Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in ...

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Article
Reconstruction of the vertebrate ancestral genome
reveals dynamic genome reorganization
in early vertebrates
Yoichiro Nakatani,1,5 Hiroyuki Takeda,2 Yuji Kohara,3 and Shinichi Morishita1,4,5
1DepartmentofComputationalBiology,GraduateSchoolofFrontierSciences,TheUniversityofTokyo,Kashiwa277-0882,
Japan;2DepartmentofBiologicalSciences,GraduateSchoolofScience,TheUniversityofTokyo,Tokyo113-0033,Japan;
3CenterforGeneticResourceInformation,NationalInstituteofGenetics,Mishima411-8540,Japan;4BioinformaticsResearch
andDevelopment(BIRD),JapanScienceandTechnologyAgency(JST),Tokyo102-8666,Japan
Although several vertebrate genomes have been sequenced, little is known about the genome evolution of early
vertebrates and how large-scale genomic changes such as the two rounds of whole-genome duplications (2R WGD)
affected evolutionary complexity and novelty in vertebrates. Reconstructing the ancestral vertebrate genome is
highly nontrivial because of the difficulty in identifying traces originating from the 2R WGD. To resolve this
problem, we developed a novel method capable of pinning down remains of the 2R WGD in the human and medaka
fish genomes using invertebrate tunicate and sea urchin genes to define ohnologs, i.e., paralogs produced by the 2R
WGD. We validated the reconstruction using the chicken genome, which was not considered in the reconstruction
step, and observed that many ancestral proto-chromosomes were retained in the chicken genome and had one-to-one
correspondence to chicken microchromosomes, thereby confirming the reconstructed ancestral genomes. Our
reconstruction revealed a contrast between the slow karyotype evolution after the second WGD and the rapid,
lineage-specific genome reorganizations that occurred in the ancestral lineages of major taxonomic groups such as
teleostfishes,amphibians,reptiles,andmarsupials.
[Supplementalmaterialisavailableonlineatwww.genome.org.]
Earlyvertebrategenomeevolutionhaslongbeeninneedofclari- identified groups of human genes (called “ohnologs” [Wolfe
fication,anditisnowofparticularinterestbecauseseveraldis- 2001]) duplicated by the 2R WGD by ensuring that individual
tantlyrelatedvertebrategenomeswererecentlysequenced.The genesinagroupweremostsimilartotheidenticalorthologous
2Rhypothesispostulatesthattworoundsofwhole-genomedu- deuterostome gene of the sea urchin (Sea Urchin Genome Se-
plication(2RWGD)occurredatthebaseofthevertebratelineage quencing Consortium 2006) or tunicate (Dehal et al. 2002;
(Ohno1970;Hollandetal.1994)becauseoftheobservationthat Shoguchietal.2006).Theidentificationofohnologsisadifficult
invertebrates have one Hox gene cluster, whereas lobe-finned task because all ohnologs and their corresponding Ciona genes
fishes and land vertebrates have four clusters. However, the 2R arerarelyconservedduetonumerouslossesandduplicationsof
hypothesishasbeenquitecontroversialuntilrecently(Skrabanek Ciona, human, and medaka genes. Given these difficulties,
andWolfe1998;GibsonandSpring2000;FriedmanandHughes ohnologsweregroupedbasedonthemethodofDehalandBoore
2001;Wolfe2001;Abi-Rachedetal.2002;FurlongandHolland (2005), which is detailed in Supplemental Figure S1 and the
2002;Guetal.2002;McLysaghtetal.2002;Durand2003;Pan- Supplemental Document. These ohnologs typically occur con-
opoulou et al. 2003; Seoighe 2003; Panopoulou and Poustka secutivelyinparalogouschromosomalregionsinthehumange-
2005;Vandepoeleetal.2004)becauseitleavesopenthepossi- nomeandarelikelytorepresentaremainingblockderivedfrom
bilityofoneroundofWGDfollowedbylarge-scaleduplications asinglegnathostome(jawedvertebrate,seephylogenetictreeof
suchassegmentalandchromosomalduplications.Recently,De- vertebrates in Fig. 6, below) proto-chromosome. To test if they
hal and Boore (2005) showed that a large part of the human are really remaining blocks of the gnathostome proto-
genome contains four-way paralogous chromosomal regions, chromosomes,theirsynteniesinthemedakagenome(Kasahara
whicharetracesofthe2RWGD. etal.2007)wereinvestigated(fordetails,seetheSupplemental
The task of reconstructing ancestral vertebrate proto- Document).Thefinalstepinournovelanalysiscombinedquali-
chromosomesbeforethe2RWGDisverydifferentfromordinary fied remaining blocks into vertebrate and gnathostome proto-
synteny analysis using orthologs because the effect of the 2R chromosomesusinginformationonohnologdistributionamong
WGD must be carefully examined. Moreover, it is necessary to theblocks.Thisseriesofstepswasnewlydevelopedforthisre-
determine human chromosome regions originating from the construction(seeMethods).
same ancestral chromosome at the second WGD and integrate Next,weattemptedtovalidatethereconstructedgnathos-
these regions to rebuild the ancestral karyotype. Thus, we first tome proto-chromosomes. If extant genomes have experienced
5Correspondingauthors. intensive interchromosomal rearrangements, they are hardly
[email protected];fax[phone]. syntenictothereconstructedancestralgenomeandarenotuse-
[email protected];fax[phone]. fulinthevalidation.Amongsequencedvertebrategenomes,we
Articlepublishedonlinebeforeprint.Articleandpublicationdateareathttp://
searched for a genome that was not used in the reconstruction
www.genome.org/cgi/doi/10.1101/gr.6316407. Freely available online
throughtheGenomeResearchOpenAccessoption. stepbutpreservedtheproto-karyotype.Thechickengenomewas
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Vertebrate genome evolution
mostpromising,giventhehypothesisthatavianmicrochromo- humanchromosomeswerecomparedwithCionaandseaurchin
somes represent archaic linkage groups of ancestral vertebrates genesasanoutgroupbecauseoftheintensiverearrangementsin
(Burt 2002). Indeed, we observed that many ancestral gnathos- the mammalian lineage. We therefore needed additional infor-
tomeproto-chromosomesinourreconstructionhadone-to-one mation.Thus,wefullyusedthemedakachromosomesasanout-
correspondence with microchromosomes in the chicken ge- group of mammalians to clarify the boundaries (see details in
nome,thusprovidingastrongvalidationofourreconstruction. Supplemental Fig. S2). The conserved vertebrate linkage (CVL)
We reconstructed the gnathostome (jawed vertebrate), os- blockswereessentialinreconstructingancestralvertebrateproto-
teichthyan(bonyvertebrate),andamniote(thegroupincluding chromosomes.
reptiles, birds, dinosaurs, and mammals) ancestral genomes,
which are located at key phylogenetic positions, leading to a
Reconstructionofthevertebrateancestralgenome
novelscenarioofgenomeevolutioninearlyvertebrates.Thetwo
roundsofWGDeventsduplicated10–13proto-chromosomesin Here,weassumedasimplifiedmodelinwhichnomajorgenome
thevertebrateancestor,producingthegnathostome(jawedver- rearrangementsoccurredbetweenthe2RWGDevents,although
tebrate) ancestor (n≈40). Subsequent chromosome fusions re- wewillpresentamoreelaboratemodellaterinthispaper.The
duced the number of chromosomes in the osteichthyan proto- expectedsignatureoftworoundsofWGDisillustratedinFigure
karyotype(n≈31)andintheamnioteproto-karyotype(n≈26). 2,A–D.The2RWGDquadruplicatedtheredandbluechromo-
Theseestimatesofchromosomenumberareconsiderablylarger somes,producingsisterchromosomeseachhavingthesameset
than previous estimates (Jaillon et al. 2004; Naruse et al. 2004; ofgenesarrangedinthesameorder.Inreality,genelossesand
Woodsetal.2005;Kohnetal.2006)andcontradictthewidely chromosomerearrangementsmusthavetakenplacebetweenthe
held hypothesis that the osteichthyan proto-karyotype was 2RWGD,butforsimplicity,thesedetailsareomittedfromFigure
n≈12(Postlethwaitetal.2000;Jaillonetal.2004;Naruseetal. 2A. Wolfe (2001) proposed calling these duplicated gene pairs
2004; Woods et al. 2005; Kohn et al. 2006) and that land- “ohnologs”afterSusumuOhno.Oneohnologisrepresentedbya
vertebrategenomeswereshapedbylineage-specificchromosome dotinatrianglewhoseX-andY-axiscoordinatesrepresentthe
“fissions”(Postlethwaitetal.2000).Onthecontrary,ourresults positions of duplicated genes in the sister chromosomes pro-
demonstrate that many lineage-specific chromosome “fusions” ducedbythe2RWGD.Becauseohnologsareproducedbychro-
shaped the ancestral karyotypes of major taxonomic groups, mosome-wide duplication and not by smaller-scale gene dupli-
suchasteleostfishes,amphibians,reptiles,andmarsupials. cation,theyareobservedbetweenpairsofdistinctsisterchromo-
somes, as illustrated by dots in the red region. In contrast, no
ohnologswouldbefoundwithinonesisterchromosome,which
Results
is shown by the absence of ohnolog dots in the green regions.
Afterthe2RWGD,chromosomebreaks(fissionsandtransloca-
Evolutionofvertebratechromosomes
tions),fusions,orinversionsmusthavealteredsomeofthesister
Figure1presentsourscenarioofvertebratechromosomeevolu- chromosomes (Fig. 2B). The accumulation of rearrangements
tion.Althoughtheancestralgenomewouldhavemorethantwo gradually disrupted the ancestral gene order and scattered the
proto-chromosomesbeforethetworoundsofWGD,forsimplic- ohnolog dots (Fig. 2C). Furthermore, intensive interchromo-
itywedisplaytwoofthereconstructedancestralvertebratechro- somal rearrangements in the mammalian lineage (Burt et al.
mosomes,coloredredandblue(Fig.1A).ThefirstroundofWGD 1999) must have distributed blocks across the human genome
doubledthesetwochromosomes,andfissionoccurredinoneof (Fig. 2D). Reconstruction of ancestral proto-chromosomes is a
theduplicatedcopiesoftheredchromosome.Afterthesecond task of reordering the CVL blocks in Figure 2D to estimate the
roundofWGD(Fig.1B),sisterchromosomesweregraduallydis- orderingsinFigure2,BandC.
rupted by genome rearrangements and broken into smaller Figure2Epresentstheactualdistributionofohnologsinthe
blocksofchromosomalsegmentsduringearlyvertebrateevolu- humangenome,inwhichCVLblocksareplacedinorderfrom
tion(Fig.1C).Eventually,theseblocksweredistributedoversev- humanchromosome1toX.Thereconstructionstepstartedwith
eral human chromosomes (Fig. 1D) because of intensive inter- categorizing the blocks into groups that were derived from a
chromosomalrearrangementsinthemammalianlineage(Burtet singleancestralvertebratechromosomebeforethe2RWGD.For
al.1999).However,intheteleostfishlineage,intensivechromo- thispurpose,anytworedCVLblockssharingseveralohnologs
some fusions and another WGD occurred after the divergence wereplacedinthesame“vertebrate”group(Fig.2F).Thesubse-
fromtheosteichthyanancestor. quentstepdividedtheblockswithineachgroupintosubgroups
As illustrated in Figure 1, conserved vertebrate linkage thatrepresentedduplicatedproto-chromosomesinthegnathos-
groups, which are groups of genes located on a single chromo- tomeancestor.Tothisend,weperformedanexhaustivesearch
someafterthesecondroundofWGD,constitutechromosomal fortheoptimalsubgroupingsothatsignificantlymoreohnologs
blocksinthehumangenome.Theseblockscanalsobeidentified weresharedbetweendistinctsubgroups,asillustratedbythered
by doubly conserved synteny (DCS) analysis with the medaka regionsinFigure2G,whereasfewohnologswereobservedwithin
genome(Jaillonetal.2004;Kellisetal.2004).Forexample,al- anysinglesubgroup,asindicatedbythegreenregions.Thede-
thoughblocks19a,19b,and19carelocatedconsecutivelyinthe tailsofthereconstructionprocedurearedescribedintheMeth-
human genome, both 19a and 19c are syntenic to each of the ods. Our reconstruction in Figure 2G shows that in five of 10
duplicatedmedakachromosomes4and17,whereasduplicatesof ancestral vertebrate chromosomes, estimating four sister chro-
19barefoundintwoduplicatedmedakachromosomes(1and8). mosomeswasmoresignificantthaninferringtwo,three,orfive
Otherneighboringblocks,i.e.,1b–1c,3a–3b,7a–7b,and9a–9b, sister chromosomes. CVL blocks with a small number of
arelocatedondistinctmedakachromosomes.Thisimpliesthat ohnologsfailtoberecognizedassisterchromosomesbecauseof
decidingtheboundariesofsuchneighboringblocksisextremely theirlowstatisticalsignificance,therebyyieldingareconstructed
important; however, the task was difficult when genes on the vertebrategroupwithlessthanfourduplicatedchromosomes.In
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Nakatani et al.
Figure1. Vertebratechromosomeevolutionscenario.(A)Forsimplicity,weillustratetwoproto-chromosomes(redandbluebars)duplicatedbythe
firstroundofWGD.Subsequently,fissiondividedoneoftheduplicatedchromosomes.(B)ThesecondroundofWGDdoubledtheproto-chromosomes.
Blocksinchromosomesarelabeledwiththeirrespectivechromosomepositionsinthehumangenome.(C)AfterthesecondWGD,earlyvertebrates
underwentslowchangesinkaryotypeoveralongevolutionaryprocess.(D)Intheancestralmammalianlineage,intensiveinterchromosomalrear-
rangementsoccurredandtheancestralchromosomeswerebrokenintosmallersegmentsthatweredistributedacrossmanyhumanchromosomes.(E)
Intheancientray-finnedfishlineage,intensivechromosomefusionsmergedtheancestralchromosomalsegmentsintoancestralteleostchromosomes.
(F)AnotherroundofWGDintheancestralteleostdoubledproto-chromosomes,butafterward,fewglobalrearrangementsshapedthepresentmedaka
genome.
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Figure2. (Legendonnextpage)
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Nakatani et al.
contrast,fiveancestralvertebratechromosomes(A–E)hadfairly proto-chromosomesbeforethedivergencefromvertebrates,pre-
largegroupsofCVLblockswith>250ohnologgenepairs,pro- sumably because of numerous chromosome rearrangements.
vidingstrongsupportforthe2Rhypothesis. Therefore,atthisstage,weestimatedthattheancestralvertebrate
had 10–13 proto-chromosomes before the 2R WGD. We assert
Assumingfusionsandfissionsbetweenthe2RWGD
thattheancestralgnathostomehad40proto-chromosomes,and
Forsimplicity,wehavethusfarassumednomajorchromosome wasnotaffectedbytheselectionofafusionorfissionscenario.
rearrangementsbetweenthe2RWGD.Here,weextendthesim-
plifiedmodeltoconsiderthepossibilityofchromosomefusions Validationofreconstructedproto-chromosomes
andfissionsbetweenthe2RWGDevents,thoughhowtodetect
Tocheckthevalidityofthereconstructedproto-chromosomes,
remains of these events is a nontrivial question. Figure 3A
wesearchedforagenomethatpreservedtheancestralchromo-
presents vertebrate groups A, B, and F in Figure 2F, and it is
somes among available deuterostome, chordate, and vertebrate
remarkabletoobservepairsoflightblueboxeswithfewohnologs
genomes.Theseaurchindraftgenome(SeaUrchinGenomeSe-
inindividualtrianglesbecausetheseboxesareexpectedtocon-
quencingConsortium2006)islargelyfragmentedintoverysmall
tainmanyohnologsaccordingtothesimplifiedmodelassumed
pieces,whereastheCionagenome(Shoguchietal.2006)experi-
sofar.Wethinkthatthesephenomenaprovidecluestotheprob-
enced numerous genome rearrangements after the divergence
lemofinferringgenomerearrangementsbetweenthe2RWGD;
from vertebrates >500 million yr ago; for example, even Hox
here,weshowthreealternativescenariosthatmayproducethese
genes are separated onto multiple chromosomes (Ikuta et al.
phenomena.
2004). Thus, these two genomes fail to preserve proto-
Figure3Bshowsthefirstscenarioinwhichduplicatesoftwo
chromosomes.Wealsoexaminedvertebrategenomesandfound
ancestralchromosomesarefused,whereasFigure3Cshowsthe
that rat, mouse, and dog genomes did not preserve the proto-
second scenario, in which one duplicate of a single ancestral
karyotypebecauseofseveralinterchromosomalrearrangements
chromosomeisdividedbyafissionevent.Bothscenariosgener-
in the mammalian lineage (Burt et al. 1999). Among tested ge-
atethesameohnologdistributionpatternamongthesixdescen-
nomes, the chicken genome significantly preserved the recon-
dent chromosomes, as illustrated in Figure 3E; note that pairs
structedancestralgnathostomechromosomes.Moreprecisely,a
A21–B22andB21–A22havenoohnologs.Thethirdscenarioin
series of CVL blocks that occur both in one ancestral gnathos-
Figure 3D indicates that neither fissions nor fusions take place
tome chromosome and in one chicken chromosome presents
between the 2R WGD events, but independent fission events
strongevidenceforthereconstruction;incontrast,occurrences
occur for duplicated sister chromosomes after the 2R WGD. As
of CVL blocks of one chicken chromosome in different sister
illustratedinFigure3F,thepairA21a–A22bisfreeofohnologs;
chromosomesofthegnathostomeancestorsuggestintensivein-
however, some ohnologs are observed in the pair A21b–A22a.
terchromosomalrearrangementsintheavianlineageorpossible
Bothpairswouldhavenoohnologsifthetwoindependentfis-
mistakes in the reconstruction. We observed that CVL blocks
sion events took place at the identical chromosomal positions,
located in the same chicken microchromosomes belonged pri-
butthiscaseislesslikely.Amongthethreescenarios,theformer
marilytothesameancestralgnathostomechromosomes(Fig.4).
twoscenariosofonefusionorfissioneventaremoreparsimoni-
Tobeprecise,chickenchromosomes7,8,11,15,19,20,21,22,
ousthanthethirdscenariooftwoindependentfissions,andthey
23,24,27,and28hadone-to-onecorrespondencetoE0,A2,B4,
better explain pairs of light blue boxes with few ohnologs in
H0,H1,B1,F0,C3,B3,J0,E2,andA1,respectively(Fig.5).Thus,
Figure3A.Thus,weestimatedthatamong10vertebrategroups,
thisclearcorrespondencebetweenchickenandgnathostomean-
A, B, and F had fusions or fissions, whereas the other seven
cestor chromosomes provides a firm grounding for our recon-
groupswerefreeofthesechromosomerearrangements.
struction.
However, it remains difficult to judge whether a fusion or
fission happened between the 2R WGD; each of the vertebrate
Reconstructionofancestralosteichthyanandamniote
groups A, B, and F may have had two ancestral chromosomes
proto-chromosomes
before the 2R WGD according to the fusion scenario, or one
proto-chromosomeiffissiontookplace.Thisargumentmaybe The reconstruction of proto-chromosomes in the gnathostome
settled by using the genomes of some invertebrates as an out- ancestorallowedustoinferthekaryotypesofevolutionarilyim-
group.However,thedraftgenomeoftheseaurchin(SeaUrchin portantancestorssuchasthebonyvertebrate(osteichthyan)an-
Genome Sequencing Consortium 2006) consists of very small cestorandtheamnioteancestor,leadingtonovelinsightsinto
genomicfragments,andtheCionadraftgenome(Shoguchietal. genomeevolutionafterthe2RWGD.Previousstudieshavesug-
2006),withanearlycompletechromosomemap,failstopreserve gestedthattheosteichthyanproto-karyotypewasn≈12bytreat-
Figure2. Modelofvertebrategenomeevolutionandreconstructionoftheancestralgenome.(A)Forsimplicity,supposethattheancestralchro-
mosomehad10genes.The2RWGDproducedohnologs(bluedotsalongthediagonallineinthetriangulardotplot)intheduplicatedchromosomes.
(B)Chromosomebreaksandinversionsmayhavealteredtheorderofohnologsonthesisterchromosomes.(C)Inthecourseofearlyvertebrategenome
evolution,theancestralgeneorderwasdisruptedbymanyinversions,resultinginscatteredohnologdots.(D)Eventually,CVLblocksweredistributed
acrossseveralhumanchromosomesbyintensiveinterchromosomalrearrangements.(a–d)Atypicalmodelofgenomeevolutioninvolvingthe2RWGD.
In the next step, we handle real human genome data. (E) This is a real instance of the dot plot in D. CVL blocks were ordered from the human
chromosomes1toX,andohnologssharedamongtheseCVLblockswereplotted.(Red)RegionsrepresentingpairsofparalogousCVLblockswitha
greatnumberofohnologs(P<10(cid:1)4,seeMethods).(F)ThiscorrespondstothestateinC.CVLblockswerereorderedinsuchawaythatparalogous
CVLblocksweregroupedsothateachgrouprepresentedoneancestralvertebratechromosome(seeMethods).(G)Thisstatecorrespondstothatin
B.CVLblockswithinindividualvertebrategroupswerefurtherreorderedtoobtainancestralgnathostomesubgroups(namely,chromosomes),which
wereduplicatedfromasingleancestralvertebratechromosomebythe2RWGDevents.Thepartitionofsubgroupsthatoptimizesthesignificance
definedintheMethods.RectanglesandtrianglesarecoloredinaccordancewiththoseinBtomakethecorrespondenceclear.(H)Thevertebrategroup
Awasdecomposedintofourgnathostomesubgroupsbystatisticalanalysis,indicatingthattheancestralchromosomeunderwent2RWGD.
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Figure3. Modelsofchromosomeevolutionduring2RWGD.(A)VertebrategroupsA,B,andEinFig.2F.Pairsoflightblueboxeswithfewohnologs
maybetheremainsofgenomerearrangementsbetweenthetwoWGDevents.(B)Twodistinctancestorchromosomes(AandB)wereduplicatedby
thefirstWGD,andduplicatedchromosomesA1andB1underwentachromosomefusion.(C)Oneancestorchromosomeconsistingoftwoparts(Aand
B)wasduplicatedbythefirstWGD,andacopyoftheduplicatedchromosomeswassplitintotwochromosomes(A1andB1)byachromosomefission
event.(D)AfterthesecondroundofWGD,twoofthefoursisterchromosomesunderwenttwoindependentchromosomefissions.Becausethetwo
fissionssplittwochromosomes(A11andA12)atdifferentchromosomepositions,blocksA21bandA22ahavesomeohnologsincommon.(E)Bothof
thefusionandfissionmodelsinBandCproducethesamedistributionofohnologs,especiallypairsoflightblueboxeswithoutohnologs.(F)The
distributionoriginatingfromindependentfissioneventsinDisdistinguishablefromthatinEasindicatedbyonelightbluebox.
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Nakatani et al.
Figure4. Reconstructedancestralchromosomes.AncestralvertebratechromosomesA,B,andFhadtwoalternativescenarios,fusionsorfissions,
betweenthe2RWGDevents,asshowninFig.3.Thus,thenumberofproto-chromosomesrangesfrom10to13dependingonthechoiceoftwo
alternatives.Thefigureillustratesthescenarioinwhichonlyfissionstookplace.Tenreconstructedproto-chromosomesinthevertebrateancestorshown
atthetopareassigneddistinctcolors,andtheirdaughterchromosomesinthegnathostomeancestoraredistinguishedbytheirrespectiveverticalbars.
Inthegenomesoftheosteichthyan,teleost,andamnioteancestors,andhuman,chicken,andmedakagenomes,genomicregionsareassignedcolors
andverticalbarsthatrepresentcorrespondencesofindividualregionstotheproto-chromosomesinthegnathostomeancestorfromwhichrespective
regionsoriginated.Unassignedblocksareshownintherightmostchromosome(Un)intheosteichthyanandamnioteancestors.
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Figure5. Syntenicchromosomecorrespondencebetweenthechickenandgnathostomeancestorchromosomes.Inthetable,thenumberinacell
indicatesthenumberoforthologousgenesinsyntenicregions(seeSupplementalmaterials)betweenthechickenchromosomeinthecolumnandthe
reconstructedgnathostomeproto-chromosomeintherow.Toemphasizechickenchromosomesthatwereprimarilyderivedfromasingleancestral
gnathostomechromosome,cellswiththemaximumnumberoforthologsarecoloredyelloworred.Inparticular,redcellsimplychickenchromosomes
thathaveone-to-onecorrespondencetotheirancestralgnathostomechromosomes.
ingtheteleostancestorofmedaka,Tetraodon,andzebrafishbe- teleostancestor,andgnathostomeancestor.Wecallthisrulethe
foreteleostWGDastheosteichthyanancestor(Postlethwaitetal. 2-of-3ruleandwedescribethedetailsandrationaleoftherulein
2000;Jaillonetal.2004;Naruseetal.2004;Woodsetal.2005)or the Methods. The resulting osteichthyan proto-karyotype was
byleavingthepossibilityofchromosomefusionsoutofconsid- n≈31, which confirms our postulate that the number of oste-
eration(Kohnetal.2006).However,thishypothesis(n≈12)ap- ichthyanproto-chromosomesismorethanthepreviousestima-
pears to disagree with our observations because many chromo- tionofn≈12.
somes in the chicken genome (n=39) have one-to-one corre- Regarding the amniote ancestor, Kohn et al. (2006) esti-
sponding proto-chromosomes in the gnathostome ancestor mated n≈18 using the teleost ancestor as an outgroup of the
(n≈40),asshowninFigure5,whereasmostchromosomesinthe human and chicken genomes. However, in our reconstruction,
teleostancestor(n≈13)havesyntenyblockswithmultiplechro- theteleostancestorgenomeunderwentseveralrearrangements,
mosomesinthegnathostomeancestor.Theseobservationsledus especiallyfusions,afterdivergencefromtheosteichthyanances-
to postulate that the osteichthyan proto-karyotype had more tor. Thus, we used the reconstructed osteichthyan ancestor, in
chromosomes than the previous estimate (n≈12), and that place of the teleost ancestor, together with the human and
manygenomerearrangementstookplacefromtheosteichthyan chicken genomes to reconstruct the amniote ancestor chromo-
proto-karyotypetotheteleostancestor. somes. Using the 2-of-3 rule, we estimated that the number of
Totestthishypothesis,weattemptedtoreconstructtheos- amniote proto-chromosomes was n≈26, which is significantly
teichthyan proto-karyotype by comparing the chicken and an- largerthanthepreviousestimateofn≈18.Overall,weconclude
cestralteleostkaryotypeswiththatofthegnathostomeancestor. thatchromosomenumbersinearlyvertebratesareconsiderably
Thisapproachissomewhatdifferentfromthetraditionalmethod larger than previous estimates (Jaillon et al. 2004; Naruse et al.
ofusinganoutgroupofthechickenandteleostancestorbecause 2004;Woodsetal.2005;Kohnetal.2006).
weinsteadusedthegnathostomeancestor,whichisanancestor
of the osteichthyan ancestor. In the reconstruction, two CVL
Karyotypeevolutionandtaxon-specificfusionmodel
blocks were expected to occur in the same osteichthyan proto-
chromosomeifthetwoCVLblockswerelocatedontheidentical ThereconstructedancestralkaryotypesinFigure4provideasce-
chromosome of at least two of the three karyotypes: chicken, narioforkaryotypeevolutioninvertebrates,asdepictedinFigure
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Figure6. Changesinchromosomenumberduringvertebratekaryotypeevolution.Thereconstructedproto-chromosomesinFig.4allowustodiscuss
howthenumberofchromosomeshaschangedinindividualvertebratelineages.(Left)Thephylogenetictreeofvertebrates,(right)thedistributionof
chromosome number in individual lineages (Gregory et al. 2007; Animal Genome Size Database, http://www.genomesize.com). Considering the
numbers of proto-chromosomes in Fig. 4, two ancient whole-genome duplication events almost quadrupled the number of chromosomes; subse-
quently,chromosomenumbersinindividuallineagestendedtodecreaseinmanylineages,althoughnotintheavianlineage.
6.BeforethefirstroundofWGD,thevertebrateancestorkaryo- model is that the ancestors of major vertebrate groups such as
typewasn≈10–13,andthesubsequent2RWGDandsomege- teleosts, amphibians, squamates, and metatherians underwent
nome rearrangements yielded the gnathostome ancestor of numerous chromosome fusions (except for birds), leading to
n≈40. After the divergence of bony vertebrates (Osteichthyes) slow changes in karyotype, as indicated by the distribution of
and cartilaginous fishes (Chondrichthyes), genome rearrange- chromosome number. Similar speculations have been made re-
mentsreducedthenumberofchromosomesintheosteichthyan gardingamphibiansandreptiles;Morescalchipostulatedthatthe
ancestorton≈31.Inthemeantime,moderncartilaginousfishes number of chromosomes decreased in the ancestral amphibian
havegenomescomprisingavarietyofkaryotypes;thenumberof based on the large number of chromosomes in some primitive
chromosomes in the haploid set, n, ranges from 25 to 50. We amphibians(GreenandSessions1991).Withrespecttoreptiles,
speculatethatsomeofextantcartilaginousfishesmayretainthe Olmo(2005)indicatedthatancestralreptileshadahigherrateof
gnathostomeancestralkaryotype(n≈40),assumingnogenome karyotypicchanges.Ourkaryotypeevolutionmodelisconsistent
duplicationeventstookplaceinthecartilaginouslineage.After withthesehypotheses.
the divergence of ray-finned and lobe-finned fishes, in the lin- Incontrast,alargeincreaseinthenumberofchromosomes
eage of ray-finned fishes (Actinopterygii), chromosome fusions was found in the avian lineage. One explanation is that fewer
reducedthenumberofchromosomesandproducedtheteleost repeatsequencesinaviangenomeswerelikelytoresultinchro-
ancestorwithn≈13.Ourfusionmodelmadeitnecessarytore- mosome fissions rather than fusions and interchromosomal re-
vise the n≈12 ancestral osteichthyan proto-karyotype hypoth- arrangements(Burtetal.1999;Burt2002).Anotherinteresting
esis,whichtreatstheteleostancestorof∼350Myaastherepre- finding involved microchromosomes in the avian lineage. Burt
sentativeoftheosteichthyanancestorof∼450Mya(Postlethwait arguedthatmanyavianmicrochromosomeswerealreadypresent
etal.2000;Jaillonetal.2004;Naruseetal.2004;Woodsetal. inthecommonancestorofbirdsandotherterrestrialvertebrates
2005;Kohnetal.2006).Subsequently,thewhole-genomedupli- andwereconservedfor>400millionyr(Burt2002).Ouranalysis
cation in the teleost ancestor doubled the number of chromo- extendsthisideaconsiderablybyshowingthatmanyofthemi-
someston≈26.Thenumberofchromosomesintheteleostlin- crochromosomes had one-to-one correspondence with proto-
eagehasremainednearlyunchangedduringevolution,andthe chromosomesinthegnathostomeancestor.
chromosome numbers of extant teleost species peak at n=24 Weconcludethatinearlyvertebrategenomeevolution,two
or25. ancientwhole-genomeduplicationeventsincreasedthenumber
Inthelineageoflobe-finnedfishes(Sarcopterygii),thedis- of chromosomes, but subsequent chromosome fusions reduced
tributionofchromosomenumberdifferslargelyacrossvertebrate the number of chromosomes in individual lineages, with the
taxa (Fig. 6). In particular, an interesting observation in our exceptionoftheavianlineage.
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Vertebrate genome evolution
Conclusions blocks were produced by independent gene duplications and
concludedthatthetwoCVLblockswereparalogous.Thecruxis
We present the first study to reconstruct the vertebrate proto- howtoselectameaningfulvalueasthethreshold.Weidentified
karyotype before the 2R-WGD events. The reconstruction was 118 CVL blocks (for details, see the Supplemental Document).
made possible by the newly developed computational method BecausethetotalnumberofallpairsofCVLblocksamountedto
outlined in Figures 2 and 3. The reconstructed genome organi- 6903,themaximumthresholdoftheprobabilitywassetto10(cid:1)4
zationofthevertebrateancestorprovidesnewinsightsintoearly so that the expected number of false-positive paralogous pairs
vertebrate genome evolution. First, some rearrangements oc- was<1.InFigure2,EandF,regionsrepresentingparalogousCVL
curred during the 2R WGD in the ancestral vertebrate. Second, blocksarecoloredrediftheP-value<10(cid:1)4.
afterthesecondWGD,thegnathostome,osteichthyan,andam-
Reconstructionofsisterchromosomesinthegnathostome
niote ancestors underwent slow karyotype evolution. Third,
ancestorbygroupingparalogousCVLblocks
rapid,lineage-specificchromosomefusionsshapedtheancestral
genomesofmajorvertebratetaxasuchasteleosts,amphibians, We began by constructing an undirected graph in which the
reptiles,andmarsupials. nodesareCVLblocksandedgesaredrawnbetweentwoparalo-
Ourreconstructionwillalsointegratenewlysequencedge- gous CVL blocks. The graph was then divided into connected
nomessuchasthepipidfrog(Xenopustropicalis),grayshort-tailed componentssothattherewasapathbetweenanytwonodesin
opossum(Monodelphisdomestica),andamphioxus(Branchiostoma aconnectedcomponent,asillustratedinSupplementalFigureS7.
floridae) into the global picture of vertebrate and chordate ge- CVLblocksrepresentedbynodesinoneconnectedcomponent
nome evolution. Our presumption is that the X. tropicalis ge- were thought to be derived from one vertebrate proto-
nomeevolvedfromtheosteichthyanancestorgenomeafterun- chromosomebeforethe2RWGD,asillustratedinFigure2H.
Subsequently, to estimate proto-chromosomes in the gna-
dergoingnumerouschromosomefusions.Becausethosefusions
thostomeancestorthatwereproducedbyWGDevents,nodesin
tookplacespecificallyintheancestralamphibianlineage,they
oneconnectedcomponentwerefurtherpartitionedintoseveral
wouldbelargelydifferentfromfusionsthattookplacefromthe
subgroupsofnodessothateachsubgrouprepresentedoneproto-
osteichthyanancestortotheteleostancestor,whichwillmakeit
chromosomeinthegnathostomeancestor.Thenumberofgna-
difficulttoidentifyone-to-onecorrespondencebetweenchromo-
thostome proto-chromosomes originating from one vertebrate
somesofX.tropicalisandtheteleostancestor.
proto-chromosomewasexpectedtobefourassumingthatthe2R
WGDeventstookplace(DehalandBoore2005),butweenumer-
Methods atedpatternsofpartitioningCVLblocksintotwo,three,four,or
fivesubgroupstosearchforthemostsignificantpattern.Specifi-
Proteinsequencesforgenesandidentificationofohnologs cally, for each connected component, all combinations of CVL
blockswereenumeratedwiththeconstraintthatanyparalogous
Human, mouse, dog, chicken, and Tetraodon protein sequences
CVLblockscouldnotbecombinedintothesamesubgroup.To
wereobtainedfromEnsembl(Birneyetal.2006)(v.36–Dec2005);
definethesignificanceofonepartition,CVLblocksinonesub-
Takifugu(version2)fromEnsembl;Cionaintestinalis(version1)
group (one gnathostome proto-chromosome) are unlikely to
from JGI (http://www.jgi.doe.gov); and Strongylocentrotus purpu-
share ohnologs, as illustrated in the green regions of Figure 2;
ratus(version2.1)fromNCBI.Ifmultiplesequencesoverlapped
however,incontrast,manypairsofCVLblocksindistinctgna-
inthegenome,theywererepresentedbythelongestsequence.
thostome proto-chromosomes are likely to share a significant
We conducted all-against-all BLASTP searches (Altschul et al.
1997) (E-value<1(cid:2)10(cid:1)10). Special treatments were necessary number of ohnologs, as shown by red boxes in Figure 2. We
therefore measured the significance of one partition with n
toidentifyohnologsproducedbythe2RWGD(seedetailsinthe
ohnologsamongdifferentsubgroups(i.e.,nohnologdotsinthe
Supplementalmaterials).
redregionsinFigure2H)bytheprobabilitythat(cid:1)nohnologsare
IdentificationofparalogousCVLblocksforgroupingCVL sharedbydifferentsubgroupsundertherandomdistributionof
blocks ohnologs in the whole triangular region. More precisely, the
probability, denoted by p, is defined by assuming that c is the
We evaluated the significance of the number of ohnologs, de- totalnumberofpairsofgenesinthefocalconnectedcomponent,
notedbyn,thattwoCVLblocksshareincommonbyevaluating misthetotalnumberofohnologsintheconnectedcomponent,
the probability that the two CVL blocks have (cid:1)n ohnologs in andristhetotalnumberofpairsofgenesindifferentsubgroups
commonundertheconditionthatallohnologsaredistributedat ofthepartition.Intuitively,cistheareaofthewholetriangular
randominallCVLblocks.Theprobabilityisdefinedasfollows: region in Figure 2H, and r is the total area of red regions. The
LetNbethetotalnumberofgenesinallCVLblocks,mbethe probability p is the total number of cases when each pair of
totalnumberofohnologsamongallCVLblocks,andxandybe paralogous CVL blocks in different subgroups of the partition
therespectivenumberofgenesinCVLblocksXandY.Assuming shares(cid:1)nohnologsdividedbythetotalnumberofcaseswhenm
arandomdistributionofohnologsamongCVLblocks,theprob- ohnologsappearinpairsoftwoarbitraryCVLblocks:
abilityofobserving(cid:1)nohnologsisapproximatedby (cid:1) (cid:2)(cid:1) (cid:2)(cid:3)(cid:1) (cid:2)
p=(cid:1)m(cid:1)mi(cid:2)qi(cid:2)1−q(cid:3)m−1,whereq=N(cid:2)N2x−y1(cid:3). p=(cid:1)im=n ri mc−−ri mc .
i=n
Here,pisthesummationoftheprobabilitythati((cid:1)n)amongm Thepartitionwiththeminimumprobabilityisthemostsignifi-
ohnologsaremembersofXandYwiththeprobabilityqthatan cantone,andthesubgroupsinthepartitionaretreatedasgna-
arbitrarypairofgenesselectedfromallN(N(cid:1)1)/2pairsbecomes thostomeproto-chromosomes.
anohnologsharedbyXandY. In the literature, some algorithms have been proposed for
If the probability was at most a reasonable threshold, we reconstructingpre-duplicatedgenomes(El-MabroukandSankoff
rejectedthenullhypothesisthatohnologsbetweenthetwoCVL 2003;Kellisetal.2004).Theideaofdoublyconservedsynteny
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