The Ecoresponsive Genomeof Daphnia pulexJohn K. Colbourne,1* Michael E. Pfrender,2† Donald Gilbert,1,3 W. Kelley Thomas,4

Abraham Tucker,3,4 Todd H. Oakley,5 Shinichi Tokishita,6 Andrea Aerts,7 Georg J. Arnold,8

Malay Kumar Basu,9‡ Darren J. Bauer,4 Carla E. Cáceres,10 Liran Carmel,9§ Claudio Casola,3

Jeong-Hyeon Choi,1 John C. Detter,7 Qunfeng Dong,1∥ Serge Dusheyko,7 Brian D. Eads,1,3

Thomas Fröhlich,8 Kerry A. Geiler-Samerotte,5¶ Daniel Gerlach,11# Phil Hatcher,4

Sanjuro Jogdeo,4** Jeroen Krijgsveld,12†† Evgenia V. Kriventseva,11 Dietmar Kültz,13

Christian Laforsch,14 Erika Lindquist,7 Jacqueline Lopez,1 J. Robert Manak,15‡‡ Jean Muller,16§§Jasmyn Pangilinan,7 Rupali P. Patwardhan,1∥∥ Samuel Pitluck,7 Ellen J. Pritham,17

Andreas Rechtsteiner,1¶¶ Mina Rho,18 Igor B. Rogozin,9 Onur Sakarya,5## Asaf Salamov,7

Sarah Schaack,3,17 Harris Shapiro,7 Yasuhiro Shiga,6 Courtney Skalitzky,15 Zachary Smith,1

Alexander Souvorov,9 Way Sung,4 Zuojian Tang,1*** Dai Tsuchiya,1 Hank Tu,7##Harmjan Vos,12††† Mei Wang,7 Yuri I. Wolf,9 Hideo Yamagata,6 Takuji Yamada,16 Yuzhen Ye,18

Joseph R. Shaw,1,19 Justen Andrews,1,3 Teresa J. Crease,20 Haixu Tang,1,18 Susan M. Lucas,7

Hugh M. Robertson,21 Peer Bork,16 Eugene V. Koonin,9 Evgeny M. Zdobnov,11,22 Igor V. Grigoriev,7

Michael Lynch,3 Jeffrey L. Boore7,23,24

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabasesand contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of geneduplication resulting in tandem gene clusters. More than a third of Daphnia’s genes have no detectablehomologs in any other available proteome, and the most amplified gene families are specific to theDaphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests thatthe maintenance of duplicated genes is not random, and the analysis of gene expression under differentenvironmental conditions reveals that numerous paralogs acquire divergent expression patterns soon afterduplication. Daphnia-specific genes, including many additional loci within sequenced regions that areotherwise devoid of annotations, are the most responsive genes to ecological challenges.

Daphnia pulex, or the water flea, is akeystone species of freshwater ecosys-tems: a principal grazer of algae, a pri-

mary forage for fish (1), and a sentinel of lentic(still water) inland ecosystems. Their populationsare defined by the boundaries of ponds and lakes,are sensitive to modern toxicants in the environ-ment, and thus are used to assess the ecologicalimpact of environmental change (2, 3). Daphniaexhibit a range of context-dependent develop-ment of specialized phenotypes, such as switchingbetween clonal and sexual reproduction in responseto environmental conditions (4). They are pheno-typically plastic, in that some species alter diurnalmigration behavior and develop exaggerated mor-phological defenses in response to predators (5).Physiological responses to abiotic environmentalfluctuations can include the rapid rise of hemoglo-bin levels when ambient oxygen levels fall (6).The genus Daphnia is speciose, with multiplelineages independently colonizing and adaptingto diverse habitats (7). Their short generation time,large brood sizes, and ease of laboratory and fieldmanipulation have assured their importance forsetting regulatory standards by environmental pro-tection agencies, for testing chemical safety, formonitoring water quality (2, 3), and as a model forecological and evolutionary research (8).

Daphnia pulex is a crustacean arthropod, thegroupmost closely allied with the insects (9), and

thus allows the cataloging of genes that likelyevolved in the pancrustacean ancestor of at leastsome lineages of insects and Crustacea (fig. S1).Although the branchiopod D. pulex representsonly a single crustacean lineage—which containsmore than 40,000 known species with strikinglevels of phenotypic diversity—the genus and itsorder (the Cladocera) date to the Permian (10).

Because Daphnia’s ecology is superbly un-derstood, access to its genome sequence (fig. S2 andtable S1) allows studying environmental influenceson gene functions in ways that are difficult in eventhe best-developed genomic model species. Traitsobserved in laboratories are likely a small subset ofthe phenotypic variation that is expressed in naturalecosystems, and a focus on laboratory studies maypartly explain why over 50% of many eukaryoticgenomes are without experimentally determinedfunctional annotations (11).

Genome sequence, assembly, and mappingto chromosomes. The D. pulex genome was as-sembled using JAZZ (12) from 1,554,564 quality-filtered nuclear sequence reads (8.7-fold coverage)from a naturally inbred isoclonal daphniid dubbed“The Chosen One” (TCO) [supporting online ma-terial (SOM) I.1]. The version 1.1 draft genomeassembly comprises 19,008 contigs arrangedwithin5191 scaffolds that sum to a genome size of ~200Mb (table S2). Two-hundred-eighty scaffolds linkto construct 118 super-scaffolds (tables S3 and

S4). Microsatellite markers (13) place 73 largescaffolds (73.9Mb total) on the 12 chromosomes(table S5). We estimate that the draft assembly ishigh quality and includes ~80% of Daphnia’snuclear genome (SOM I.2, tables S6 and S7, andfigs. S3 to S5). We determine that 3598 missingregions (59%) contain duplicated genes, whereasothers are heterochromatic regions, includingthe centromeres and telomeres. We estimate that25% of the genome may be heterochromatic(table S8 and fig. S6). The ends of D. pulex chro-mosomes appear to consist of long stretches ofTTAGG repeats with flanking regions (30 to 40Kb) internal to these repeats consisting of repet-itive sequences, including at least two kinds ofsatellite sequences (SOM 1).

Gene inventory. A minimum set of 30,907protein-encoding genes was predicted forD. pulex,with 26,867 gene models having the followingsupport (tables S9 to S14 and fig. S7): (i) 145,578expressed sequence tags (ESTs) from 37 separateconditions validating 10,578 genes; (ii) whole-genome tiling microarrays examining gene ex-pression under six different conditions that detect186,269 transcriptionally active regions (TARs)validating 57,294 exons from 14,135 genes (ad-ditional TARs suggest gene models not yetincluded within the minimum set); (iii) similarityto proteins from other (non-daphniid) genomesthat detect 19,641D. pulex genes (blast e < 10−5);(iv) 18,765 genes identified in protein similaritysearches against a preliminary draft genome se-quence forD. magna (SOM 2), which belongs toa separate subgenus (7); (v) more than 11,000D. pulex peptide sequences detected by tandemmass spectrometry, of which 93% map to 1273gene models in the minimum set; (vi) 716 highlyconserved single-copy eukaryotic genes, of whichD. pulex ismissing only two (table S15), confirmingthat expected genes are included in the assembly;and (vii) 13,105 loci identified as paralogs bynucleotide sequence similarity searches for eachpredicted gene against the complete gene list (e <10−20). Measures of the relative rate of nonsyn-onymous nucleotide substitutions to the substitu-tion rate at synonymous sites (Ka/Ks) indicate thatthe paralogs within our gene set generally showevidence of purifying selection (fig. S8).

To ensure that the gene count was not inflatedby the erroneous assembly of alleles of the samelocus as unique gene copies, we conducted com-parative genomic hybridizations of labeled TCODNA on microarrays. We detected no correlationbetween the read coverage and the mean fluo-rescing units of probes representing genes (fig.S9). Counts can also be inflated by inclusion ofpseudogenes. However, manual annotations sug-gest that pseudogenes account for only 4 to 6% oflarge paralogous family members in Daphnia[see companion studies (14)].

Many non–protein-encoding genes were alsoidentified in theD. pulex genome (SOM 3). FiftymicroRNA genes are annotated, and 27 are val-idated using tiling microarrays (table S16 and fig.

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S10). We estimate 468 ribosomal RNA loci andfind 3798 transfer RNA genes. As in Drosophilamelanogaster and Caenorhabditis elegans, theseloci are clustered (fig. S11). Transposable elementsconstitute 9.4% of the assembled genome (tableS17), consisting of 275 families of retrotransposons(Class I) and DNA transposons (Class II) (tableS18). Intra-element pair-wise divergence amongtermini for intact elements of long terminal re-peat retrotransposons ranges from 0 to 25.3%among the three superfamilies, BEL, gypsy, andcopia (averaging 2%), indicatingmany recent trans-positions (fig. S12).

Attributes of a compact genome. Compar-ison with gene-structure statistics for insects, nem-atode, and mouse, reveals reduced intron size inDaphnia (table S19 and fig. S13), resulting in amean gene span of ~1000 base pairs (bp) shorterthan the mean Drosophila gene length. However,average protein length is similar in these two spe-cies. Aside from introns, most other structures ofthe D. pulex genome are approximately equal insize or in number to those of the nematode, orexceed measurements in other species. The re-duced intergenic regions compared with insectsmay partly be attributed to smaller repeated ele-ments (table S19).

The average length of EST-validatedD. pulexintrons is 170 bp; only 10% of introns are largerthan 210 bp. The intron density ofDaphnia pulexgenes is similar to that of Apis mellifera, havingmore than twice as many introns per gene asDrosophila. About 50% of introns are sharedamong respective orthologs in Daphnia andApis (tables S19 to S23 and fig. S14). TheDaphnialineage shows an estimated intron gain/loss ratiosubstantially greater than 1 (table S24 and fig.

S15). We estimate that 78% of these intron gainsare unique to this lineage and that 22% occurredin parallel with gains in other lineages (fig. S16).

Origin and preservation of Daphnia pulexgenes. Daphnia’s gene catalog shows more uni-versal bilaterian genes than other arthropods(8096; black in Fig. 1A) and thus shares thehighest number of genes with human (table S25).Only 1383 genes (4.5%) appear pancrustacean(green in Fig. 1A). Remarkably, over 36% of theminimal set ofD. pulex genes have no detectablehomology to those in the other species (Fig. 1A),which can partly be explained by the dispropor-tionate expansion of gene families distinctive tothis crustacean lineage (c2 = 450.55, P < 0.0001)(table S26 and Fig. 1B) and fast divergence forsome genes (enlarged beige fraction in Fig. 1A).A phylogenetic accounting of the expansions andcontractions of all gene families within pancrus-tacean and representative deuterostome genomes(tables S27 and S28) suggests a net increase inthe number of paralogs within the lineage leadingto Daphnia (Fig. 1C). By reconstructing genefamily histories across a phylogeny (SOM IV.2),we count 17,424 new and 1079 lost genes in thebranch leading to Daphnia. By contrast, the sumof inferred gains and loss along the longest seriesof branches in the insect phylogeny—originatingfrom the shared pancrustacean ancestor withDaphnia—only reaches 8981 gained loci with3040 gene losses. Therefore, the overall elevatedDaphnia gene count appears to result from bothgaining and retaining more genes.

To better understand gene duplication in theDaphnia genome, we examined the age distribu-tion of gene duplicates by estimating Ks for 66,502pair-wise combinations of paralogs showing >40%

sequence similarity and by comparing this distribu-tion to that of 12,570 nematode and 64,783 humangene pairs (Fig. 1D). The single-pair duplicateswithin the youngest cohort (Ks < 0.01) suggest thatD. pulex genes duplicate at a rate three times as highas those measured for fly and nematode and 30%greater than human, evenwhenwe exclude nearlyidentical gene copies that may be biased by geneconversion (table S29 and figs. S17 and S18).

In the genomes of many species, new duplicategenes are found in clusters (fig. S19) (15). TheD. pulex genome shows ~20% (table S30) of allgenes tightly arranged in clusters of 3 to 80 paralogsand with elevated numbers of tandemly duplicatedgenes at intervening intervals of 1000 to 2000 bp(fig. S20). The age distribution and positioning ofgene duplicates indicate that Daphnia has notexperienced whole-genome duplication, but thegenome is instead characterized by a high andhistorically steady rate of tandem duplication(Fig. 1D).

Nine gene families have expanded indepen-dently in Daphnia and other aquatic lineages, in-cluding vertebrates (tables S26 and S31). Theseinclude photoreactive or photoresponsive genefamilies (cryptochromes, opsins, and G proteins).TheD. pulex genome shows 46 opsins (table S32and figs. S21 and S22), of which 42 derive fromtwo rhabdomeric subfamilies, one ciliary pterop-sin subfamily, and a newly discovered lineage thatforms a sister group to rhabdomeric opsins that wehave named arthropsins (SOM 4). Arthropsinsare ancestral to the chordate melanopsin lineageand thus appear to have been retained in Daphnia,despite their loss from all other available bilateralanimal genomes. The expansion of these genefamilies suggests that adaptations to a more com-

1Center for Genomics and Bioinformatics, Indiana Univer-sity, 915 East Third Street, Bloomington, IN 47405, USA.2Department of Biology, Utah State University, 5305 OldMain Hill Road, Logan, UT 84322–5205, USA. 3Departmentof Biology, Indiana University, 1001 East Third Street,Bloomington, IN 47405, USA. 4Hubbard Center for GenomeStudies, University of New Hampshire, 35 Colovos Road,Durham, NH 03824, USA. 5Department of Ecology, Evolu-tion, and Marine Biology, University of California–SantaBarbara, Santa Barbara, CA 93106, USA. 6Laboratory ofEnvironmental and Molecular Biology, Environmental Sci-ences Division, School of Life Sciences, Tokyo University ofPharmacy and Life Sciences, 1432-1 Horinouchi, Hachiouji,Tokyo, 192-0392, Japan. 7Department of Energy Joint GenomeInstitute (JGI), 2800 Mitchell Drive, Walnut Creek, CA 94598,USA. 8Laboratory for Functional Genome Analysis (LAFUGA),Gene Center, Ludwig-Maximilians-Universität München, Ger-many. 9National Center for Biotechnology Information,National Library of Medicine, National Institutes of Health,8600 Rockville Pike, Bethesda, MD 20894, USA. 10School ofIntegrative Biology, University of Illinois, 515 Morril Hall,Urbana, IL 61801, USA. 11University of Geneva MedicalSchool and Swiss Institute of Bioinformatics, 1 rue Michel-Servet, 1211 Geneva, Switzerland. 12Biomolecular Mass Spec-trometry and Proteomics Group, Bijvoet Centre for BiomolecularResearch and Utrecht Institute for Pharmaceutical Sciences,and Netherlands Proteomics Center, Utrecht University,Sorbonnelaan 16, 3584 CA, Utrecht, Netherlands. 13Depart-ment of Animal Science, University of California, Davis,Meyer Hall, One Shields Avenue, Davis, CA 95616, USA.14Department of Biology II and GeoBio Center Munich,Ludwig-Maximilians-University Munich, Großhadernerstrasse

2, 82152 Planegg-Martinsried, Germany. 15Gene Expres-sion, Roche NimbleGen Inc., 504 South Rosa Rd, MadisonWI 53719, USA. 16Structural and Computational Biology Unit,European Molecular Biology Laboratory (EMBL) Heidelberg,Meyerhofstrasse 1, D-69117 Heidelberg, Germany. 17Depart-ment of Biology, University of Texas, Arlington, Box 19498,Arlington, TX 76019, USA. 18School of Informatics and Com-puting, Indiana University, Informatics Building, 901 EastTenth Street, Bloomington, IN 47408–3912, USA. 19Schoolof Public and Environmental Affairs, Indiana University,1315 East Tenth Street, Bloomington, IN 47405, USA. 20De-partment of Integrative Biology, University of Guelph,Guelph, Ontario N1G 2W1 Canada. 21Department of En-tomology, University of Illinois at Urbana-Champaign, 505South Goodwin Avenue, Urbana, IL 61801, USA. 22ImperialCollege London, South Kensington Campus, SW7 2AZLondon, UK. 23Genome Project Solutions, 1024 PromenadeStreet, Hercules, CA 94547, USA. 24Department of Integra-tive Biology, University of California, Berkeley, CA 94720,USA.

*To whom correspondence should be addressed. E-mail:jcolbour@indiana.edu†Present address: Department of Biological Sciences, Uni-versity of Notre Dame, 109B Galvin Life Sciences, NotreDame, IN 46556, USA.‡Present address: J. Craig Venter Institute, 9704 MedicalCenter Drive, Rockville, MD 20850, USA.§Present address: Department of Genetics, The AlexanderSilberman Institute of Life Sciences, The Hebrew University ofJerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem91904, Israel.

||Present address: Departments of Biological Sciences andComputer Science and Engineering, University of NorthTexas, Denton, TX 76203, USA.¶Present address: Department of Organismic and Evolu-tionary Biology, Harvard University, 16 Divinity Avenue,Cambridge, MA 02138, USA.#Present address: Research Institute of Molecular Pathology(IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.**Present address: Center for Genome Research and Biocomput-ing, Oregon State University, Corvallis, OR 97331, USA.††Present address: Genome Biology Unit, EMBL Heidelberg,Meyerhofstrasse 1, 69117 Heidelberg, Germany.‡‡Present address: Department of Biology and Roy J. CarverCenter for Genomics, University of Iowa, Iowa City, IA 52242, USA.§§Present address: Laboratoire de Diagnostic Génétique,CHU Strasbourg Nouvel Hôpital Civil, 1 place de l’hôpital,67000 Strasbourg, France, and IGBMC (Institut de Génétiqueet de Biologie Moléculaire et Cellulaire), CNRS/INSERM/Universitéde Strasbourg, 67404 Illkirch cedex, France.||||Present address: Department of Genome Sciences, Universityof Washington, Seattle, WA 98195, USA.¶¶Present address: Department of Molecular, Cell, and De-velopmental Biology, University of California, Santa Cruz,Santa Cruz, CA 95064, USA.##Present address: Life Technologies Corporation, Foster City,CA 94404, USA.***Present address: Center for Health Informatics and Bio-informatics, New York University Langone Medical Center,333 East 38th Street, New York, NY 10016, USA.†††Present address: Department of Molecular Cancer Re-search, University Medical Center Utrecht, Utrecht University,Universiteitsweg 100, 3584 CG Utrecht, Netherlands.

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plex light regime in aquatic environments (16, 17)can be influential in shaping the gene content ofthese organisms.

Implications of Daphnia’s genome struc-ture. Tandemly duplicated gene clusters are pre-disposed to homogenization by gene conversionand unequal crossing-over (18). If common, con-certed evolution canmaintain sequence and func-tional similarities among paralogs. We examinedcopied DNA segments among all paralogs inthe genome (SOM V.1) and observed that 47%of the genes show tracts of nonallelic gene con-version comparedwith 12 to 18%of genes in fiveDrosophila species (tables S33 to S38 and figs.S23 and S24). Thus, concerted evolution is affect-ing more than 1 Mb (8%) of all protein-codingsequences in Daphnia, especially when duplicatesare oriented on the same strand, with a similarconversion rate (converted pairs of paralogs/totalpairs of paralogs analyzed) and number of eventsper pair as Drosophila. The greater proportion ofconverted genes in D. pulex is mainly attributed

to the greater number of targets for gene conver-sion within the genome, including tandemly du-plicated gene clusters with intervening genes.Conversion events in Daphnia are less commonamong the youngest duplicates and within genefamilies containing only two paralogs.

One example of widespread gene conversionis found in the di-domain hemoglobin genes.Hemoglobin levels in the hemolymph of daph-niids can rise by more than one order of mag-nitude in response to reduced oxygen availabilityin aquatic habitats, which fluctuates in diurnaland seasonal cycles (Fig. 2A). In Daphnia, atandemly duplicated gene cluster of hemoglobin(Hb) genes contributes to the protein’s varyingcomposition (19). We sequenced and assembledthe full D. magna cluster to compare with thearrangement of eight clustered D. pulex hemo-globin genes (figs. S25 and S27). (D. pulex alsohas three nonclustered Hb genes.) Notably, thetwo species show almost identical gene arrange-ments within an interval of ~23.5 Kb (table S39)

except for the obvious absence of Hb6 from theD. magna cluster (Fig. 2B). In both species, anoncoding RNA gene interrupts the cluster be-tween Hb4 and Hb5, and hypoxia response ele-ments plus ancillary sequences are preservedwithin the regulatory regions of each gene. Thus,the duplication and subsequent divergence of he-moglobins must have occurred before the diver-gence time of D. pulex and D. magna.

However, a phylogenetic analysis of protein-coding sequences (SOM V.2) suggests that mosthemoglobin genes have duplicated independentlywithin each species (Fig. 2C). A separate phylo-genetic reconstruction using sequences from in-tergenic regions recovers a tree that is consistentwith duplication before speciation (Fig. 2D). Be-cause the support values at nodes for both treesare equally strong, we conclude that gene con-version tracts are homogenizing the proteincoding regions. The hemoglobin gene clustersin both species are homologs because of ancestralgene duplications, yet the duplication history of

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Fig. 1. The Daphnia pulex gene repertoire. (A) Comparison of genes among D.pulex, Drosophila melanogaster, Pediculus humanus, Tribolium castaneum, andStrongylocentrotus purpuratus (urchin), and Gallus gallus, Xenopus tropicalis,and Homo sapiens, showing the core bilaterian genes (black), vertebrate (blue),insect (aqua), and pancrustacean (green) specific genes, patchy or ancientorthologs present in at least one arthropod and one deuterostome genomebut lost in other lineages (pink), multiple copy homologs (yellow and beige),and species-specific genes (white). (B) Distribution of D. pulex gene familysizes comparing genes with and without detectable homology to other ge-nomes. (C) History of gene family expansions and losses among pancrustaceanplus representative deuterostome genomes with the outgroup Nematostellavectensis. Tree topology is fixed from the assumed species phylogeny and

used to map gene family histories by a combination of gene similarity andcharacter-state optimization with Dollo parsimony (SOM IV.2). Branch lengthsscaled to differences between inferred gene gains and losses. Scale bar corre-sponds to 1000 genes gained. Gene gains along each branch of the tree arescaled by the maximum value along the branch leading to D. pulex (blue);gene losses along each branch are scaled by the maximum loss along thebranch leading to Caenorhabditis elegans (yellow). (D) Frequency of pair-wisegenetic divergence at silent sites (Ks) among all gene duplicates in the D. pulex,C. elegans, and H. sapiens genomes, for genes with >100 aligned aminoacids and percent identity >40% (66,502, 12,570, and 64,783 pair-wisecomparisons for the three genomes, respectively). The vertical axis differs forD. pulex.

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genes is obfuscated by independent gene con-versions facilitated by their ordered arrangementin the genomes.

Evolutionary diversification of duplicatedgenes. Gene duplication is an important sourceof evolutionary novelty. After duplication, onecopy is commonly disabled by mutation andbecomes a pseudogene. This fate may be avoidedif selection maintains both copies through genedosage, novel function, or by subdividing the

gene’s original function into multiple components(20). We conducted microarray experiments todetermine the magnitude of functional divergenceamong paralogs, then traced (21) and tested (22)whether their patterns of gene transcription differin 1 to 12 ecologically relevant conditions as afunction of Ks (table S40 and SOM VI.1). Asexpected, many recent duplicates (Ks < 0.05)have indistinguishable gene expression patternsfor the tested conditions (47%) (Fig. 3A). Within

many gene families, divergence in expressionpatterns correlates with age (figs. S28 and S29).We found that long-wavelength opsins most sim-ilar in sequence have the same expression pat-terns (correlation > 0.9) but then diverge in theirresponse to shared conditions as they age, at anestimated rate of 0.6% per 10% synonymousnucleotide substitutions. A similar pattern is ob-served for the di-domain hemoglobins, albeit withmore rapid divergence in expression.

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Fig. 2. Evolution of Daphnia di-domain hemoglobin (Hb) genes. (A) When de-prived of oxygen, many species (here D. magna) increase hemoglobin concen-tration in the hemolymph by a factor of 15 to 20 within a single molting, coloringthe body red. (B) Organization of the Hb gene cluster in the D. magna and theD. pulex genomes. Black boxes are exons. Gray boxes represent an RNA gene.Vertical bars are hypoxia response elements (HREs), and asterisks show ancillaryelements. Conserved HREs are linked by hatches. Open boxes represent highlysimilar sequences. The lengths of intergenic regions are shown in parentheses.

Daphnia pulex genes Hb9 to Hb11 are located on separate sequence scaffolds.(C) Phylogenetic tree (SOM V.2) from nucleotide sequences of Hb genes inD. pulex (red) and inD. magna (black). Outgroup Hb cDNA sequences are fromAscaris suum and Pseudoterranova decipiens. Scale bar shows mean numberof differences (0.1) per nucleotide along each branch. Posterior probabilitynode support <100% are shown. (D) Phylogenetic tree based on nucleotidesequences of intergenic regions between the stop codons and the downstreamTATA of the neighboring gene. Posterior probabilities <100% are shown.

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In contrast to the steady expression divergenceof many duplicates, we observed an equally largefraction of recently arisen paralogs—with nearlyidentical sequences—that differ in their expressionin at least one condition (Fig. 3A). Although wecould confidently detect locus-specific expressionfor only a fraction of the youngest duplicatesrepresented on the microarray (table S40), a plotof the maximum difference in the expressionresponse of paralogs to an identical conditionsuggests that, on average, newly duplicated genesmay differ in expression by asmuch as a factor of1.9 (Fig. 3B). These may be cases in which newregulatory programs were created by the geneduplication itself through a failure to copy reg-ulatory elements or when a duplicate is integratedwithin a new genomic location (23).

Gene conversion, homogenizing nonregula-tory nucleotide sequences, can contribute to thisclass of highly differentially expressed (DE) para-logs at low sequence divergence (Ks). We testedwhether gene conversion accounts for the dif-ferences in the evolutionary rates of expressiondivergence by comparing duplicates (Ks < 2) onthe basis of their structural arrangements in thegenome (SOM VI.2). Neighboring paralogs with-in tandem gene clusters were just as likely todiverge in expression as dispersed duplicatesoutside of clusters (c2 = 0.027, P = 0.87). Glob-ally, gene conversion reduces the expression-level divergence of paralogs (c2 = 11.9, P =0.0005) (table S41), yet we detected no signif-icant impact on the observed fractions of di-vergently expressed paralogs when we removedduplicated genes with signatures of gene con-version (table S42). Although adjacent genesare often coexpressed (24), the local placementof genes within tandem gene clusters has no cleareffect on gene expression divergence in D. pulex.

We thus conclude that paralogs, even in tandem,frequently acquire divergent expression patternsat, or soon after, the time of duplication.

Functional importance of expanded genefamilies. To investigate the functional role ofparalogs and their preservation, we examinedinteracting genes with known function. A total of1908 genes representing 563 enzymes werecharted onto the global metabolic pathway forD. pulex by referencing the metabolic enzymenetworks of three insects and four vertebrates(Fig. 4) (SOM VII.1). Of these, 38 gene familieswere amplified in Pancrustacea, and 32 are ex-panded in the lineage leading to Daphnia (figs.S30 and S31 and tables S43 and S44). Half (19 of38) of the amplified genes are nonrandomlyclustered within seven distinct pathways (P <0.03 by exact binominal test and P < 0.03 bynetwork permutation analysis) (Fig. 4, A to G,and fig. S32). These data, showing coexpansionof genes within pathways, suggest that duplicatedgenes can be interdependent.

A study of the expression patterns of du-plicated genes from this metabolic network (SOMVII.2) reveals greater average similarity betweengenes from coexpanding and interacting families(sameKyoto Encyclopedia of Genes and Genomesmap ID in table S43) than between genes fromnonassociating families (t = 3.30, P = 0.0025).This pattern suggests nonindependent functionaldivergence of expanding genes within pathways(e.g., tables S45 to S48 and figs. S33 and S34).One example involves nine phylogroups of fucosyl-transferase paralogs that share 95% amino acidsimilarity (colored lines in fig. S35) and have inde-pendently diversified to express seven transcription-al profiles sharedwith interacting glycosyltransferaseparalogs. Such a pattern of codivergence suggestsa decoupling of duplication history and functional

association. To test this prediction, we estimatedthe ratio of among-group variance to total variancein differential expression (Dst) for phylogroups offucosyltransferase paralogs and for expressionprofile clusters (SOMVII.2). We detect no signifi-cant subdivision of expression patterns for fucosyl-transferase paralogs based on phylogeny (bluenodes in fig. S35; Dst = 0.0042, P = 0.89). Bycontrast, clusters based on transcriptional profiles,and including distantly related paralogs and inter-acting glycosyl transferase paralogs, show signif-icant subdivision (Dst = 0.0836, P = 0.002).

Ecoresponsive genes. The D. pulex genomecontains many duplicated genes with unknownhomology. Although this may diminish with theavailability of more crustacean genomes, theseunknown genes appear to play important roles inthe animal’s ecology. ESTs from 37 cDNA librariesrepresenting transcriptomes of daphniids exposedto biotic ecological challenges, abiotic ecologicalstressors, and different life-history stages in labora-tory environments (table S10) show that genesunique to the Daphnia lineage, and genes thatreside within tandemly duplicated gene clusters, aresignificantly over-representedwithin transcriptomesunder ecological conditions (Fig. 5A and table S49;c2 = 265.1, P = 2.66 × e−58 and c2 = 41.0, P =1.23 × e−09, respectively). Whole-genome tiling-expression microarray experiments show differ-ential expression to be twice as frequent in genomicregions devoid of gene models (intergenic) whenD. pulex are exposed to environmental challengescompared with conditions of life history (tableS50 and fig. S36).

We count 34,844 transcriptionally active re-gions (TARs) within unannotated regions of thegenome, showing predictable exon-intron inter-vals supporting additional gene models not yetincluded within the minimum set (TAR-genes)(table S12) and that are condition-dependent intheir regulation. By partitioning the differentiallyexpressed genome by experimental conditions, be-tween 72% and 85% of the transcriptome uniquelyresponded to one of the three conditions (Fig. 5B).In all, 73% of differential regulation under bioticor abiotic stressors requires additional gene mod-els or extensions.

Evolutionary perspectives. Daphnia pulexparalogs follow different evolutionary trajectoriesthat are determined, in part, by their initial tran-scriptional expression patterns. At least half ap-pear to acquire divergent expression patterns at ornear the time of origin. Interacting and coexpand-ing genes can also appear to be codiverging intheir responses to environmental conditions. Theseobservations suggest that the persistence of thisdistinctive class of functionally divergent geneduplicates is due to preservation by entrainment(PBE). Entrainment is defined as the process ofincreasing the initial probability of preserving aduplicated gene through its functional interactionwith existing or newly interacting genes sharingregulatory programs. Because biological processescan be governed by interdependent regulation ofinteracting genes, there are three likely evolutionary

A B

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0 1 2 3

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loge

) in

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Fig. 3. Functional diversification of duplicated genes from 12 microarray experiments. (A) The fraction ofduplicated genes with similar versus divergent DE patterns as a function of their pair-wise divergence atsilent sites (Ks). (B) Regression (r = 0.29) of the maximum observed difference (treatment versus control)between duplicated genes among the 12 conditions as a function of the age of duplicated genes inferredfrom Ks. Red points are significant values (P< 0.05, analysis of variance). The regression line y-axis intercept(ln 0.642 T 0.009) suggests that, on average, newly duplicated genes may differ in expression by as much asa factor of 1.9 at particular conditions, which is significantly different from zero (t = 68.7, P < 2 × e−16) andvalidated by tiling microarray data (r = 0.16; t = 75.3, P < 2 × e−16).

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outcomes for these interacting duplicated genes(Fig. 6). Geneswith expression patterns unchangedat the time of duplication may continue to sharethe condition-specific regulation of existing in-teracting genes (Fig. 6A). In this scenario, selec-tion for gene dosage may increase the probabilitythat gene duplicates are preserved (25). Alter-natively, duplicates may initially have divergentexpression patterns but have inappropriate tran-scriptional responses to environmental conditionsor lack appropriately coregulated interacting genes(Fig. 6B). Duplicates within this category are mostlikely lost. In contrast, genes with divergent ex-pression patterns at the time of duplication, yet

with regulation sufficiently similar to the expres-sion patterns of a different interacting gene, mayhave combined products that are beneficial un-der a distinct environmental condition (Fig. 6C).In this scenario, the likelihood for preservationof these new gene duplicates is increased. Thus,when genes are advantageous at the time of du-plication, their coding regions are subject to puri-fying selection from the start and are entrained toa distinct regulatory pattern dictated by condition-specific gene-gene interactions. Although thelikelihood of converging on a beneficial geneexpression profile near the time of duplication isvery small, in the case of Daphnia, PBE is facil-

itated by the high rate of gene duplication, re-sulting in co-regulated interacting genes that canpotentially define environment-specific transcrip-tomes, which may increase with the complex-ity of interactions between organisms and theirenvironments.

In conclusion, by examining genome struc-ture and the functional responses of genes toenvironmental conditions within species with trac-table ecologies, we further our understanding ofgene-environment interactions in an evolutionarycontext. Many responsive genes to ecological con-ditions have unknown function, and informationfrom laboratory model species may be insuf-

Fig. 4. Map of globalKEGG metabolic pathwayin D. pulex showing sig-nificantly expanded or con-tracted gene families inmetabolic pathways. Nodesand edges represent com-pounds and enzymes, re-spectively. Expanded genefamilies in D. pulex (red);expanded gene familiesin Pancrustacea (yellow);independently expandedgene families in D. pulexand in insects (purple);contracted gene familiesin Pancrustacea (blue); andgenes present in D. pulex(green). Amplification ofgene families encodingeach highlighted enzymeis supported by the Fisherexact test (thick edges aresupported by Bonferronicorrection), on the basisof the distribution of thenumber of genes encod-ing corresponding enzymesamong Homo sapiens,Mus musculus, Gallusgallus, Tetraodon nigro-viridis, Drosophila mela-nogaster, Apis mellifera,and Anopheles gambiae.Emphasized pathways(A to G) include at leasttwo cases of expandedinteracting enzymes. Thenonrandom coexpansionof interacting enzymes issupported by exact bino-mial test (P < 0.03) andby the node permutationtest on 1000 randomizedmetabolic networks (P <0.03).

Daphnia

Present in Daphnia

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Pancrustacea

Hexapoda & Daphnia

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Chitin

Chitin

GlcNAcUDP-N-acetylglucosamine

Chitinase & chitin synthase

DOPA decarboxylase & dopamine hydroxylase

Glyoxylate

L-proline

Androgen and estrogen metabolismE

Testosterone

Testosteroneglucuronide

Androstenedione

Estrone3-sulfate

Estrone

glucuronosyltransferase : c450

d i h d l

Estrone glucuronide

D Sulfur metabolism

Sphingolipid biosynthesis

Arachidonic acid biosynthesis

PAP

PAPS

Arginine and proline metabolism

GF

ArachidonicacidPGH2

fucosyl-transferase

galactosyl-transferase

beta-1,3-acetylglucosaminyl-transferase

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ficient because of a lack of homology or experi-mentally demonstrated functions in response tothe environment. Thus, ecological genomics re-quires empirical annotations of new genome se-quences from a broader diversity of species, testedunder a variety of natural conditions.

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H. Yamagata, J. Biol. Chem. 274, 10649 (1999).20. H. Innan, F. Kondrashov, Nat. Rev. Genet. 11, 97

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36, 577 (2004).23. V. Katju, M. Lynch, Mol. Biol. Evol. 23, 1056 (2006).24. M. J. Lercher, T. Blumenthal, L. D. Hurst, Genome Res.

13, 238 (2003).25. F. A. Kondrashov, I. B. Rogozin, Y. I. Wolf, E. V. Koonin,

Genome Biol. 3, research0008 (2002).26. We thank M. Frazer (JGI), P. Cherbas (CGB), R. Green,

and T. Takova (Roche NimbleGen, Inc.). The workconducted by the U.S. Department of Energy JointGenome Institute (JGI) was supported by the Office ofScience of the U.S. Department of Energy undercontract DE-AC02-05CH11231 and in collaborationwith the Daphnia Genomics Consortium (DGC). Thisproject was also supported by NSF grants 0221837and 0328516 and NIH grant R24GM07827401.Coordination infrastructure for the DGC is provided bythe Center for Genomics and Bioinformatics (CGB) atIndiana University, which is supported in part by theMETACyt Initiative of Indiana University, funded in partthrough a major grant from the Lilly Endowment, Inc.Additional contributions and acknowledgments areprovided in the SOM. Our work benefits from andcontributes to the Daphnia Genomics Consortium.Daphnia pulex genome assembly version 1.1 andannotations are deposited at DNA Data Bank of Japan,European Molecular Biology Laboratory, and GenBankdatabases under accession ACJG00000000. ESTs(FE274839 to FE425949) are in GenBank. Microarrayplatforms GPL11200 to GPL11201 and data GSE25823are deposited at National Center for BiotechnologyInformation Gene Expression Omnibus database.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/331/6017/555/DC1Materials and MethodsSOM TextFigs. S1 to S36Tables S1 to S50References

14 September 2010; accepted 16 December 201010.1126/science.1197761

Fig. 5. Function of genesunique to the D. pulex lin-eage. (A) Pie charts showthedistributionofexpressedgenes both with and with-out detectable homologyto other sequenced ge-nomes, sampled under ex-posure tobacterial infection,predators, hormones, vary-ingdiets (biotic challenges),environmental toxicants,elevated ultraviolet radia-tion, hypoxia, acid, salinity,andcalciumstarvation (abi-otic challenges), in additionto various stages of lifehistory within a controlledlaboratory environment(standard conditions). (B)Differential expression ofthe genome upon exposure to Chaoborus kairomone (Kair), cadmium (Cad), and by sex, measured asnucleotides in kilobases (Kb) on genome tiling microarrays. Comparing three experimental conditions, 79%,72%, and 83%of transcriptomes are condition-specific (Venn diagram) and twice as pronounced in genomicregions that are currently void of gene models (yellow) when D. pulex are exposed to ecological conditions.

No homologyHomology

Biotic challenges

Abiotic challenges

Standard conditions

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89 Kb

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Kair Cad SexIntergenicIntronsGenes

Condition specific fractions

Expression pattern is divergent at the time of duplication, may be condition-specific, yet without an appropriate interacting gene: Loss of the duplicate gene

Expression pattern is unchanged at the time of duplication and the new gene retains the conditon-dependent expression of an existing interacting gene: Beneficial increase in dosage and preservation of the duplicate gene

Expression pattern is divergent at the time of duplication, is condition-specific and matches the environmental expression pattern of a different interacting gene: Functional entrainment and preservation of the duplicate gene

Interacting genes

A B C

1 2 3 4 5

Up-regulated expresionDown-regulated expression

Environments

Interacting genes

Duplicating gene ( ) ( )

Fig. 6. Model of gene duplicationunder the PBE model. (A) B2BA (Bornto be Alike) shows duplicated geneswith unaltered expression patterns that are preserved because of beneficialincrease in dosage (20) in associationwith the condition-dependent expression ofan interacting gene. (B) B2BU (Born to be Useless) genes with initially divergent expression patterns and withinappropriate condition-dependent responses or interacting genes are most likely lost. (C) B2BD (Born to beDifferent). When the derived expression pattern of a paralog at the time of duplication is shared with a differentinteracting gene (white negative sign), andwhen the effect of their combined products is beneficial under a distinctenvironmental condition, the likelihood for preservation is increased. Color-coding represents condition-dependentexpression patterns across multiple environments. Lines represent the process of functional entrainment.

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Daphnia pulexThe Ecoresponsive Genome of

Evgeny M. Zdobnov, Igor V. Grigoriev, Michael Lynch and Jeffrey L. BooreShaw, Justen Andrews, Teresa J. Crease, Haixu Tang, Susan M. Lucas, Hugh M. Robertson, Peer Bork, Eugene V. Koonin, Tang, Dai Tsuchiya, Hank Tu, Harmjan Vos, Mei Wang, Yuri I. Wolf, Hideo Yamagata, Takuji Yamada, Yuzhen Ye, Joseph R.Sarah Schaack, Harris Shapiro, Yasuhiro Shiga, Courtney Skalitzky, Zachary Smith, Alexander Souvorov, Way Sung, Zuojian

Salamov,Patwardhan, Samuel Pitluck, Ellen J. Pritham, Andreas Rechtsteiner, Mina Rho, Igor B. Rogozin, Onur Sakarya, Asaf Christian Laforsch, Erika Lindquist, Jacqueline Lopez, J. Robert Manak, Jean Muller, Jasmyn Pangilinan, Rupali P.Geiler-Samerotte, Daniel Gerlach, Phil Hatcher, Sanjuro Jogdeo, Jeroen Krijgsveld, Evgenia V. Kriventseva, Dietmar Kültz, Casola, Jeong-Hyeon Choi, John C. Detter, Qunfeng Dong, Serge Dusheyko, Brian D. Eads, Thomas Fröhlich, Kerry A.Tokishita, Andrea Aerts, Georg J. Arnold, Malay Kumar Basu, Darren J. Bauer, Carla E. Cáceres, Liran Carmel, Claudio John K. Colbourne, Michael E. Pfrender, Donald Gilbert, W. Kelley Thomas, Abraham Tucker, Todd H. Oakley, Shinichi

DOI: 10.1126/science.1197761 (6017), 555-561.331Science 

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