Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of

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Reconstruction of ancestral chromosome architecture
and gene repertoire reveals principles of genome
evolution in a model yeast genus
Nikolaos Vakirlis, Véronique Sarilar, Guénola Drillon, Aubin Fleiss, Nicolas
Agier, Jean-Philippe Meyniel, Lou Blanpain, Alessandra Carbone, Hugo
Devillers, Kenny Dubois, et al.
To cite this version:
Nikolaos Vakirlis, Véronique Sarilar, Guénola Drillon, Aubin Fleiss, Nicolas Agier, et al.. Reconstruc-
tion of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution
in a model yeast genus. Genome Research, 2016, 26 (7), pp.918-932. ￿10.1101/gr.204420.116￿. ￿hal-
01331620￿
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Research
Reconstruction of ancestral chromosome architecture
and gene repertoire reveals principles of genome
evolution in a model yeast genus
Nikolaos Vakirlis,1,6 Véronique Sarilar,2,6 Guénola Drillon,1,6 Aubin Fleiss,1
Nicolas Agier,1 Jean-Philippe Meyniel,3 Lou Blanpain,2 Alessandra Carbone,1
Hugo Devillers,2 Kenny Dubois,4 Alexandre Gillet-Markowska,1 Stéphane Graziani,3
Nguyen Huu-Vang,2 Marion Poirel,3 Cyrielle Reisser,5 Jonathan Schott,4
Joseph Schacherer,5 Ingrid Lafontaine,1 Bertrand Llorente,4 Cécile Neuvéglise,2
and Gilles Fischer1
1SorbonneUniversités,UPMCUniv.Paris06,CNRS,InstitutdeBiologieParis-Seine,LaboratoryofComputationalandQuantitative
Biology,F-75005,Paris,France;2MicalisInstitute,INRA,AgroParisTech,UniversitéParis-Saclay,78350Jouy-en-Josas,France;3ISoft,
Routedel’Orme,Parc“LesAlgorithmes”BâtimentEuclide,91190Saint-Aubin,France;4CRCM,CNRS,UMR7258,Inserm,U1068;
InstitutPaoli-Calmettes,Aix-MarseilleUniversité,UM105,F-13009,Marseille,France;5DepartmentofGenetics,Genomicsand
Microbiology,UniversityofStrasbourg/CNRS,UMR7156,67083Strasbourg,France
Reconstructinggenome history iscomplexbut necessary to reveal quantitative principles governing genome evolution.
Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture
andgenerepertoire.Here,wereconstructedthegenomehistoryofthegenusLachanceathatappearedtocoveracontinuous
evolutionaryrangefromcloselyrelatedtomoredivergedyeastspecies.Ourapproachintegratedthegenerationofahigh-
qualitygenomedataset;thedevelopmentofAnChro,anewalgorithmforreconstructingancestralgenomearchitecture;and
acomprehensiveanalysisofgenerepertoireevolution.WefoundthattheancestralgenomeofthegenusLachanceacontained
eightchromosomesandabout5173protein-codinggenes.Moreover,wecharacterized24horizontalgenetransfersand159
putativegenecreationeventsthatpunctuatedspeciesdiversification.Weretracedallchromosomalrearrangements,includ-
inggenelosses,geneduplications,chromosomalinversionsandtranslocationsatsinglegeneresolution.Geneduplications
outnumberedlossesandbalancedrearrangementswith1503,929,and423events,respectively.Genecontentvariations
betweenextantspeciesaremainlydrivenbydifferentialgenelosses,whilegeneduplicationsremainedgloballyconstant
in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to
14%ofallgenelossesbydisruptinggenesattheirbreakpoints.Finally,wefoundthatnonsynonymoussubstitutionsreached
fixationatacoordinatedpacewithchromosomalinversions,translocations,andduplications,butnotdeletions.Overall,we
provideagranularviewofgenomeevolutionwithinanentireeukaryoticgenus,linkinggenecontent,chromosomerear-
rangements,andproteindivergenceintoasingleevolutionaryframework.
[Supplementalmaterialisavailableforthisarticle.]
Eukaryoticgenomesevolvethroughtheaccumulationofpointmu- Hurst2007;Berthelotetal.2015),theyhavethepotentialtomodify
tationsandchromosomalrearrangementsthatultimatelycontrib- geneexpression,createnewgenecombinations,anddisruptgenes
utetotheevolutionofthegenerepertoire.Pointmutationscan attheirbreakpoints(Rowley1998;Perez-Ortinetal.2002;Avelar
promote gene inactivation by pseudogenization of coding se- etal.2013;Quintero-Riveraetal.2015).Unbalancedchromosomal
quences (Mighell et al. 2000; Lafontaine and Dujon 2010) but rearrangementsincludedeletionsandduplicationsofthechromo-
alsoparticipateingenegainbydenovogenecreationfromnoncod- somesegments,whichpromotereductionandexpansionofthe
ing sequences (Khalturin et al. 2009; McLysaght and Guerzoni gene repertoire, respectively (Llorente et al. 2000; Dujon et al.
2015).Balancedrearrangements—includingtranslocations,inver- 2004;Wapinskietal.2007;Butleretal.2009;Soucietetal.2009;
sions,andchromosomefusion/fission—modifygeneorderandori- Scannelletal.2011;Gabaldonetal.2013).Whole-genomeduplica-
entation. Although these rearrangements are often thought to tion(WGD)andhybridizationeventsalsoaffectgenerepertoire,as
occurmostlyinintergenicregions(Pengetal.2006;Poyatosand well-documentedinyeasts(SemonandWolfe2007;Louisetal.
© 2016 Vakirlis et al. This article is distributed exclusively by Cold Spring
6Theseauthorsequallycontributedtothiswork. HarborLaboratoryPressforthefirstsixmonthsafterthefull-issuepublication
Corresponding authors: [email protected], ncecile@ date(seehttp://genome.cshlp.org/site/misc/terms.xhtml).Aftersixmonths,it
grignon.inra.fr,[email protected] isavailableunderaCreativeCommonsLicense(Attribution-NonCommercial
Articlepublishedonlinebeforeprint.Article,supplementalmaterial,andpubli- 4.0 International), as described at http://creativecommons.org/licenses/
cationdateareathttp://www.genome.org/cgi/doi/10.1101/gr.204420.116. by-nc/4.0/.
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Vakirlis et al.
2012; Morales and Dujon 2012; Marcet-Houben and Gabaldon Basedongenomecomparisonbetweenthreepreviouslyse-
2015).Theimpactofhorizontalgenetransfers(HGTs),although quencedLachanceaspecies,wepredictedthatthenumberofrear-
seeminglyimportantinPezizomycotina,islimitedinSaccharomy- rangements that reached fixation in this genus was sufficiently
cotina,withonlyafewdozenreportedeventssofar(Rollandetal. high,butnottoohigh,toprovidekeyinformationonthedynam-
2009; Galeote et al. 2010; Marcet-Houben and Gabaldon 2010; icsofchromosomeevolution(Fischeretal.2006;Payenetal.2009;
Wisecaveretal.2014;Marsitetal.2015). Soucietetal.2009;DrillonandFischer2011;Gordonetal.2011).
Comparativegenomicshasbeeninstrumentalinidentifying Therefore,weundertookthereconstructionofgenomehistoryin
thesemechanismsanddecipheringtheircontributiontogenome thisgenustoseekforquantitativerulesthatgoverntheevolution
evolution.Notably,thestudyofsyntenyconservationacrossmul- ofgenomes.First,wesequenced,assembled,andannotatedthege-
tiple species allowed critical conceptual advances in the under- nomes of seven additional Lachancea species. With 10 fully se-
standingof genome dynamics.Comparativestudieson synteny quenced, assembled, and annotated genomes, the Lachancea
conservationrevealedhighlyvariableratesofchromosomerear- cladeisthemostdenselysampledyeastgenusatthegenomiclevel
rangementsbetweenindividuallineagesbothinvertebratesand within the Saccharomycetaceae family. Second, we developed a
inyeasts(Bourqueetal.2005;Fischeretal.2006).Interestingly, newcomputationalmethodcalledAnChro,toreconstructances-
a comparative study between 12 Drosophila genomes reported tral genome organization and identify all balanced rearrange-
thatthedisruptionofsyntenyregionsviachromosomalinversions ments that accumulated during evolution. We combined these
approximatedalinearprocessovertime(Bhutkaretal.2008).Ata reconstructionswithanexhaustivesurveyofthegenerepertoire
broaderevolutionaryscale,reconstructionsofancestralgenecon- andrevealedgeneralprinciplesthatgoverngenomeevolutionin
tentinProteobacteriaandArchaeashowedthatgenelossesand/or thisyeastgenus.
duplications correlated with amino acid substitution rates (Snel
etal.2002;CsurosandMiklos2009).Similarly,linearcorrelations
Results
betweentheratesofgenomicrearrangementssuchasgenedupli-
cationsandlosses,HGTsandgenecreations,andtherateofnon-
High-qualityreferencegenomesoftheLachanceagenus
synonymous substitutions were recently reported in bacteria
(Puigboetal.2014).Byanalogytothetraditionalmolecularclock Wesequencedandassembledintoonescaffoldperchromosome
(ZuckerkandlandPauling1962),theinvestigatorscoinedtheterm the nuclear genomes of seven Lachancea species (see Methods).
“genomicclock”todescribethecoordinatedpaceoffixationbe- Haploidgenomesizesrangefrom10.2to11.3 Mb(Fig.1A).All
tweenpointmutationsandlarge-scalerearrangements.Thefirst Lachanceaspecieshaveeightchromosomes,eachcontainingone
attempttodefineagenomicclockinyeastwasbasedonthecorre- centromere with the three typical elements CDEI, CDEII, and
lationbetweensyntenyconservationandaminoacididentitybe- CDEIII (Supplemental Fig. S1; Supplemental Table S1), except
tweenorthologousgenes(RollandandDujon2011). Lachancea fantastica that has only seven chromosomes because
Reconstructing genome history is a rather difficult task re- of a telomere-to-telomere fusion (Supplemental Fig. S2; Supple-
quiringefficientreconstructionofancestralgenomeorganization mentalTableS2).ThegenomicGCcontentrangesfrom41.2%–
andprecisecharacterizationofthechromosomalrearrangements 47.3%(SupplementalTableS2).InL.kluyveri,aregionof1Mbcon-
thatoccurredalongdifferentlineages.Reconstructionofancestral tainingtheMATlocusandcoveringalmostthewholeleftarmof
genome architecture has benefited from the development of chromosomeC,hasanaverageGCcontentof52.9%,whichissig-
several computational models (Ma et al. 2006; Faraut 2008; nificantlyhigherthanthe40.4%globalGCcontentoftherestof
Muffato and Roest Crollius 2008; Alekseyev and Pevzner 2009; thegenome(Payenetal.2009;Soucietetal.2009).Theortholo-
Ouangraouaetal.2011;Gagnonetal.2012).However,integrating gouscounterpartofthischromosomalregionisfoundinallother
thereconstructionofancestralgenomearchitectureintoanevolu- Lachancea species, but none of them presented the GC content
tionaryframeworkhasonlybeenachievedinalimitednumberof heterogeneitycharacterizedinL.kluyveri,reinforcingthehypoth-
cases.Inyeast,Wolfeandcolleaguesmanuallyreconstructedthe esisofanintrogressioneventattheoriginofthischromosomal
ancestralgenomeofSaccharomycescerevisiaeasitwasbeforethe arm(Friedrichetal.2015).
WGDeventandidentifiedatleast144structuralrearrangements, Weannotatedthecodingandnoncodingelementsofthesev-
as well as 124 genes that are present in the actual S. cerevisiae ennewlysequencedgenomesandreannotatedthethreeprevious-
genomebutabsentfromitsancestor(Gordonetal.2009).These lysequencedgenomes.Protein-codinggenesrangefrom4997in
investigators also traced the complete rearrangement history of Lachanceameyersiito5378inL.kluyveri,andpseudogenesrange
the Lachancea kluyveri genome since its common ancestor with from52inLachanceacidrito104inLachanceanothofagi(excluding
S. cerevisiae (Gordon et al. 2011). In vertebrates, a recent study Lachanceawaltiiwheregapsintheoriginalsequence[Kellisetal.
usedancestralgenomereconstructiontoexplainthedistribution 2004] artificially increase the number of pseudogenes to 295)
patternofrearrangementbreakpointsinBoreoeutheriangenomes (SupplementalTableS2).Onaverage,codingsequencesrepresent
(Berthelotetal.2015).Theinvestigatorsfoundastrongpositive between67%and77%ofthegenome(Fig.1A),similartomost
correlationbetweengenedensityandevolutionaryrearrangement Saccharomycotina sequenced genomes (Dujon et al. 2004).
breakpoints and showed that this property could be extended Finally, we found a small number of Class I retrotransposons
toyeastgenomes.Finally,Wengetal.(2014)recentlyreconstruct- (fromone to17) inall speciesexceptinL. cidriandL.meyersii,
ed the ancestral genome organization of highly rearranged while Class II elements are more widespread, with at least one
Geraniaceaeplastidgenomesandcharacterizedtherearrangements copyinL.cidriandLachanceafermentati andupto41copiesin
unique to each genus. They found that the degree of plastid L.fantastica(SupplementalTableS2;Sarilaretal.2014).
genome rearrangements was correlated with nonsynonymous Wefoundnocorrelationbetweengenomesizeandeitherthe
substitution rates but not with synonymous substitution rates, numberofgenes,thecumulativesizeofcodingsequences,orthe
compatible with the existence of a genomic clock in plastid transposableelementcontent.However,wefoundaclearpositive
genomes. correlation between genome sizes and the cumulative sizes of
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Reconstruction of genome history in Lachancea
Figure1. (A)Cumulativesequencelengthoftheannotatedgeneticelementsinthe10Lachanceagenomes.Thepercentagesofprotein-codingsequenc-
esareinparentheses.(B)GenomesizeinLachanceapositivelycorrelateswithintergenelength(top)butnotwithcumulativegenelength(bottom).(C)
Phylogenyof34Saccharomycetaceaespeciesinferredfromamaximumlikelihoodanalysisofaconcatenatedalignmentof756familiesofsyntenichomo-
logs.ThetreetopologywithintheLachanceacladeremainsidenticalforseveralreconstructionmethods:concatenationtree,majoritytree,andextended
majorityruleconsensus(eMRC)tree(seeMethods).InternalbrancheswithintheLachanceacladearenamedb tob .Thecorrespondinginternodecer-
1 9
tainty(IC)values,indicatingtherobustnessoftheeMRCtreetopology,aregiven.(WGD)Whole-genomeduplication.(D)Relationshipbetweenorthol-
ogousproteinsimilarityandthenumberofconservedsyntenyblockswithindifferentyeastgenera.TheLachanceagenusistheonlycladeshowinga
continuumofgenomereorganizationandpairwiseproteinsimilaritiesoveralargeevolutionaryrange.
intergenicregionsandintrons(Fig.1B;SupplementalFig.S3).The betweengenomesizesaremainlyduetovariationsinnoncoding
largestsizevariationinintergenicregionsequalsatotalof1.38Mb sequencelengthandnottodifferentialgeneortransposableele-
betweenL.fermentatiandL.meyersii,showingthatthedifferences mentcontent.Interestingly,otherstudiesalsoreportedgenome
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Vakirlis et al.
sizechangestargetedtowardintergenicregions.Adecreaseingene tionandthemajoritytopologies(seeMethods).Weappliedthein-
densitywithincreasinggenomesizewasobservedinAscomycota ternodecertainty(IC)measure(SalichosandRokas2013;Salichos
genomes,(KelkarandOchman2012),andasimilarcorrelationwas etal.2014)toestimatetherobustnessoftheeMRCtopology.Sixof
observedfor81Saccharomycotinamitochondrialgenomes(Freel eightinternalbrancheshavegoodsupportingvalues (IChigher
etal.2015b). than0.8)(Fig.1C).Branchesb andb havethelowestICvalues
4 8
(0.47and0.22,respectively);however,theircorrespondingbipar-
Arobustspeciestreetoreliablyreconstructgenomehistory titionsare7.2and3.2timesmorefrequentthantheirsecondmost
prevalentbipartitions,indicativeofaweakerbutstillexploitable
Establishingarobustspeciesphylogenetictreeisacrucialprerequi-
phylogenetic signal (Fig. 1C). Altogether these analyses show
sitetoanyevolutionaryreconstruction.Thephylogeneticposition
that the Lachancea phylogenyis robustand reliablefor genome
oftheLachanceacladewasfirstinferredfromamaximumlikeli-
historyreconstruction.
hoodanalysisofaconcatenatedalignmentof756familiesofsyn-
tenichomologs(seeMethods)sharedby36species(Supplemental
Table S3). The resulting tree shows that all Lachancea species
HGTsandgenecreationeventscontributedtothegene
shareamonophyleticorigin,supportingtheexistenceofthege-
repertoireevolution
nus(Fig.1C).
Wefurtherreconstructedallgenetreesforthe3598setsof Wecharacterized24eventsofputativeHGTthatcorrespondtoato-
orthologspresentinthe10LachanceaspeciesandinS.cerevisiae talof85codingsequences(CDS)inLachancea(0.2%oftheCDSand
(seeMethods).Atotalof796differenttopologieswereobserved pseudogenes;seeMethods).Twenty-threeeventsarenovelcom-
amongthe3598individualgenetrees.Themostprevalenttopol- pared to previously reported HGT cases (Rolland et al. 2009;
ogywassharedby472genetrees(majoritytopology).Thesameto- Morel et al. 2015). The 24 HGT families are similar to proteins
pologywassystematicallyretrievedfromtheconcatenationsofthe fromPezizomycotinaspecies(10cases),fromothereukaryotes(three
corresponding472alignments,the3598alignments,orthe756 cases)andfrombacteria(11cases)(SupplementalTableS4).The
families of syntenic homologs (Supplemental Fig. S4). We also phyletic patterns show that eight HGTs are common to several
showed that the extended majority rule consensus (eMRC) tree Lachanceaspecies;therefore,thetransferswouldhavehappened
(Felsentein1995)topologywasidenticaltoboththeconcatena- alonginternalbranchesofthetree(Fig.2).NineHGTfamiliesare
Figure2. EvolutionoftheLachanceagenerepertoire.Thenumberofgeneduplications(inblue)andlosses(inred)wereestimatedoneachbranchofthe
treeunderabirth–deathevolutionarymodel(Methods).Thetotalnumberofgenelossesindicatedonthefigure(1036)comprisesthe107casesofdubious
loss(seetext).Thestatisticalsignificanceoftheenrichmentofgenelossesassociatedtorearrangementscomparedtotheproportionofgenesassociatedto
rearrangementsisindicatedbyanasterisk(P<0.05)ordoubleasterisk(P<10−4).Notableexamplesofgenelossesareinparenthesesbelowtheircorre-
spondingbranches.Thephyleticpatternsofthe51,110CDSand1018pseudogeneswereusedtomapHGTsandgenecreationevents(ingreen)in
thedifferentbranchesofthetree(Methods).Thenumberofspecies-specificsingletonsisingrayatthetipofeachterminalbranch.Thetotalnumber
ofgenesineachancestralgenomeandthenumberofgenesandpseudogenesinextantspeciesareindicatedbetweensquaredbrackets.
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Reconstruction of genome history in Lachancea
similartoproteinsofunknownfunction;threearesimilartopro- atelevelsofgenomereorganization,rangingfromhighlycollinear
teinsinvolvedinDNAmetabolism,i.e.,atranscriptionregulator genomes down to significantly reordered chromosome maps
(pseudogene),anendonuclease,andaserinerecombinaseprevi- (Fig.1D).Thiscontinuousrangeofrelatednessisalsorecognizable
ouslydescribed(Rollandetal.2009);andthe12othersaresimilar through the pairwise protein similarities shared between
toproteinswithcatalyticactivitiesmostlybelongingtooxidation- Lachanceaorthologs,rangingfrom69%–83%(Fig.1D).Moreim-
reductionprocesses(SupplementalTableS4).Interestingly,homo- portantly,divergenceinthegenusLachancea,inbothtermsofpro-
logs of the polysaccharide lyase family 3 of the phytopathogen tein sequences and chromosome reorganization, remains below
fungusBotrytiscinerea(noblerotfungus)arepresentinL.fantastica, the thresholds beyond which the accumulation of too many
L.meyersii,Lachanceathermotolerans,andL.waltii(FamilyID4751) mutations and rearrangements leads to the progressive loss of
(SupplementalTableS4).Polysaccharidelyasesaremostlyfoundin detectable synteny blocks, which prevents any reliable recon-
phytopathogensbecausetheycatalyzetheeliminativecleavageof struction of genome history (Drillon and Fischer 2011). Such
pectin, whichis a majorcomponent of the primarycell wallof continuousevolutionaryrangeissofaruniqueamongsequenced
manyplants.L.fantastica,L.waltii,andL.thermotoleranswereiso- Saccharomycotina species (Fig. 1D) and makes the genus
latedfromplant-associatedhabitats,whileL.meyersiiwasisolated Lachanceaanidealcandidatefortheevolutionaryreconstruction
fromseawater.Consistentlywithecologicaldistribution,theho- ofgenomehistory.
mologinL.meyersiiishighlydivergedandonlypartiallysimilar
tothemembersinthethreeotherspecies.Overall,ourstudysug- AnChro,anewcomputationaltooltoreconstructancestralgenome
geststhatthecontributionofHGTsontheevolutionofthegene architecture
repertoireinyeastisprobablyunderestimated.
Wedevelopedanewcomputationalmethodofancestralgenome
We also characterized 596 taxonomically restricted gene
reconstructionnamedAnChro. Thistoolis partof anintegrated
(TRG)familiesthatarespecifictotheLachanceacladewithoutany
suiteofsoftwarenamedCHROnicle(freelyavailableatwww.lcqb
detectable homolog in the nonredundant sequence database or
.upmc.fr/CHROnicle/).Briefly,inthefirststepofthereconstruc-
conserveddomaininthePFAMdatabase(seeMethods).Sixty-six
tion, SynChro identifies conserved synteny blocks between pair-
TRGfamilies(encompassing316CDSandfourpseudogenes)com-
wise combinations of genomes (Drillon et al. 2014). In the
prisemembersinatleasttwodifferentLachanceaspeciesand/orsev-
second step, ReChro constructs cycles of linked breakpoints be-
eralparalogswithinagivenLachanceaspecies(SupplementalTable
tweenadjacentsyntenyblocks,andinthelaststep,AnChroinfers
S5). Therefore, they could result from de novo gene formation
theancestralgeneorderbycomparisonwithexternalreferencege-
eventsthatoccurredintheLachanceaclade.Theirphyleticpatterns
nomes(seeSupplementalInformation).
were used together with a birth–death–innovation evolutionary
There are nine ancestralgenomesintotalinthe Lachancea
model(seeMethods)tomaptheseeventsontheLachanceatree
phylogenetictree(namedL.A1toL.A9)(Figs.2,3).Genomerecon-
(Fig.2).Theevolutionaryratesforthese66TRGfamiliesaregener-
structionsforthesenineancestorsresultedineightancestralge-
allyabovethemedianevolutionaryrateofthesetoforthologous
nomes composed of eight chromosomes and in one composed
genes(seeMethods)butremainwithinthedistribution,suggesting
of nine scaffolds,probably because one ancestraladjacency was
thatnoremotehomologwouldhavebeenmissedbecauseofunusu-
notreconstructed(Fig.3,L.A4).Thenumberofgenesperancestral
allyhighdivergence.Fourfamiliesshowanonsaturatedrateofsyn-
genomevariesbetween4446forL.A1and4799forL.A9(Fig.3).
onymous substitutions (d <1), and all of them have a mean
S Each ancestral genome is provided as a list of ordered ancestral
pairwiseratioofnonsynonymousoversynonymoussubstitution
geneswiththeircorrespondingorthologousgenesinall10extant
ratesofd /d <1,suggestingthattheycouldbeunderpurifyingse-
N S Lachancea species (Supplemental Table S6). The robustness of
lection(SupplementalTableS5).
AnChro’sreconstructionswascomprehensivelytestedby(1)cal-
Theremaining530singletonsbeartheusualcharacteristicsof
culatingtheprobabilityofreconstructingancestralgenomeorga-
TRG(Khalturinetal.2009);theygloballyhavealowerGCcontent,
nization with a single centromere by chromosome, given that
asmallersize,andalowercodonadaptationindex(CAI)valuethan
AnChrodoesnotuseanyinformationofcentromerepositiontore-
orthologousgenesequences.With131predictedgenes,theL.kluy-
construct ancestral genome organization (see Supplemental
veribranchencompassesthehighestnumberofspecies-specificsin-
Information); (2) comparing the reconstructions to previously
gleton genes. We used the available population genomic data
publishedancestralgenomes(Fig.4;SupplementalInformation;
(Friedrichetal.2015)tocheckwhethertheseCDSareconservedbe-
Gordonetal.2009,2011;Jonesetal.2012);and(3)benchmarking
tweenthegenomesofL.kluyveriisolateswerecentlysequenced.We
AnChro against four existing reconstruction software tools on
foundthat114CDShavehomologsconservedinseveralstrains
bothrealandsimulated genomedata sets (Fig.4;Supplemental
and,therefore,probablycorrespondtorealgenes.Theremaining
Information). All these tests showed that AnChro achieved the
17 CDS are absent or pseudogenized in all other sequenced ge-
most reliable and complete reconstruction of ancestral chromo-
nomes,suggestingthatthesegenesshouldbeconsidereddubious.
somearchitecture.
Fortheotherninespeciesforwhichnopopulationdataareavail-
able,all403species-specificsingletonsarealsopresentlyconsidered
Unbalancedrearrangementsoutnumberedbalanced
asdubiousgenes.Altogether,thenonverticallyinheritedgenesin
rearrangements
Lachanceawouldresultfromaminimumof24HGTand159gene
creationevents,whichhaveenrichedthegenus’generepertoire. We performed two independent analyses to identify both
balancedchromosomalrearrangements,i.e.,translocations,inver-
sions,andchromosomefusion/fission,andunbalancedrearrange-
ThegenusLachanceacoversauniquecontinuousevolutionary
ments, i.e., duplications and deletions, that occurred since
rangeinSaccharomycotina
divergencefromthelastcommonancestorofthegenus.
The number and size of conserved synteny blocks between To identify balanced rearrangements, we used SynChro
Lachanceaspeciesrevealthattheyshareacontinuumofintermedi- (Drillon et al. 2014) to compute the synteny blocks between
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Figure3. ChromosomalhistoryoftheLachanceagenomes.Thechromosomalstructuresofthe10extantspeciesandtheancestralgenomesL.A2toL.A9
arerepresentedasafunctionofthegenomestructureofL.A1,thelastcommonancestoroftheclade.Thenumberofgenesofeachancestralgenomeis
indicated with brackets. The total number of translocations and inversions accumulated between two genomes is indicated above each branch.
RearrangementsinvolvingMAT,HML,HMR,rDNA,orcentromeresareindicatedbeloweachbranch.TherelocationoftherDNAarrayoccurredinthe
branchbetweenL.A4andL.A5.Thistranspositioneventoccurredintra-chromosomallyfromanancestralsite,representedasapurpleregioninL.A4,to
anewgenomiclocationclosetothegreencentromereinL.A5.TherelativeorientationoftherDNAandthecentromerewasinvertedbetweenL.A4
andL.A5,suggestingthattherDNArelocationresultedfromtwointra-chromosomalinversionsinvolvingonebreakpointreuse(SupplementalFig.S5;
SupplementalTableS6).TheintervalbetweenMATandHMLwasneverbrokenandwasinheritedintactfromL.A1inallextantspeciesexceptL.kluyveri,
whichlostbothHMLandHMR.TheHMRcassetteunderwentmanyrearrangements(threetranslocations,oneinversion,andoneduplication)butalways
remainedsubtelomeric.TheHML,HMR,andtheMATlociwerelocatedonthesamechromosomeinthelastcommonancestorofthegenus,L.A1,withone
silentcassetteateachchromosomeend.TheonlyLachanceaspeciesthatalsoharborsthethreesexuallocionasinglechromosomeisL.fermentati,butthis
organizationisnotinheritedfromL.A1asitresultsfromadditionaltranslocationsinthebranchbetweenL.A3andL.fermentati.Therefore,noneofthe
present-dayLachanceaspecieshasretainedtheoriginalchromosomalorganizationofthesexualloci.Theinsetplotrecapitulatesthetotalnumberoftrans-
locationsandinversionsthataccumulatedsinceeachextantspeciesdivergedfromthelastcommonancestor,L.A1.
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Figure 4. (A) Comparison between the two versions of the L.A1 ancestral genome reconstructed by AnChro and by ANGES (Jones et al. 2012).
Chromosome painting representations of ancestral genomes are colored relatively to the L. kluyveri chromosomes. The black triangles indicate the
same12ancestraladjacenciesthatresultedfromsixtranslocationsidenticallyreconstructedbythetwotools.(B)Comparisonbetweenthemanuallyre-
constructed(Gordonetal.2009,2011)andtheAnChroversionofthepre-WGDancestralgenomerelativetotheL.kluyverigenome.Theonlyinter-chro-
mosomaldifferencebetweenthetworeconstructionsisindicatedbytheredtriangles.(C)ComparisonofthenineLachanceaancestors(L.A1toL.A9)
reconstructedbyAnChro,ANGES,GapAdj,MGRA,andPMAG+.SyntenyblockswerecomputedwithI-ADHoReforthefivereconstructiontools.For
AnChro,asingledefaultreconstructionispresented.Eachcolumnrepresentstheancestralchromosomesofagivenancestorasanalternationofgray
andbeigeboxes,withsizebeingproportionaltothenumberofreconstructedancestralgenes.Thesmallblackcirclesindicatethecentromereposition.
Thesmallredcirclesindicatethecentromerepositionswhenanancestralchromosomewasreconstructedwithtwocentromeres.(D)Ancestralgenome
reconstructionsonsimulatedgenomes.Thefigurepresents900reconstructedancestralgenomescorrespondingtoninedifferentancestorspersimulation
and100simulations,performedwithAnChro(defaultreconstructions),ANGES,GapAdj,MGRA,andPMAG+.Eachgenomereconstructionisrepresented
byadot.Thequalityofthereconstructionsisassessedbythreecriteria:thenumberofancestralscaffolds(ideallyeight),thenumberofancestralgenes
(ideally5000),andtheproportionofscaffoldsperreconstructionwithasinglecentromere(ideally100%).(E)Averageproportionsofcorrectlyandincor-
rectlyreconstructedadjacenciesforthe900reconstructionsobtainedbythefivetools.Incorrectadjacenciesaredecomposedinsingleblockinversionsand
intra-andinter-chromosomalcontradictions.Theaverageproportionofadjacenciesthatwerenotreconstructedbythedifferentsoftwareisalsoindicated.
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Vakirlis et al.
consecutiveancestralgenomesininternalbranchesofthetreeand Byintegratingallthesegeneexpansionsandreductions,we
between an ancestor and an extant genome in the terminal estimatedthatthelastcommonancestorofthegenusLachancea,
branches.TheSynChrostringencyparameterwassettozerotoal- L.A1,containedabout5173genes(Fig.2).Thenumberofgenes
lowbuildingsyntenyblockscomprisingasingleinvertedortholo- inextantgenomesrangesfrom4768inL.waltiito5378inL.kluy-
gous gene-pair (see Supplemental Information). These blocks veri, not very different from the estimated ancestral number of
subsequently served as inputs for ReChro to identifyall the bal- genes.Thesecomparablefiguresmightgivetheimpressionthat
ancedrearrangementsthatoccurredineachbranchofthetree,in- thetotalgenenumberinLachanceagenomeshasreachedequilib-
cluding single gene inversions. We identified a total of 423 riumwheregeneexpansioneventsroughlyequalgenereduction
balancedrearrangements(Fig.3).Thenumberofrearrangements events. However, weobserved between 200 and 400 gene gains
accumulated between L.A1 and the different Lachancea species per lineage since the divergence from L.A1, while gene losses
washighlyvariable,from22inL.kluyveriupto152inLachancea werehighlyvariable,rangingfrom31inL.kluyverito466inL.fan-
dasiensis(Fig.3,insetplot).Theserearrangementscorrespondto tastica.Asaresult,wefoundanegativecorrelationbetweenthe
136inversions,includingninewithendpointsattelomeres;147 numberofgenesinextantgenomesandthenumberofgenelosses
translocations,including140reciprocaltranslocations;seventelo- thatoccurredsincethedivergencefromthelastcommonancestor
meric nonreciprocal translocations; and 140 rearrangements for oftheclade(R2=0.85,P=1.4×10−4)(Fig.5B),butnotwiththe
which it was not possible to discriminate between inversions number of gene duplications or HGTs/de novo creations.
and translocations because of breakpoint reuse. We identified Therefore, despite more abundant gene gains, the variation of
102casesofinversioncorrespondingtoindividualchromosomal the number of genes between extant genomes mainly results
eventswithnooverlaporbreakpointreusewithotherrearrange- fromthenumberofgenelossesthatoccurredalongthedifferent
ments.Thesizedistributionofthese102inversionsfitsapower branchesofthetree.Remarkably,theespeciallylowlevelofgene
law,clearlyshowingthatsmallinversionsarefavoredoverlonger lossesinL.kluyveriraisestheexcitingpossibilitythatmostgene
ones(Fig.5A).Onlytwoverylargeinversionsof318and351genes lossesoccurbynonhomologousendjoining(NHEJ),sinceessen-
werefound. tialcomponentsofthisDSBrepairpathwayhavebeenspecifically
We used a birth–death evolutionary model on the gene lostinL.kluyveri(Gordonetal.2011,andseebelow).
familyclassificationofthecompletesetofprotein-codinggenes Finally,wefoundanegativecorrelationbetweenthenumber
fromthe10Lachanceagenomestoidentifyunbalancedrearrange- of genes and pseudogenes present in extant genomes and the
ments (see Methods). We characterized 1503 gene duplications number of balanced rearrangements (inversions and transloca-
and1036genelosses.Wecheckedallgenelossesbylookingfor tions)thataccumulatedsincethedivergencefromthelastancestor
syntenichomologsthatwouldhavebeenmissedduringeitherge- L.A1(R2=0.70,P=1.7×10−5)(Fig.5B).Thisrelationshipwasrath-
nomeannotationorgenefamilyclusteringbecauseofalevelof erunexpectedandsuggestedthatfixedbalancedrearrangements
divergence that could have exceeded the threshold. We filtered couldberesponsibleforasignificantproportionofgenelosses.
out107casesofsuchdubiouslosses,leavingatotalof929gene
losses, clearly outnumbered by the 1503 gene duplications. We
Balancedrearrangementsfrequentlydisruptgenes
then determined their positions in the phylogenetic tree (Fig.
attheirbreakpoints
2).For132genefamilieswherethephyleticpatternsclearlyindi-
cated which members of the family corresponded to the dupli- Wetestedwhethersomegenelossescouldresultfromgenedisrup-
cated copies, we found 94 inter- and 38 intra-chromosomally tioncausedbyinversionsortranslocationswithendpointswithin
duplicated copies.The distribution of the distances between in- codingsequences.Fromourinitialestimateof929genelosses,we
tra-chromosomallyduplicatedcopiesisbimodal,with20events excludedthe125lossesspecifictoL.waltiibecausethesequencing
separatedby<10kb,possiblyresultingfromtandemduplications gapsinthecurrentgenomeassemblyartificiallyincreasethenum-
(SupplementalFig.S6). berofgenelossesinthisbranch(Kellisetal.2004;DiRienzietal.
Attheleveloftheentireclade,unbalancedrearrangements 2011),yielding804genelosses,whichwerepositionedinthedif-
aresixtimesmoreabundantthaninversionsandtranslocations. ferentbranchesofthetree.Foreachlostgene,weconsideredits
Notethatthisratiomightbeoverestimatedbecausethenumber twoflankinggenesinthespeciesthatdidnotundergotheloss.
of gene duplications and losses characterized in this work does Wethendeterminedthepositionsoftheorthologsoftheseflank-
notnecessarilycorrespondtothenumberofeventsthatoccurred inggenesinthegenomethatunderwenttheloss.Foreachposition,
sincesomeduplicationsanddeletionscouldhaveinvolvedseveral welookedatwhetheratleastoneoftheseorthologswasattheex-
genesatthesametime.Altogether,thisdetailedandexhaustive tremityofasyntenyblockinvolvedinabalancedrearrangement
catalogofbalancedandunbalancedchromosomalrearrangements predictedbyReChroonthecorrespondingbranch.Wefoundthat
positionedonthedifferentbranchesofthephylogenetictreepro- 109lossescolocalizedwitharearrangementbreakpointinagiven
videstheopportunitytoidentifyquantitativeprinciplesgovern- branchofthetree(Fig.2).Thisresultsuggeststhatupto14%of
inggenomeevolution. allgenelosses(109/804)couldresultfromthedisruptionofacod-
ingsequencebyaninversionoratranslocationevent.Wecalculat-
ed the statistical significance of this result by testing the global
Thenumberofgenesinextantgenomesisdriven
enrichmentofgenelossesassociatedtorearrangementscompared
bythenumberofancestralgenelosses withtheproportionofgenesassociatedtorearrangements(χ2test,
TheLachanceagenerepertoireunderwent1686expansionevents P<1.9×10−30).Thesamecalculationperformedoneachbranchof
due to 1503 gene duplications, 159 putative gene creations, 24 thetreeshowedsignificativityinonlysevenoutofthe17branches
HGTs,and1947reductionevents,correspondingto1018pseudo- because of the small sample size in each branch (Fig. 2).
genizationsbypointmutationsorsmallindelsand929genelosses Remarkably,wefoundthreegenerelics,i.e.,highlydegenerated
bydeletionordisruptionofthecodingsequencebyarearrange- remnantsofgenes(Lafontaineetal.2004),withinintergenicse-
mentbreakpoint. quencescorrespondingtorearrangementbreakpoints.Suchrelics
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Reconstruction of genome history in Lachancea
B
A
C D
E F
Figure5. (A)Distributionofinversionsizes(innumberofgenes)accumulatedsincethedivergencefromthelastcommonancestorofthegenus(L.A1).
Theredlinesymbolizesapowerlawfittothedata(P(s)=C∗s−a,withC=106.4andα=1.86),whichrepresentstheprobabilityofaninversionhavingits
twoend-pointsatsgenesapart.Theinsetplotshowsacumulativehistogramofthesamedataplottedwithlogarithmicscale.(B)Correlationsbetweenthe
numberofgenesandpseudogenesinextantgenomesandnumberofbalancedrearrangements,i.e.,inversionsandtranslocations(lefty-axis)andwiththe
numbersofgenelosses,i.e.,genedisruptionsanddeletions(righty-axis).(∗∗)P<10−4.Nocorrelationwasfoundbetweenthenumberofgenesinextant
genomesandthenumberofgeneduplications(datanotshown).(C)Correlationbetweenthenumberofbalancedrearrangements(inversionsandtrans-
locations)andthecorrespondingindividualbranchlengthsfromtheLachanceaspeciestreebasedontheconcatenationof3598orthologousgenescor-
respondingto1,983,702alignedpositions(SupplementalFig.S4).(∗∗)P<10−4.(D)Correlationbetweenthenumberofgenedisruptionsresultingfrom
balancedrearrangementsandbranchlengthsestimatedasinC.(∗∗)P<10−4.(E)Correlationbetweenthenumberofgeneduplicationsandbranchlengths
estimatedasinC.(∗)P<0.05.(F)NosignificantcorrelationbetweenthenumberofgenedeletionsandbranchlengthsestimatedasinC.
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