Nature 442, 684–687 (2006). ABRM00000000 and ACZU00000000. The Curvibacter sp. genome sequence 17. Ryan, J. F. et al. Pre-bilaterian origins of the Hox cluster and the Hox code: has been deposited in GenBank under accession numbers FN543101, FN543102, evidence from the sea anemone, Nematostella vectensis. PLoS One 2, e153 (2007). FN543103, FN543104, FN543105, FN543106, FN543107 and FN543108. Reprints 18. Mokady, O., Dick, M. H., Lackschewitz, D., Schierwater, B. & Buss, L. W. Over one- and permissions information is available at www.nature.com/reprints. This paper half billion years of head conservation? Expression of an ems class gene in is distributed under the terms of the Creative Commons Attribution-Non- Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa). Proc. Natl Acad. Sci. USA 95, Commercial-Share Alike licence, and is freely available to all readers at 3673–3678 (1998). www.nature.com/nature. The authors declare no competing financial interests. 19. Hobmayer, B. et al. WNT signalling molecules act in axis formation in the Correspondence and requests for materials should be addressed to R.E.S. diploblastic metazoan Hydra. Nature 407, 186–189 (2000). ([email protected]) or D.S.R. ([email protected]). ©2010 Macmillan Publishers Limited. 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The dynamic genome of Hydra

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Copyright © 2010 by The Author(s)
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Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
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Vol 464 |25 March 2010 |doi:10.1038/nature08830 LETTERS 1 2 3,4 5 4 Jarrod A. Chapman *, Ewen F. Kirkness *, Oleg Simakov *, Steven E. Hampson {, Therese Mitros , 6 6 3 2 2 Thomas Weinmaier , Thomas Rattei , Prakash G. Balasubramanian , Jon Borman , Dana Busam , 2 2 2 2 2 Kathryn Disbennett , Cynthia Pfannkoch , Nadezhda Sumin , Granger G. Sutton , Lakshmi Devi Viswanathan , 2 1 1 4 1 Brian Walenz , David M. Goodstein , Uffe Hellsten , Takeshi Kawashima , Simon E. Prochnik , 1,4 1 7,8 8,9 7,8 5 Nicholas H. Putnam {, Shengquiang Shu , Bruce Blumberg , Catherine E. Dana , Lydia Gee , Dennis F. Kibler , 7,8 7,8 10 7,8 11 3 Lee Law , Dirk Lindgens , Daniel E. Martinez , Jisong Peng , Philip A. Wigge {, Bianca Bertulat , 3 3 3 3 12 12 Corina Guder , Yukio Nakamura , Suat Ozbek , Hiroshi Watanabe , Konstantin Khalturin , Georg Hemmrich , 12 12 12 13 13 13 ´ ´ Andre Franke , Rene Augustin , Sebastian Fraune , Eisuke Hayakawa , Shiho Hayakawa , Mamiko Hirose , 13 13 13 13 14 Jung Shan Hwang , Kazuho Ikeo , Chiemi Nishimiya-Fujisawa , Atshushi Ogura {, Toshio Takahashi , 15 16 17 17 17 Patrick R. H. Steinmetz , Xiaoming Zhang , Roland Aufschnaiter , Marie-Kristin Eder , Anne-Kathrin Gorny {, 17 18 19 18 20 Willi Salvenmoser , Alysha M. Heimberg , Benjamin M. Wheeler , Kevin J. Peterson , Angelika Bo¨ttger , 6 20 13 2 2 Patrick Tischler , Alexander Wolf , Takashi Gojobori , Karin A. Remington {, Robert L. Strausberg , 2 15 17 12 3 J. Craig Venter , Ulrich Technau , Bert Hobmayer , Thomas C. G. Bosch , Thomas W. Holstein , 13 7,8 20 1,4 8,9 Toshitaka Fujisawa , Hans R. Bode , Charles N. David , Daniel S. Rokhsar & Robert E. Steele The freshwater cnidarian Hydra was first described in 1702 and has (71% A1T), and includes ,57% transposable elements (see below). been the object of study for 300 years. Experimental studies of Hydra Although the sequenced strain reproduces clonally in the laboratory between 1736 and 1744 culminated in the discovery of asexual repro- by asexual budding, it is diploid with substantial heterozygosity duction of an animal by budding, the first description of regeneration (,0.7% single nucleotide polymorphism between alleles), which in an animal, and successful transplantation of tissue between ani- we find is distributed along the genome as expected if it were drawn mals .Today, Hydra is an important model for studies of axial pat- from a randomly mating population (Supplementary Information 3 4 5 terning , stem cell biology and regeneration .Herewereportthe section 3). These features complicate shotgun sequencing and assembly. genome of Hydra magnipapillata and compare it to the genomes of Two complementary assemblies (CA and RP) were generated (Sup- the anthozoan Nematostella vectensis and other animals. The Hydra plementary Information section 3) and deposited in GenBank. The genome has been shaped by bursts of transposable element expan- CA assembly (1.5 gigabases (Gb)) has contig and scaffold N50 values sion, horizontal gene transfer, trans-splicing, and simplification of of 12.8 kilobases (kb) and 63.4 kb, respectively. The RP assembly (1.0 gene structure and gene content that parallel simplification of the Gb) has a contig N50 length of 9.7 kb and a scaffold N50 length of Hydra life cycle. We also report the sequence of the genome of a novel 92.5 kb. The CA assembly gives an estimated non-redundant genome bacterium stably associated with H. magnipapillata. Comparisons of size of 1.05 Gb. The RP assembly gives an estimated non-redundant genome size of 0.9 Gb (see Supplementary Information section 3 for a the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated discussion of genome size calculations). For analysis, we chose the transcription factors, the Spemann–Mangold organizer, pluripotency assembly that minimized sequence redundancy owing to the separate genes and the neuromuscular junction. assembly of haplotypes (see Supplementary Information section 3 for The genomic basis of cnidarian evolution has so far been viewed further discussion). Approximately 99% of known Hydra genes are found in both assemblies, attesting to their completeness with respect from the perspective of an anthozoan, the sea anemone Nematostella vectensis . Hydra is a medusozoan that diverged from anthozoans to protein-coding genes. at least 540 millions year ago. Features of Hydra and Nematostella Although the present Hydra assembly is too fragmented for a are compared in Supplementary Table 1. We generated draft chromosome-scale analysis, we found evidence for synteny with other assemblies of the Hydra magnipapillata genome using a whole- metazoans. Of the 33 longest gene-rich Hydra scaffolds (that is, those genome shotgun approach (Supplementary Information sections containing genes from at least 10 Hydra/Nematostella orthologue 1–3 and Supplementary Figs 1–3). The Hydra genome is (A1T)-rich groups), 15 (45%) were significantly enriched (P, 0.01) for genes from 1 2 3 US Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA. The J. Craig Venter Institute, Rockville, Maryland 20850, USA. Institute of Zoology, Department of Molecular Evolution and Genomics, University of Heidelberg, D-69120 Heidelberg, Germany. Center for Integrative Genomics, Department of Molecular and Cell 5 6 Biology, University of California, Berkeley, California 94720, USA. Department of Computer Science, University of California, Irvine, California 92697-3435, USA. Department of 7 8 Genome-Oriented Bioinformatics, Technische Universita¨tMu¨nchen, D-85354 Freising, Germany. Department of Developmental and Cell Biology, Developmental Biology Center, 9 10 University of California, Irvine, California 92697-2275, USA. Department of Biological Chemistry, University of California, Irvine, California 92697-1700, USA. Department of 11 12 Biology, Pomona College, Claremont, California 91711, USA. The Salk Institute, La Jolla, California 92037, USA. Zoologisches Institu¨t, Christian-Albrechts-University, D-24098 Kiel, 13 14 15 Germany. National Institute of Genetics, Yata 1, 111, Mishima 411-8540, Japan. Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan. Department of Molecular Evolution and Development, University of Vienna, A-1090 Vienna, Austria. Department of Anatomy and Cell Biology, The University of Kansas Medical Center, Kansas City, Kansas 17 18 66160, USA. Institute of Zoology and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria. Department of Biological Sciences, Dartmouth College, 19 20 Hanover, New Hampshire 03755, USA. Department of Computer Science, North Carolina State University, Raleigh, North Carolina 27695, USA. Department of Biology II, Ludwig- Maximilians-University, D-82152 Planegg-Martinsried, Germany. {Present addresses: Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK (P.A.W.); Institute of Human Genetics, University of Heidelberg, D-69120 Heidelberg, Germany (A.-K.G.); Center for Bioinformatics and Computational Biology, National Institute of General Medical Sciences, Bethesda, Maryland 20892-6200, USA (K.A.R.); Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas 77251-1892, USA (N.H.P.); Ochadai Academic Production, Ochanomizu University, Ohtsuka, Bunkyo, 1128610 Tokyo, Japan (A.O.). *These authors contributed equally to this work. {Deceased. ©2010 Macmillan Publishers Limited. All rights reserved NATURE | Vol 464 |25 March 2010 LETTERS CR_Y a b H. viridissima (380 Mb) H. oligactis (1,450 Mb) H. vulgaris (1,300 Mb) III H. magnipapillata (1,300 Mb) 8 II Inferred ‘bursts’ of TE activity Transib 0.0 0.1 0.2 0.3 0.4 0.5 1.5 Chapaev Jukes–Cantor distance CR1 hAT Penelope 1.0 Mariner Gypsy Other III 0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 I II Jukes–Cantor distance from ReAS consensus Figure 1 | Dynamics of transposable element expansion in Hydra reveals is per cent dissimilarity on the nucleotide level from the repeat consensus. several periods of transposon activity. a, The top panel shows phylogenetic This substitution level for transposons is equivalent to Nei-Gojobori relationships between four Hydra species based on ESTs (using Nei- synonymous substitution rates in the ESTs. Three element expansions are Gojobori synonymous substitution rates; see Supplementary Fig. 8). The inferred, the most distinct are the most ancient at ,0.4 and the most recent bottom panel shows the fraction of the genome that is occupied by a specific at 0.05 divergence levels. The middle expansion at about ,0.2 is not well repeat class at a given divergence from the repeat consensus generated by the synchronized and is more clearly seen for individual element classes in ReAS (recovery of ancestral sequences) algorithm (see Supplementary Supplementary Figs 5 and 6. b, c, Example of periods of activity of a single Information section 9). Substitution levels are corrected for multiple Hydra CR1 retrotransposon family (b) and the maximum likelihood substitutions using the Jukes–Cantor formula K523/4ln(12i4/3), where i phylogeny of the family (c). specificeumetazoanlinkagegroups ,indicatingthatvestigesoftheancestral Most individual Hydra transposable element families show discrete eumetazoan genome organization persist in Hydra.Thisisincontrastto bursts of expansion (Fig. 1b, c) that are possibly associated with popu- the highly diverged genomes of Drosophila and Caenorhabditis elegans, lation bottlenecks . The correspondence between speciation times in which show no synteny with other metazoans by these methods. the genus Hydra and the timing of transposon activity may have been We estimate that the Hydra genome contains ,20,000 bona fide associated with the approximately threefold increase in genome size protein-coding genes (excluding transposable elements), based on (Fig. 1a) in H. magnipapillata, H. vulgaris and H. oligactis relative to H. expressed sequence tags (ESTs), homology and ab initio gene prediction viridissima (380 megabases (Mb)) . (Supplementary Information section 6). The amino acid substitution Addition of short RNA leader sequences to the 59 ends of messenger rate in the Hydra lineage is enhanced relative to the Nematostella RNAs by trans-splicing occurs in a subset of metazoans and unicellular lineage; the sequence divergence between a Hydra peptide and its human eukaryotes . Transcripts from at least one-third of EST-supported orthologue is typically greater than the sequence divergence between genes in Hydra undergo trans-spliced leader addition (Supplemen- Nematostella and human (Supplementary Information section 8 and tary Information section 10). Hydra has multiple spliced leader genes Supplementary Fig. 4) as expected based on the longer branch leading to (Supplementary Table 9), and a given transcript may be trans-spliced Hydra in peptide-based phylogenies . Similarly, the rate of intron loss with several different spliced leaders. Notably, trans-splicing is has been higher in the Hydra lineage; we find that 22% (126 out of 575) absent from Nematostella (Supplementary Information section 10). of the introns shared by Nematostella and human in well-aligned coding It now seems likely that trans-splicing has evolved multiple times regions have been lost in Hydra. Conversely, only 6% (28 out of 476) of independently . the introns shared by Hydra and human are absent in Nematostella. Trans-splicing occurs in Hydra viridissima (N. A. Stover and R.E.S., Transposable elements make up ,57% of the Hydra genome and unpublished data; GenBank accession number DQ092354) and in represent over 500 different families (Supplementary Information several other hydrozoans (Supplementary Table 10), and may be an section 9). The most abundant element, comprising ,15% of the ancestral feature of the class. Spliced leader addition gives a eukaryotic genome (Fig. 1 and Supplementary Table 3), is a non-long-terminal- cell the opportunity to combine genes into operons, the multi-cistronic repeat (non-LTR) retroelement of the chicken repeat 1 (CR1) family. To transcripts of which can be resolved into individual mRNAs by trans- our knowledge, elements of this family are more abundant in the Hydra splicing. We found 32 potential Hydra operons (Supplementary genome than in any other sequenced animal genome (in comparison, Information section 10, Supplementary Table 11 and Supplementary the CR1 family occupies only,1% of the Nematostella assembly and 3% Fig. 9), but no obvious evidence for functional relationships between of the chicken assembly). This retrotransposon is still active in Hydra,as genes in these operons. indicated by its representation in 105 ESTs. We also found 789 cases of Bacteria are stably associated with Hydra . Electron micrographs intronless genes that were derived recently from multi-exon genes, most reveal bacterial cells underneath the glycocalyx, the coat that overlies probably through retrotransposition. DNAtransposons (predominantly the apical surface of the ectodermal epithelial layer of Hydra (Sup- ‘cut-and-paste’ elements of the mariner, Transib and hAT (hobo- plementary Fig. 10). Our assembly yielded eight large putative bacterial Ac-Tam3) types) occupy ,20% of both the Hydra and Nematostella scaffolds as evidenced by: (1) high G1C content (in contrast to the low genomes, and are also active in Hydra based on the presence of ESTs. G1C content of the Hydra genome); (2) no high-copy repeat Timing of transposable element activity using sequence divergence sequences typical of Hydra scaffolds; and (3) closely spaced single-exon of extant copies reveals at least three periods of element expansion (at open reading frames with best hits to bacterial genes (Supplemen- ,5%, ,20% and ,40% nucleotide substitutions; Fig. 1 and Sup- tary Information section 11, Supplementary Fig. 11 and Supplemen- plementary Figs 5 and 6). In marked contrast, comparable expansions tary Table 12). These scaffolds span a total of 4 Mb encoding 3,782 are absent from the Nematostella genome (Supplementary Fig. 7). single-exon genes and represent an estimated 98% of the bacterial ©2010 Macmillan Publishers Limited. All rights reserved Percentage of the current genome Counts LETTERS NATURE |Vol 464 | 25 March 2010 (Supplementary Fig. 13). This pathway could modify endogenous glycoproteins or proteoglycans in Hydra. We also identified 90 transposable elements that were potentially hori- zontally transferred into the Hydra genome. These elements have expanded recently (less than 10% nucleotide divergence from their con- sensus) and have no older copies in the genome. The most frequent nv nv nv element class consists of hAT transposons with 34 different families, although all major classes of transposable element (DNA transposon, LTR and non-LTR elements) are represented. Transposable elements have been shown previously to be horizontally transferred in metazoans . emc emc emc We identified 51 unique non-tRNA/non-rRNA transcripts that correspond to putative non-coding RNA genes based on 454 sequencing of short transcripts from Hydra (Supplementary Information section 13 Ac-CoA + Ch Nerve and Supplementary Table 16). At least 17 of these are microRNAs cell (miRNAs), compared to 40 identified miRNAs in Nematostella . Surprisingly, only a single miRNA gene in the available data sets, miR- 2022, is common to both cnidarian species. ChT Hox and ParaHox gene families arose from a megacluster that AChE Ch included a number of other homeobox genes (for example, NK genes) . Agrin With the exception of engrailed, descendants of all of the classes of 16,17 homeobox genes in the megacluster are found in Nematostella . Muscle cell Hydra is missing a substantial fraction of megacluster descendants , Rapsyn Rapsyn indicating secondary loss. For example, the eve and emx genes are absent from Hydra, although they are present in Nematostella and several Figure 2 | The neuromuscular junction in Hydra.a, Electron micrograph of hydrozoans (Supplementary Table 17). The loss of these genes from a nerve synapsing on a Hydra epitheliomuscular cell. emc, epitheliomuscular Hydra is therefore recent in relation to the diversification of hydro- cell; nv, nerve cell. Three vesicles are located in the nerve cell at the site of contact with the epitheliomuscular cell. Scale bar, 200 nm. b, Schematic zoans. These genes are expressed in a cell-type-specific manner in larvae 17 18 diagram of a canonical neuromuscular junction. Yellow indicates presence and adults of Nematostella and Hydractinia ;itisintriguingthatthe in Hydra. Choline acetyltransferase (ChAT) is shown in red because it is not loss of these genes correlates with the absence of a larval stage in Hydra clear whether Hydra has an enzyme that prefers choline (Ch) as a substrate. (Supplementary Table 17). The absence of these genes in Hydra indi- Acetylcholine (ACh) molecules are shown as blue circles. The nicotinic cates that despite their near-universal presence in animals, it is possible acetylcholine receptor (nAChR) is shown in the open state with to construct a metazoan without either of them. In addition to the loss acetylcholine bound (left), and in the closed state in the absence of bound of emx and eve genes, Hydra has undergone several other marked gene acetylcholine (right). AChE, acetylcholinesterase; ChT, choline transporter; losses; for example, it lacks fluorescent protein genes and key circadian MuSK, muscle-specific kinase; VAChT, vesicular acetylcholine transporter. rhythm genes (Supplementary Information section 14). All major bilaterian signalling pathways, including Wnt, transform- chromosome. Phylogenetic analysis of 16S rRNA (Supplementary ing growth factor-b, Hedgehog, receptor tyrosine kinase and Notch, Fig. 12) and conserved clusters of orthologous groups of proteins are present in Hydra and Nematostella. An important signalling centre (COGs) indicate that this bacterium is a novel Curvibacter species in Hydra is the head organizer, which uses the Wnt signalling pathway belonging to the family Comamonadaceae (order Burkholderiales) . 19,20 to establish positional values along the body column . The head About 60% of annotated Curvibacter sp. genes have an orthologue in organizer, which is located at the apical tip of the adult polyp, is another species of Comamonadaceae (Supplementary Table 13). derived from the gastrula blastopore in cnidarians. A transplanted Notably, the Curvibacter sp. genome encodes nine different ABC sugar head organizer has the capacity to induce axis formation , similar transporters, compared to only one or two in other species of to the Spemann–Mangold organizer in Xenopus. Orthologues of a Comamonadaceae (Supplementary Table 14), possibly reflecting an number of genes known to act in the Spemann–Mangold organizer adaptation to life in association with Hydra. in Xenopus are present in the Hydra and Nematostella genomes. Non-metazoan genes among cnidarian ESTs have been reported Moreover, several of the secreted signalling molecules and trans- previously , and we have now found further examples of such genes cription factors encoded by these genes are expressed specifically in the Hydra genome assembly. These genes are candidates for hori- in the Hydra head organizer and the blastopore organizer in the zontal gene transfer (HGT) (Supplementary Information section 12). Nematostella gastrula (Supplementary Information section 15 and Seventy-one Hydra gene models showed closer relationships to bac- Supplementary Table 18). Thus, the Hydra head organizer and the terial genes than to metazoan genes based on sequence similarity and Xenopus Spemann–Mangold organizer may share common descent phylogenetic analysis (Supplementary Table 15). Of these, 51 have no from an organizer in the ancestor of cnidarians and bilaterians. 22,23 blast hits to other metazoans, except in a few cases to Nematostella. The extracellular portions of two Hydra receptor tyrosine kinases Potential donors of these HGT candidates are widely distributed contain a novel protein domain, sweet tooth (SWT). The SWT domain among different bacterial phyla (Supplementary Table 15) and show is also present in ESTs from the hydrozoan Clytia, but is absent from all no enrichment for close relatives of Curvibacter. Approximately 70% other sequenced genomes, including that of Nematostella (Supplemen- of the HGT candidates have EST support, and transcripts from 30% tary Fig. 15). SWT is among the most abundant protein domains of the genes have spliced leaders, indicating unambiguously that they encoded in the Hydra genome. The SWT domain is present in one are derived from Hydra and not from associated bacteria (Sup- or more copies in predicted secreted proteins. Given its presence in plementary Table 15). The HGT candidates generally have fewer receptors and secreted proteins, we deduce that the SWT domain introns than Hydra genes and nearly one-half are single-exon genes defines a large, diverse and novel set of signalling proteins. (Supplementary Fig. 14), as expected if they were relatively recently Hydra contains a pluripotent stem cell type that gives rise to germ acquired by Hydra. A number of the HGT candidates encode sugar- cells, nerve cells, nematocytes and secretory cells . Of the five genes that modifying enzymes. Three genes encode enzymes in the branch of the have been shown to induce pluripotency in differentiated somatic cells bacterial lipopolysaccharide synthesis pathway that leads to formation of mammals (Myc, Nanog, Klf4, Oct4 and Sox2) , homologues of three of the activated heptose precursor of the lipopolysaccharide inner core (Nanog, Klf4 and Oct4) are clearly not present in the Hydra genome. ©2010 Macmillan Publishers Limited. All rights reserved ChAT VAChT nAChR nAChR MuSK MuSK nAChR nAChR NATURE | Vol 464 |25 March 2010 LETTERS Hydra has four Myc homologues. There are two members of the Sox B ac b group in Hydra. The Sox B group includes Sox2, but the evolutionary relationship between vertebrate Sox2 genes and Hydra Sox B genes is not clear . We conclude that the stem cell genetic network in Hydra probably has an evolutionary origin independent from the network Ecto used in mammalian stem cells. Studies of diverse cnidarians support this scenario (see Supplementary Information section 14 for details). Hydra’s shape is formed by epitheliomuscular cells, a cell type unique to cnidarians. A survey of genes that encode muscle structural and d e regulatory proteins in Hydra and Nematostella reveals a conserved eumetazoan core actin-myosin contractile machinery shared with bilaterians (Supplementary Table 19). Both cnidarians, however, lack Endo crucial, specific regulators associated with vertebrate striated (troponin complex) or smooth muscles (caldesmon), indicating that these spe- cializations arose after the cnidarian–bilaterian split. Hydra also shows secondary simplifications relative to Nematostella, which has a greater degree of muscle-cell-type specialization, including specialized retrac- tor muscle cells. Hydra lacks several components of the dystroglycan complex (a/e-sarcoglycan and b-sarcoglycan, a/b-dystroglycan and c-syntrophin), which may lead to a less robust tethering of actin to the cell membrane than in Nematostella. Similarly, the absence of a bona fide myosin light chain kinase and phosphatase in Hydra indicates adivergenceorlossofregulationbymyosinregulatorylight chain phosphorylation. The greater degree of muscle-cell-type specialization in Nematostella is also mirrored in the higher number of myosin light Adherens junction/ Structural Classic cadherin ? desmosome Associated β-Catenin chain genes in this species. Thus, even among cnidarians, we see sub- p120/δ-catenin stantial variation in muscle-associated components superimposed on α-Catenin the eumetazoan core, with the Hydra muscular system representing a Vinculin α-Actinin secondary simplification from a more complex cnidarian ancestor. Afadin Ultrastructural studies show that nerve cells in Hydra form synapses Nonclassic cadherin on contractile epitheliomuscular cells (Fig. 2a), and that these synapses Tight junction Structural Occludin contain dense core vesicles, paramembranous densities and cleft fila- Claudin Associated ZO-1 ments similar to canonical neuromuscular junctions in bilaterians. Stardust/PALS1 Several components of the bilaterian neuromuscular junction (choline PATJ transporter, nicotinic acetylcholine receptor) are encoded in the Hydra Par3 Par6 genome (Supplementary Information section 16 and Supplementary Septate junction Structural Neurexin Table 20) and their expression is consistent with a role in neuromus- Neuroglian cular signalling (Supplementary Figs 16 and 17). Other components, Contactin Gliotactin however, are found only in a possibly primitive form (putative carni- Associated Discs large tine acetyltransferases that lack the diagnostic residues for choline Scribble selectivity), and some components are absent (the vesicular acetylcholine Coracle Gap junction Structural Innexin/Pannexin transporter; Fig. 2b). Together, these data indicate that a canonical bila- Connexin terian neuromuscular junction was probably not present in the last Hemidesmosome Structural Integrin-α common ancestor of cnidarians and bilaterians. Hydra is known to Integrin-β Associated Talin use neuropeptides for the control of behaviour , and these may be Paxillin contained in the dense-core vesicles seen at Hydra synapses. Tensin In Hydra and Nematostella, epitheliomuscular cells have an apical Integrin-linked kinase Focal adhesion kinase junctional belt in the form of a septate junction, clear apical–basal polar- ity, and hemidesmosome-like contact sites with the extracellular matrix Figure 3 | Hydra cell junctions. a, Schematic diagram of the positions of (mesoglea) on their basal surface (Fig. 3a). The Hydra and Nematostella cell–cell and cell–matrix contacts in Hydra epitheliomuscular cells. Septate genomes encode almost all of the proteins known from bilaterians to be junction, red; gap junctions, green; spot desmosomes, blue; hemidesmosome- involved in the establishment of cell–cell and cell–substrate contacts like cell–matrixcontact,yellow.Ecto,ectodermal cell; Endo, endodermal cell; (Fig. 3b and Supplementary Fig. 18). This indicates that the common M, mesoglea. For simplicity the nervous system has been omitted. b–e,Electron micrographs of cell–cell and cell–matrix contacts in Hydra. b,Apical septate cnidarian–bilaterian ancestor possessed a genetic inventory for the junction. c, Spot desmosome between basal muscle processes. d, Gap junction formation of all types of eumetazoan cell–cell and cell–substrate junc- 28 in the lateral cell membrane. e, Hemidesmosome-like cell–mesoglea contact tions. The presence of innexin genes in the Hydra and Nematostella site. Scale bars in b–e indicate 100 nm. f, Phylogenetic distribution of cell–cell genomes (Fig. 3b and Supplementary Fig. 19) combined with the lack of and cell–substrate contact proteins. A filled box indicates the presence of an connexin genes in non-chordate genomes clearly support the view that orthologue from the corresponding protein family as identified by SMART/ innexin-based gap junctions are an ancestral eumetazoan feature, and Pfam analysis or conserved cysteine patterns. See Supplementary Information that gap junctions formed by connexins arose later in animal evolution. section 17 and Supplementary Table 21 for details. Similarly, the lack of occludin genes in cnidarians and other non- chordates (Fig. 4) indicates that occludins and their function in tight bilaterians. For example, Hydra and Nematostella have classic cadherins junction formation first arose in the deuterostome lineage. exhibiting a highly conserved, bilaterian-type cytoplasmic (CCD) Although some gene families associated with cell–cell and cell– domain (Fig.3band SupplementaryFig.18)thatisabletointeractwith substrate interactions are also found in placozoans, demosponges b-and p120/d-catenin (Supplementary Information section 17). So far, and choanoflagellates, it is important to note that there are cell- only one sponge cadherin gene that encodes a cytoplasmic domain with adhesion-associated protein domains specific to cnidarians and weak similarity to the eumetazoan CCD domain has been detected. ©2010 Macmillan Publishers Limited. All rights reserved Monosiga Amphimedon Trichoplax Nematostella Hydra Drosophila Human LETTERS NATURE |Vol 464 | 25 March 2010 20. Broun, M., Gee, L., Reinhardt, B. & Bode, H. R. Formation of the head organizer in The sequencing of the Hydra genome has revealed unexpected rela- hydra involves the canonical Wnt pathway. Development 132, 2907–2916 (2005). tionships between the genetic makeup of the animal and its biology. 21. Browne, E. The production of new hydranths in Hydra by the insertion of small The genes encoding the proteins that form epithelial junctions in grafts. J. Exp. Zool. 7, 1–23 (1909). bilaterians are present in Hydra yet there are obvious differences in 22. Bridge, D. M., Stover, N. A. & Steele, R. E. Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in structures of the junctional complexes. Despite the morphological patterning at the oral and aboral ends of hydra. Dev. Biol. 220, 253–262 (2000). similarity of neuromuscular junctions in bilaterians and Hydra,several 23. Reidling, J. C., Miller, M. A. & Steele, R. E. Sweet Tooth, a novel receptor protein- of the key genes required to make this junction in bilaterians are absent tyrosine kinase with C-type lectin-like extracellular domains. J. Biol. Chem. 275, from Hydra. Hydra has a complete set of muscle genes but lacks meso- 10323–10330 (2000). 24. Feng, B., Ng, J. H., Heng, J. C. & Ng, H. H. Molecules that promote or enhance derm and forms muscles only in epithelial cells. Most of the genes reprogramming of somatic cells to induced pluripotent stem cells. Cell Stem Cell 4, required for stem cell pluripotency in mammals are absent from 301–312 (2009). Hydra,yet Hydra has a multipotent stem cell system that functions 25. Jager, M., Queinnec, E., Houliston, E. & Manuel, M. Expansion of the SOX gene family similarly to stem cell systems in bilaterians. The availability of the predated the emergence of the Bilateria. Mol. Phylogenet. Evol. 39, 468–477 (2006). provide an 26. Westfall, J. A., Yamataka, S. & Enos, P. D. Ultrastructural evidence of polarized Hydra genome sequence and methods to manipulate it synapses in the nerve net of Hydra. J. Cell Biol. 51, 318–323 (1971). opportunity to understand how this remarkable animal evolved. 27. Takahashi, T. et al. Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families. Proc. Natl Acad. Sci. USA 94, METHODS SUMMARY 1241–1246 (1997). 28. Alexopoulos, H. et al. Evolution of gap junctions: the missing link? Curr. Biol. 14, The genome of Hydra magnipapillata strain 105 was sequenced at the J. Craig Venter R879–R880 (2004). Institute using the whole genome shotgun approach. Two different assemblies were 29. Yen, M. R. & Saier, M. H. Jr. Gap junctional proteins of animals: the innexin/ generated and deposited in GenBank (accession numbers ABRM00000000 and pannexin superfamily. Prog. Biophys. Mol. Biol. 94, 5–14 (2007). ACZU00000000). Complementary DNA libraries were prepared using standard 30. Wittlieb, J., Khalturin, K., Lohmann, J. U., Anton-Erxleben, F. & Bosch, T. C. G. methods and ESTs were generated at the National Institute of Genetics (Mishima, Transgenic Hydra allow in vivo tracking of individual stem cells during Japan) and the Genome Sequencing Center (Washington University, St Louis). ESTs morphogenesis. Proc. Natl Acad. Sci. USA 103, 6208–6211 (2006). have been deposited in the dbEST database at the National Center for Biotechnology Supplementary Information is linked to the online version of the paper at Information. The Curvibacter sp. genome sequence has been deposited in GenBank www.nature.com/nature. (accession numbers FN543101, FN543102, FN543103, FN543104, FN543105, FN543106, FN543107 and FN543108). Acknowledgements We aregratefultoS.Clifton,R.Wilsonand theEST sequencing group at the Genome Sequencing Center at the Washington University School of Received 9 April 2009; accepted 11 January 2010. Medicine for their efforts in generating the Hydra ESTs and to the National Science Published online 14 March 2010. Foundation for its support of the Hydra EST project (grant number IBN-0120591). Funding for the sequencing of the Hydra genome was provided by the National 1. van Leeuwenhoek, A. in The Collected Letters of Antoni van Leeuwenhoek Vol. XIV Human Genome Research Institute. We thank J. Gerhart who, as co-chair of the (ed. Palm, L. C.) 169–173 (Swets and Zeitlinger, 1996). National Human Genome Research Institute Working Group on Comparative 2. Trembley, A. Me´moires, pour servir a ` l’historie d’un genre de polypes d’eau douce, a` Genome Evolution, advocated sequencing of the Hydra genome. The septate junction bras en forme de cornes (Jean and Herman Verbeek, 1744). electron micrograph in Fig. 3 was provided by G. Rieger.K.J.P.thanks N.Margulisfor 3. Bode, P. M. & Bode, H. R. in Pattern Formation, A Primer in Developmental Biology technical assistance and the NSF for support (grant number DEB-0716960). U.T. was (eds Malacinski, G. M. & Bryant, S. V.) 213–241 (Macmillan, 1984). supported by the Austrian Science Fund and the Norwegian Research Council. B.H. 4. David, C. N. & Murphy, S. Characterization of interstitial stem cells in Hydra by was supported by Austrian Science Fund grants FWF P16685 and FWF P20734. cloning. Dev. Biol. 58, 372–383 (1977). D.S.R. was supported by R. Melmon and the Gordon and Betty Moore Foundation. 5. Holstein, T. W., Hobmayer, E. & Technau, U. Cnidarians: an evolutionarily Work at the US Department of Energy Joint Genome Institute was supported by the conserved model system for regeneration? Dev. Dyn. 226, 257–267 (2003). Office of Science of the US Department of Energy under Contract No. 6. Putnam, N. H. et al. Sea anemone genome reveals ancestral eumetazoan gene DE-AC02-05CH11231. T.F. and T.G. were supported by Grants-In-Aid for Scientific repertoire and genomic organization. Science 317, 86–94 (2007). Research from the Ministry of Education, Culture, Sports, Science, and Technology of 7. Lynch, M. The evolution of genetic networks by non-adaptive processes. Nature Japan. T.C.G.B. was supported by grants from the Deutsche Forschungsgemeinschaft Genet. 8, 803–813 (2007). (DFG SFB617-A1) and from the DFG Cluster of Excellence programs ‘The Future 8. Zacharias, H., Anokhin, B., Khalturin, K. & Bosch, T. C. G. Genome sizes and Ocean’ and ‘Inflammation at Interfaces’. T.W.H. was supported by grants from the chromosomes in the basal metazoan Hydra. Zoology 107, 219–227 (2004). Deutsche Forschungsgemeinschaft including SFB488-A12 and the DFG Cluster of 9. Douris, V., Telford, M. J. & Averof, M. Evidence for multiple independent origins of Excellence program ‘CellNetworks’. Support for H.W. was provided by the TOYOBO trans-splicing in Metazoa. Mol. Biol. Evol. doi:10.1093/molbev/msp286 (25 Biotechnology Foundation and the Alexander von Humboldt Foundation. Support for November 2009). A.B., B.B., C.G., C.N.D., H.W., P.G.B., S.O., T.F. (Mercator Professor) and Y.N. was 10. Fraune, S. & Bosch, T. C. Long-term maintenance of species-specific bacterial microbiota provided by the Deutsche Forschungsgemeinschaft. in the basal metazoan Hydra. Proc. Natl Acad. Sci. USA 104, 13146–13151 (2007). Author Contributions J.A.C., E.F.K., O.S., H.R.B., C.N.D., D.S.R. and R.E.S. directed 11. Ding,L.& Yokota, A.Proposals of Curvibacter gracilis gen. nov., sp. nov. and the project and wrote the manuscript. E.F.K., K.A.R., R.L.S. and J.C.V. directed Herbaspirillum putei sp. nov. for bacterial strains isolated from well water and genome sequencing and assembly at JCVI. J.B., D.B., K.D., C.P., N.S., G.G.S., L.D.V. reclassification of [Pseudomonas] huttiensis,[Pseudomonas] lanceolata,[Aquaspirillum] and B.W. were responsible for library construction, sequence production and delicatum and [Aquaspirillum] autotrophicum as Herbaspirillum huttiense comb. nov., genome assembly at JCVI. D.S.R., J.A.C., O.S., T.M., D.M.G., U.H., T.K., S.E.P., S.S. Curvibacter lanceolatus comb. nov., Curvibacter delicatus comb. nov. and Herbaspirillum and N.H.P. carried out genome assembly and gene annotation at UC Berkeley. autotrophicum comb. nov. Int. J. Syst. Evol. Microbiol. 54, 2223–2230 (2004). Construction of cDNA libraries and analysis of ESTs was carried out by H.R.B., 12. Technau, U. et al. Maintenance of ancestral complexity and non-metazoan genes R.E.S., D.F.K., S.E.H., L.G., D.L., L.L., J.P., B.B., P.A.W., T.F., C.N.-F., T.G., J.S.H., E.H., in two basal cnidarians. Trends Genet. 21, 633–639 (2005). S.H., M.H., K.I., A.O., T.T., T.C.G.B., K.K., G.H., A.F., R.A., S.F., T.W.H., C.G., P.G.B., 13. Pace, J. K. II, Gilbert, C., Clark, M. S. & Feschotte, C. Repeated horizontal transfer of B.B., Y.N., S.O., H.W. and D.E.M. T.W., T.R., P.G.B., C.E.D., P.T. and C.N.D. carried a DNA transposon in mammals and other tetrapods. Proc. Natl Acad. Sci. USA 105, out analysis of the Curvibacter genome and HGT candidate genes. A.M.H., B.M.W., 17023–17028 (2008). O.S. and K.J.P. carried out the microRNA analyses. U.T., B.H., A.W., P.R.H.S., X.Z., 14. Grimson, A. et al. Early origins and evolution of microRNAs and Piwi-interacting R.A., M.-K.E., A.-K.G., W.S., T.F. and A.B. carried out analyses of genes involved in RNAs in animals. Nature 455, 1193–1197 (2008). various biological processes. J.A.C., E.F.K. and O.S. are joint first authors. 15. Butts, T., Holland, P. W. & Ferrier, D. E. The urbilaterian Super-Hox cluster. Trends Genet. 24, 259–262 (2008). Author Information Two different assemblies of the Hydra magnipapillata strain 16. Chourrout, D. et al. Minimal ProtoHox cluster inferred from bilaterian and 105 genome were generated and deposited in GenBank under accession numbers cnidarian Hox complements. Nature 442, 684–687 (2006). ABRM00000000 and ACZU00000000. The Curvibacter sp. genome sequence 17. Ryan, J. F. et al. Pre-bilaterian origins of the Hox cluster and the Hox code: has been deposited in GenBank under accession numbers FN543101, FN543102, evidence from the sea anemone, Nematostella vectensis. PLoS One 2, e153 (2007). FN543103, FN543104, FN543105, FN543106, FN543107 and FN543108. Reprints 18. Mokady, O., Dick, M. H., Lackschewitz, D., Schierwater, B. & Buss, L. W. Over one- and permissions information is available at www.nature.com/reprints. This paper half billion years of head conservation? Expression of an ems class gene in is distributed under the terms of the Creative Commons Attribution-Non- Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa). Proc. Natl Acad. Sci. USA 95, Commercial-Share Alike licence, and is freely available to all readers at 3673–3678 (1998). www.nature.com/nature. The authors declare no competing financial interests. 19. Hobmayer, B. et al. WNT signalling molecules act in axis formation in the Correspondence and requests for materials should be addressed to R.E.S. diploblastic metazoan Hydra. Nature 407, 186–189 (2000). ([email protected]) or D.S.R. ([email protected]). ©2010 Macmillan Publishers Limited. All rights reserved

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Published: Mar 14, 2010

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