A high-resolution, chromosome-assigned Komodo dragon ... · 71 lizards are all present in the...
Transcript of A high-resolution, chromosome-assigned Komodo dragon ... · 71 lizards are all present in the...
Ahigh-resolution,chromosome-assignedKomododragongenomerevealsadaptationsinthe1
cardiovascular,muscular,andchemosensorysystemsofmonitorlizards2
3
AbigailL.Lind1,YvonneY.Y.Lai2,YuliaMostovoy2,AlishaK.Holloway1,AlessioIannucci3,Angel4
C.Y.Mak2,MarcoFondi3,ValerioOrlandini3,WalterL.Eckalbar4,MassimoMilan5,Michail5
Rovatsos6,7,,IlyaG.Kichigin8,AlexI.Makunin8,MartinaJ.Pokorná6,MarieAltmanová6,Vladimir6
A.Trifonov8,ElioSchijlen9,LukášKratochvíl6,RenatoFani3,TimS.Jessop10,TomasoPatarnello5,7
JamesW.Hicks11,OliverA.Ryder12,JosephR.MendelsonIII13,14,ClaudioCiofi3,Pui-Yan8
Kwok2,4,15,KatherineS.Pollard1,4,16,17,18,&BenoitG.Bruneau1,2,199
10
1.GladstoneInstitutes,SanFrancisco,CA94158,USA.11
2.CardiovascularResearchInstitute,UniversityofCalifornia,SanFrancisco,CA94143,USA.12
3.DepartmentofBiology,UniversityofFlorence,50019SestoFiorentino(FI),Italy13
4.InstituteforHumanGenetics,UniversityofCalifornia,SanFrancisco,CA94143,USA.14
5.DepartmentofComparativeBiomedicineandFoodScience,UniversityofPadova,3502015
Legnaro(PD),Italy16
6.DepartmentofEcology,CharlesUniversity,12800Prague,CzechRepublic17
7.InstituteofAnimalPhysiologyandGenetics,TheCzechAcademyofSciences,2772118
Liběchov,CzechRepublic19
8.InstituteofMolecularandCellularBiologySBRAS,Novosibirsk630090,Russia20
9.B.U.Bioscience,WageningenUniversity,6700AAWageningen,TheNetherlands21
10.CentreforIntegrativeEcology,DeakinUniversity,WaurnPonds3220,Australia22
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
11.DepartmentofEcologyandEvolutionaryBiology,SchoolofBiologicalSciences,Universityof23
California,Irvine,CA92627,USA.24
12.InstituteforConservationResearch,SanDiegoZoo,Escondido,CA92027,USA25
13.ZooAtlanta,AtlantaGA30315,USA.26
14.SchoolofBiologicalSciences,GeorgiaInstituteofTechnology,Atlanta,GA,30332,USA.27
15.DepartmentofDermatology,UniversityofCalifornia,SanFrancisco,CA94143,USA.28
16.DepartmentofEpidemiologyandBiostatistics,UniversityofCalifornia,SanFrancisco,CA29
94158,USA.30
17.InstituteforComputationalHealthSciences,UniversityofCalifornia,SanFrancisco,CA31
94158,USA.32
18.Chan-ZuckerbergBioHub,SanFrancisco,CA94158,USA.33
19.DepartmentofPediatrics,UniversityofCalifornia,SanFrancisco,CA94143,USA. 34
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
Summary35
Monitorlizardsareuniqueamongectothermicreptilesinthattheyhaveahighaerobiccapacity36
anddistinctivecardiovascularphysiologywhichresemblesthatofendothermicmammals.We37
havesequencedthegenomeoftheKomododragon(Varanuskomodoensis),thelargestextant38
monitorlizard,andpresentahighresolutiondenovochromosome-assignedgenomeassembly39
forV.komodoensis,generatedwithahybridapproachoflong-rangesequencingandsingle40
moleculephysicalmapping.ComparingthegenomeofV.komodoensiswiththoseofrelated41
speciesshowedevidenceofpositiveselectioninpathwaysrelatedtomuscleenergy42
metabolism,cardiovascularhomeostasis,andthrombosis.Wealsofoundspecies-specific43
expansionsofachemoreceptorgenefamilyrelatedtopheromoneandkairomonesensinginV.44
komodoensisandseveralotherlizardlineages.Together,theseevolutionarysignaturesof45
adaptationrevealgeneticunderpinningsoftheuniqueKomodosensory,cardiovascular,and46
muscularsystems,andsuggestthatselectivepressurealteredthrombosisgenestohelp47
Komododragonsevadetheanticoagulanteffectsoftheirownsaliva.Astheonlysequenced48
monitorlizardgenome,theKomododragongenomeisanimportantresourcefor49
understandingthebiologyofthislineageandofreptilesworldwide.50
51
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Introduction52
Theevolutionofformandfunctionintheanimalkingdomcontainsnumerousexamples53
ofinnovationanddiversity.Withinvertebrates,non-avianreptilesareaparticularlyinteresting54
lineage.Thereareanestimated10,000reptilespeciesworldwide,foundoneverycontinent55
exceptAntarctica,withadiverserangeofmorphologiesandlifestyles1.Thistaxonomic56
diversitycorrespondstoabroadrangeofanatomicandphysiologicaladaptations.57
Understandinghowtheseadaptationsevolvedthroughchangestobiochemicalandcellular58
processeswillrevealfundamentalinsightsinareasrangingfromanatomyandmetabolismto59
behaviorandecology.60
Thevaranidlizards(genusVaranus,ormonitorlizards)areanunusualgroupof61
squamatereptilescharacterizedbyavarietyoftraitsnotcommonlyobservedwithinnon-avian62
reptiles.Varanidlizardsvaryinmassbyclosetofiveordersofmagnitude(8grams–10063
kilograms),comprisingthegenuswiththelargestrangeinsize2.Amongthesquamatereptiles,64
varanidshaveauniquecardiopulmonaryphysiologyandmetabolismwithnumerousparallels65
tothemammaliancardiovascularsystem.Forexample,theircardiacanatomyallowshigh66
pressureshuntingofoxygenatedbloodtosystemiccirculation3.Furthermore,varanidlizards67
canachieveandsustainveryhighaerobicmetabolicratesaccompaniedbyelevatedblood68
pressureandhighexerciseendurance4–6,whichfacilitateshigh-intensitymovementswhile69
huntingprey7.Thespecializedanatomical,physiological,andbehavioralattributesofvaranid70
lizardsareallpresentintheKomododragon(Varanuskomodoensis).Asthelargestextantlizard71
species,Komododragonscangrowto3metersinlengthandrunatspeedsofupto2072
kilometersmilesperhour,whichallowsthemtohuntlargepreysuchasdeerandboarintheir73
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nativeIndonesia8.Komododragonshaveahighermetabolismthanpredictedbyallometric74
scalingrelationshipsforvaranidlizards9,whichhelpsexplainstheirextraordinarycapacityfor75
dailymovementtolocateprey10.Theirabilitytolocateinjuredordeadpreythroughscent76
trackingoverseveralkilometersisenabledbyapowerfulolfactorysystem8.Additionally,77
serrateteeth,sharpclaws,andsalivawithanticoagulantandshockinducingpropertiesaid78
Komododragonsinhuntingprey11,12.Komododragonshaveaggressiveintraspecificconflicts79
overmating,territory,andfood,andwildindividualsoftenbearscarsfrompreviousconflicts8.80
81
TounderstandthegeneticunderpinningsofthespecializedKomododragonphysiology,82
wesequenceditsgenomeandusedcomparativegenomicstodiscoverhowtheKomododragon83
genomediffersfromotherspecies.Wepresentahighqualitydenovoassembly,generatedwith84
ahybridapproachoflong-rangesequencingusing10xGenomics,PacBio,andOxfordNanopore85
sequencing,andsingle-moleculephysicalmappingusingtheBioNanoplatform.Thissuiteof86
technologiesallowedustoconfidentlyassembleahigh-qualityreferencegenomeforthe87
Komododragon,whichcanserveasatemplateforothervaranidlizards.Weusedthisgenome88
tounderstandtherelationshipofvaranidstootherreptilesusingaphylogenomicsapproach.89
WeuncoveredKomodo-specificpositiveselectionforasuiteofgenesencodingregulatorsof90
musclemetabolism,cardiovascularhomeostasis,andthrombosis.Further,wediscovered91
multiplelineage-specificexpansionsofafamilyofchemoreceptorgenesinseveralsquamates,92
includingsomelizardsandasnake,aswellasintheKomododragongenome. Finally,we93
generatedahigh-resolutionchromosomalmapresultingfromassignmentofscaffoldto94
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chromosomes,providingapowerfultooltoaddressquestionsaboutkaryotypeandsex95
chromosomeevolutioninsquamates.96
97
Results98
Denovogenomeassembly99
WeobtainedDNAfromperipheralbloodoftwomaleindividualshousedatZooAtlanta:100
Slasher,amaleoffspringofthefirstKomododragonsgivenasgiftsin1986toUSPresident101
ReaganfromPresidentSuhartoofIndonesia,andRinca,ajuvenilemale(Figure1A).TheV.102
komodoensisgenomeisdistributedacross20pairsofchromosomes,comprisingeightpairsof103
largechromosomesand12pairsofmicrochromosomes13,14.Denovoassemblywasperformed104
using57Xcoverageof10xGenomicslinked-readsequencingdatatogenerateaninitial105
assemblywithascaffoldN50of10.2MbandacontigN50of95kb.Separately,80Xcoverageof106
Bionanophysicalmappingdatawasdenovoassembledtocreateanassemblywithascaffold107
N50of1.2Mb.Thesetwoassembliesweremergedintoahybridassemblyandthenscaffolded108
furtherusing6.3XcoveragefromPacBiosequencingand0.75XcoveragefromOxfordNanopore109
MinIonsequencing,foratotalcoverageof144X.Thefinalassemblycontained1,403scaffolds110
(>10kb)withanN50of29Mb(longestscaffold:138Mb)(Table1).Theassemblyis1.51Gbin111
size,~32%smallerthanthegenomeoftheChinesecrocodilelizard(Shinisauruscrocodilurus),112
theclosestrelativeoftheKomododragonforwhichasequencedgenomeisavailable,and113
~15%smallerthanthegreenanole(Anoliscarolinensis),amodelsquamatelizard(TableS1).An114
assembly-freeerrorcorrectedk-mercountingestimateofgenomesizeestimatestheKomodo115
dragongenometobe1.69Gb,makingourassembly89%complete.TheGCcontentofthe116
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PacBio long reads
Oxford Nanopore MinIon
Hybrid assembly
10X Genomics linked-readassembly
Bionano physical mappingassembly
Scaffolding
Final assembly
A
B
Figure 1. (A) Komodo dragons (left, Slasher; right, Rinca) sampled for DNA in this study. Photos courtesy of Adam K Thompson/Zoo Atlanta. (B) Genome assembly workflow. Two separate de novo assemblies were generated with 10x genomics and Bionano physical mapping data and merged into an intermediate hybrid assembly. Long reads from PacBio and Oxford Nanopore MinIon were used to scaffold the hybrid assembly into a final version.
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Komododragongenomeis44.0%,similartotheGCcontentoftheS.crocodilurusgenome117
(44.5%)buthigherthantheGCcontentofA.carolinensis(40.3%)(TableS1).Repeatswere118
annotatedusingRepeatMaskerandthesquamaterepeatdabatase15.Repetitiveelements119
accountedfor32%ofthegenome,mostofwhichweretransposableelements(TableS2).As120
repetitiveelementsinS.crocodilurusaccountfor49.6%ofthegenome16,mostofthe121
differenceinsizebetweentheKomododragongenomeanditsclosestsequencedrelative122
genomecanbeattributedtodifferencesinrepetitiveelementcontent.123
124
Chromosomescaffoldcontent125
Weisolatedchromosome-specificDNApoolsfromafemaleembryoofV.komodoensis126
(VKO)fromPraguezoostockthroughflowsorting14. WeobtainedIlluminashort-read127
sequencesofthese15DNApoolscontainingallchromosomesofV.komodoensis(TableS3).For128
eachchromosomewedeterminedscaffoldcontentandhomologytoA.carolinensisandG.129
galluschromosomes(Table2andTableS4).Foreachchromosome,wedeterminedscaffold130
contentandhomologytoA.carolinensisandG.galluschromosomes(Table2andTablesS4-5).131
Forthosepoolswherechromosomesweremixed(VKO6/7,VKO8/7,VKO9/10,VKO11/12/W,132
VKO17/18/Z,VKO17/18/19)wedeterminedpartialscaffoldcontentofsinglechromosomes.133
HomologytoA.carolinensismicrochromosomeswasdeterminedusingscaffoldassignmentsto134
chromosomesperformedinAnolischromosome-specificsequencingproject17.Atotalof243135
scaffoldscontaining1.14Gb(75%oftotal1.51Gbassembly)wereassignedto20chromosomes136
ofV.komodoensis.Assexchromosomesshareshomologousregions(pseudoautosomal137
regions),scaffoldsthatwereenrichedinboth17/18/Zand11/12/Wsamplesmostlikely138
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containedsexchromosomeregionsofV.komodoensis.Consideringthatourreferencegenome139
wasfromamaleindividual,theywereassignedtotheZchromosome.Allthesescaffoldswere140
homologoustoA.carolinensischromosome18,andmostlytochickenchromosome28as141
recentlydeterminedbytranscriptomeanalysis18.142
143
Geneannotation144
AsKomododragonshaveauniquecardiovascularphysiology,weusedhearttissueas145
thesourceforRNAsequencingtoincreasetheaccuracyofcardiovasculargeneprediction,146
increasingourpowertodetectinterestingchangestothecardiovascularsystemencodedinthe147
genome.RNAsequencingwasassembledintotranscriptswithTrinity19.Aftersoftmasking148
repetitiveelements,geneswereannotatedusingtheMAKERpipelinewithproteinhomology,149
assembledtranscripts,anddenovopredictionsasevidence,andstringentlyqualityfiltered(see150
Methods).Atotalof18,462proteincodinggeneswereannotatedintheKomodogenome,151
17,194(93%)ofwhichhaveoneormoreannotatedInterprofunctionaldomain(Table1).Of152
theseprotein-codinggenes,63%wereexpressed(RPKM>1)intheheart.Atotalof89%of153
Komododragonprotein-codinggenesareorthologoustogenesinthemodellizardA.154
carolinensisgenome.Themedianpercentidentityofsingle-copyorthologsbetweenKomodo155
andA.carolinensisis68.9%,whereasitis70.6%betweensingle-copyorthologsinKomodoand156
S.crocodilurus(FigureS1).157
158
PhylogeneticplacementofKomodo159
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0.06
Leopard gecko
Saltwater crocodile
Chinese alligator
Japanese gecko
Australian dragon lizard
Chicken
Mouse
American alligator
Gharial
Green sea turtle
Asian glass lizard
Green anole
Western painted turtle
Chinese softshell turtle
Chinese crocodile lizard
Burmese python
Komodo dragon
100
90
100
100
100
100
100
100
100
100
89
100
100
100
Figure 2. Estimated species phylogeny of 15 non-avian reptiles species and 2 additional vertebrates. Maximum likelihood phylogeny was constructed from 2,752 single-copy orthologous proteins. Support values from 10,000 bootstrap replicates are shown. All images obtained from PhyloPic.org.
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AstheKomododragongenomeisthefirstmonitorlizard(FamilyVaranidae)tohavea160
completegenomesequence,previousphylogeneticanalysesofvaranidlizardshasbeenlimited161
tomarkersequences20,21.WeusedtheKomododragongenometoestimateaspeciestree162
using2,752singlecopyorthologs(seeMethods)presentintheKomododragonand14163
representativenon-avianreptilespecies,including7squamates,3turtles,and4crocodilians,164
alongwithoneavianspecies(chicken)andonemammalianspecies(mouse)(Figure2).The165
placementofKomododragonandthemonitorlizardgenususingthisgenome-widedataset166
agreeswithpreviousmarkergenestudies20,21.167
168
Expansionofvomeronasalgenesacrosssquamatereptiles169
Thevomeronasal,ortheJacobson’s,organisachemosensorytissuethatdetects170
chemicalcuessuchaspheromonesandkairomones.Itissharedacrossamphibians,mammals,171
andreptilesthoughithasbeensecondarilylostinsomegroups,includingbirds22,23.Squamate172
reptilessuchassnakesandlizardshaveapparentlyfunctionalvomeronasalorganswiththe173
abilitytosenseprey-derivedchemicalsignals,aswellasspecificassociatedbehaviorssuchas174
tongue-flickingtodeliverolfactorycuestothesensorytissue,anditisclearthatthe175
vomeronasalorganplaysanimportantroleinsquamatereptileecology24.Twotypesof176
chemosensoryreceptors,bothofwhichareseven-transmembraneG-proteincoupled177
receptors,functionassensorsinthevomeronasalorgan.ThenumberofType1vomeronasal178
receptors(V1Rs)hasexpandedthroughgeneduplicationsincertainmammalianlineages,while179
thenumberofType2receptors(V2Rs)hasexpandedinamphibiansandsomemammalian180
lineages22.CrocodilianandturtlegenomescontainfewtonoV1RandV2Rgenes25.Snakes,in181
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contrast,haveasignificantlyexpandedV2Rrepertoirethathasarisenthroughgeneduplication182
26.183
Toclarifytherelationshipbetweenvomeronasalorganfunctionandevolutionof184
vomeronasal-receptorgenefamilies,weanalyzedthecodingsequencesof15reptiles,including185
Komodo,forpresenceofV1RandV2Rgenes(Figure3A).WeconfirmedthattherearefewV1R186
genesacrossreptilesgenerallyandfewtonoV2Rgenesincrocodiliansandturtles(TableS6).187
ThelownumberofV2Rgenesingreenanole(Anoliscarolinensis)andAustraliandragonlizard188
(Pogonavitticeps)suggestthatV2Rgenesareinfrequentlyexpandediniguanians,thoughmore189
iguaniangenomesareneededtotestthishypothesis.Incontrast,wefoundalargerepertoire190
ofV2Rs,comparableinsizetothatofsnakes,intheKomododragonandotherlizards.191
Toinferthedetailsofthedynamicevolutionofthisgenefamily,webuiltaphylogenyof192
allV2Rgenesequencesacrosssquamates(Figure3B).Thetopologyofthisphylogenysupports193
that,aspreviouslyhypothesized,V2Rsexpandedinthecommonancestorofsquamates,as194
therearecladesofgenesequencescontainingmembersfromallspecies26.Inaddition,there195
arealargenumberofwell-supportedsinglespeciesclades(i.e.,Komododragononly)dispersed196
acrossthegenetree,whichisconsistentwithmultipleduplicationsofV2Rgeneslaterin197
squamateevolution,includingintheKomodoandgeckolineages(Figure3B).198
BecauseV2Rshaveexpandedinrodentsthroughtandemgeneduplicationsthat199
producedclustersofparalogs27,weexaminedclusteringofV2RgenesinourKomodoassembly200
todetermineifasimilarmechanismislikelydrivingthesegeneexpansions.Of151V2Rs,99are201
organizedinto26geneclustersranginginsizefrom2to14genes(Figure4A,TableS7).To202
understandifthesegeneclustersarosethroughtandemgeneduplication,weconstructeda203
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Japanese gecko
Leopard gecko
Burmese python
Asian glass lizard
Komodo dragon
Chinese crocodile lizard
Green anole
Australian dragon lizard
163251
12254
7185
197150
A B
Figure 3. Type 2 vomeronasal receptors have expanded in Komodo dragons and several other squamate reptiles. (A) Type 2 vomeronasal gene counts in squamate reptiles. (B) Unrooted gene phylogeny of 1,093 vomeronasal Type 2 receptor transmembrane domains across squamate reptiles. The topology of the tree supports a gene expansion ancestral to squamates (i.e., clades containing representatives from all species) as well as multiple species-specific expansions through gene duplication events (i.e., clades containing multiple genes from one species). Branches with bootstrap support less than 60 are collapsed. Colors correspond to species in (A). Clades containing genes from a single species are collapsed.
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0.09
B
A
m
l
f
kigcjh
2 genes
8 genes
7 genes
2 genes
7 genes
6 genes
75 genes
deba
a b c mlkjihgfed
Figure 4. Gene clusters of Type 2 vomeronasal receptors evolved through gene duplication. (A) Genes in a cluster of vomeronasal Type 2 receptor genes in the Komodo dragon genome containing 14 V2R genes. Pink genes are V2R genes and gray genes are non-V2R genes. Gene labels correspond to labels in (B). (B) Unrooted phylogeny of 151 vomeronasal Type 2 receptor genes in Komodo. As most of the genes in this gene cluster group together in a gene phylogeny of all Komodo dragon V2R genes, it is likely that this cluster evolved through gene duplication events. Branches with bootstrap support less than 80 are collapsed. Clades without genes in this V2R gene cluster are collapsed. Genes in the V2R cluster are colored pink and labeled as in (A).
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phylogenyofallKomododragonV2Rgenes(Figure4B).ThelargestV2Rclustercontains14V2R204
genes,whichgrouptogetherinagenetreeofKomodoV2Rgenes(Figure4).Oftheremaining205
52V2Rgenes,38areonscaffoldslessthan10Kbinsize,soourestimateofV2Rclusteringisa206
lowerboundduetofragmentationinthegenomeassembly(TableS7).207
208
Positiveselection209
TotestforadaptiveproteinevolutionintheKomododragongenome,weidentified210
single-copyorthologsacrosssquamatereptiles,builtcodonalignments,andrantestsof211
positiveselectionusingabranch-sitemodeltodeterminegenesthathavediversifiedinthe212
varanidlineage(seeMethodsandTableS8).Ouranalysisrevealed201geneswithsignaturesof213
positiveselectioninKomododragons(TableS9).Manyofthegenesunderpositiveselection214
pointtowardsimportantadaptationsoftheKomododragon’smammalian-likecardiovascular215
andmetabolicfunctions,whichareuniqueamongnon-varanidectothermicreptiles,though216
25%ofpositivelyselectedgeneswerenotdetectablyexpressedintheheartandlikely217
representadaptationsinotheraspectsofKomododragonbiology.Pathwayswithpositively218
expressedgenesincludemitochondrialregulationandcellularrespiration,hemostasisandthe219
coagulationcascade,innateandadaptiveimmunity,andangiotensinogen(acentralregulatorof220
cardiovascularphysiology).ManyofthesehaveimplicationsforKomodophysiology,andfor221
varanidlizardphysiologygenerally.Weidentifiedseveralfunctionalcategorieswithmultiple222
positivelyselectedformoredetailedanalysis.Ineachcase,thegenesarelocatedindifferent223
partsoftheKomodogenomeandthereforelikelyrepresentrecurrentselectiononthese224
functionsduringKomodoevolution.225
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226
Positiveselectionofgenesregulatingmitochondrialfunction227
Mitochondriaregulateenergyproductionincellsthroughtheoxidativephosphorylation228
process,whichismediatedthroughtheelectrontransportchain.Multiplesubunitsand229
assemblyfactorsoftheType1NADHdehydrogenaseandcytochromecoxidaseprotein230
complexes,whichperformthefirstandlaststepsoftheelectrontransportchainrespectively,231
showevidenceofpositiveselectionintheKomododragongenome(Figure5,FigureS2,Table232
S9).TheseincludethegenesNDUFA7,NDUFAF7,NDUFAF2,NDUFB5fromtheType1NADH233
dehydrogenasecomplexandCOX6CandCOA5fromthecytochromecoxidasecomplex.234
Beyondtheelectrontransportchain,otherelementsofmitochondrialfunctionhave235
signaturesofpositiveselectionintheKomodolineage(Figure5).Ofnote,wealsodetected236
positiveselectionforACADL,whichencodesLCAD-acyl-CoAdehydrogenase,longchain—a237
memberoftheacyl-CoAdehydrogenasefamily.LCADisacriticalenzymeformitochondrial238
fattyacidbeta-oxidation,themajorpostnatalmetabolicprocessincardiacmyocytes28.239
Further,twogenesthatpromotemitochondrialbiogenesis,TFB2MandPERM1,have240
undergonepositiveselectionintheKomododragon.TFB2MregulatesmtDNAtranscriptionand241
dimethylatesmitochondrial12srRNA29,30.PERM1regulatestheexpressionofselective242
PPARGC1A/BandESRRA/B/Gtargetgeneswithrolesinglucoseandlipidmetabolism,energy243
transfer,contractilefunction,musclemitochondrialbiogenesisandoxidativecapacity31.244
PERM1alsoenhancesmitochondrialbiogenesis,oxidativecapacity,andfatigueresistancewhen245
over-expressedinmice32.Finally,wealsoidentifiedMDH1,encodingmalatedehydrogenase,246
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whichtogetherwiththemitochondrialMDH2,regulatesthesupplyofNADPHandacetyl-CoAto247
thecytoplasm,thusmodulatingfattyacidsynthesis33. 248
Multiplefactorsregulatingtranslationwithinthemitochondriahavealsoundergone249
positiveselectionintheKomododragon(Figure5).Thisincludesthemitochondrialribosome,250
includingfourcomponentsof28Ssmallribosomalsubunit(MRPS15,MRPS23,MRPS31,and251
AURKAIP1)andtwocomponentsofthe39Slargeribosomalsubunit(MRPL28andMRPL37).We252
alsofoundevidenceforpositiveselectionontheELAC2andTRMT10Cgenes,whichare253
requiredformaturationofmitochondrialtRNA,andMRM1,whichencodesamitochondrial254
rRNAmethyltransferase34–36.255
Overall,theseinstancesofpositiveselectioninalargerangeofgenesencodingproteins256
importantformitochondrialfunctionandbiogenesisclearlypointtoacoordinatedgenetic257
pathwaythatcouldexplaintheremarkableaerobiccapacityoftheKomododragon.Whileitis258
notpossibletodeterminewhethertheseadaptationsarepresentinothermonitorlizardsinthe259
absenceofasequencedgenome,changesincellularrespirationlikelyplayaroleinthehigh260
aerobiccapacityofmostvaranidlizards.261
262
Positiveselectionofangiotensinogen263
Wedetectedpositiveselectionforangiotensinogen(AGT),whichencodestheprecursor264
ofseveralimportantpeptideregulatorsofcardiovascularfunction,themostwell-studiedbeing265
angiotensinII(AII)andangiotensin1-7(A1-7).AIIhasmultipleimportantandpotentactivitiesin266
cardiovascularphysiology.Thetwomostnotable,andperhapsmostrelevanttoKomodo267
dragonphysiology,areitsvasoactivefunctioninbloodvessels,anditsinotropiceffectsonthe268
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
MDH1Electron transport chain
Structural proteinsNDUFA7
NDUFAF2NDUFB5
Structural proteinsCOX6C
ComplexI
ATPsynthase
ComplexII
ComplexIII
ComplexIV
Fatty acidbeta-oxidation
ACADL
PERM1
Nuclear transcriptionof mitochondrial genes
TCA cycle
Assembly proteinsNDUFAF7
Assembly proteinsCOA5
TFB2M
mtDNA transcription
MRPS15MRPS23
MRPL28MRPL37
Mitochondrial translation
MRPS31AURKAIP1
39S
28S
ELAC2TRMT10CMRM1
Malate oxidation
Figure 5. Mitochondrial genes under positive selection in the Komodo dragon. Genes in the Komodo dragon genome under positive selection include components of the electron transport chain, regulators of transcription, regulators of translation, and fatty acid beta-oxidation.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
heart.Inmammalsduringintensephysicalactivity,Allincreasesandcontributestoarterial269
bloodpressureandregionalbloodregulation37,38.ThepositiveselectionforAGTpointsto270
importantadaptationsinthesephysiologicalparameters.Reptileshaveafunctionalrenin-271
angiotensinsystemthatisimportantfortheircardiovascularphysiology39–41.Itislikelythat272
positiveselectionforAGTisrelatedtoamammalian-likecardiovascularfunctionintheKomodo273
dragon.274
275
Positiveselectionofthrombosis-relatedgenes276
Wefindevidenceforpositiveselectionacrossdifferentelementsofthecoagulation277
cascade,includingregulatorsofplateletsandfibrin.Thecoagulationcascadecontrols278
thrombosis,orbloodclotting,preventingbloodlossduringinjury.Fourgenesthatregulate279
plateletactivities,MRVI1,RASGRP1,LCP2,andCD63haveundergonepositiveselectioninthe280
Komododragongenome.MRVI1isinvolvedininhibitingplateletaggregation42,RASGRP1281
coordinatescalciumdependentplateletresponses43,LCP2isinvolvedinplateletactivation44,282
andCD63playsaroleincontrollingplateletspreading45.Inadditiontoregulatorsofplatelets,283
twocoagulationfactors,F10(FactorX)andF13B(CoagulationfactorXIIIBchain)have284
undergonepositiveselectionintheKomodogenome.FactorXiscentrallyimportanttothe285
coagulationcascadeanditsactivationisthefirststepininitiatingcoagulation46.Factor13Bis286
thebetasubunitofFactor13,whichisthefinalcoagulationfactoractivatedinthecoagulation287
cascade47.Further,FGB,whichencodesoneofthethreesubunitsoffibrinogen,themolecule288
whichisconvertedtotheclottingagentfibrin48,hasundergonepositiveselectioninthe289
Komodogenome.290
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Komododragons,alongwithotherspeciesofmonitorlizards,produceanticoagulants291
andhypotension-inducingproteinsintheirsalivawhicharehypothesizedtoaidinhunting11,12.292
Inadditiontohunting,Komododragonsusetheirserrateteethduringintraspecificconflict,293
whichcanbeaggressiveandinflictseriouswounds8.Becauseitislikelythattheirsalivaenters294
thebloodstreamofKomododragonsduringtheseconflicts,wehypothesizethatthepositive295
selectionthatwedetectedinmanyKomododragoncoagulationgenesmayresultfrom296
selectivepressureforKomododragonstoevadetheanticoagulanteffectsofconspecifics.297
298
Discussion299
Wehavesequencedandassembledahigh-qualitygenomeoftheKomododragon.The300
combinationofplatformsthatweusedallowedthedenovoassemblyofagenomethatwill301
serveasatemplateforanalysisofothervaranidgenomes,andforfurtherinvestigationof302
genomicinnovationsinthevaranidlineage.Moreover,weassigned75%ofthegenometo303
chromosomes.AssignmentoftheKomododragongenometochromosomesprovidesa304
significantcontributiontocomparativegenomicsofsquamatesandvertebratesingeneral.305
306
Ourcomparativegenomicanalysisidentifiedpreviouslyundescribedspecies-specific307
expansionofType2vomeronasalreceptorsacrossmultiplesquamates,includinglizardsandat308
leastonesnake.ItwillbeexcitingtoexploretherolethisexpansionofV2Rsplaysinbehavior309
andecologyofKomododragons,includingtheirabilitytolocatepreyatlongdistances8.310
Komododragons,likeothersquamates,areknowntopossessasophisticatedlingual-311
vomeronasalsystemsforchemicalsamplingoftheirenvironment49.Thissensoryapparatus312
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
allowsKomododragonstoperceivechemicalsfromtheenvironmentforavarietyofsocialand313
ecologicalactivities,includingkinrecognition,matechoice50,51,predatoravoidance52,53,314
huntingprey54,55,andforlocatingandtrackinginjuredordeadprey.Komododragonsare315
unusualastheyadoptbothforagingtacticsacrossontogenywithsmallerjuvenilespreferring316
activeforagingforsmallpreyandlargeadultdragonstargetinglargerungulatepreyviaambush317
predation10.However,retentionofahighlyeffectivelingual-vomeronasalsystemacross318
ontogenyseemslikely,giventheexceptionalcapacityforKomododragonsofallsizestolocate319
injuredordeadprey.320
321
Wefindevidenceforpositiveselectionacrossmanygenesinvolvedinregulating322
mitochondrialbiogenesis,cellularrespiration,andcardiovascularhomeostasis.Komodo323
dragons,alongwithothermonitorlizards,haveahighaerobiccapacityandexerciseendurance,324
andourresultsrevealselectivepressuresonbiochemicalpathwaysthatarelikelytobethe325
sourceofthishighaerobiccapacity.Futuregenomicworkonadditionalvaranidspecies,and326
othersquamateoutgroups,willtestthesehypotheses.Theseselectiveprocessesareconsistent327
withtheincreasedoxidativecapacityinpythonheartsafterfeeding28.Reptilemuscle328
mitochondriatypicallyoxidizesubstratesatamuchlowerratethanmammals,partlybasedon329
substrate-typeuse56.ThefindingsthatKomodohaveexperiencedselectionforseveralgenes330
encodingmitochondrialenzymes,includingoneinvolvedinfattyacidmetabolism,points331
towardsamoremammalian-likemitochondrialfunction.Inadditiontoaclearindicationof332
adaptivemusclemetabolism,wefoundpositiveselectionforAGT,whichencodestwopotent333
vasoactiveandinotropicpeptideswithcentralrolesincardiovascularphysiology.Acompelling334
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
hypothesisisthatthispositiveselectionisanimportantcomponentintheabilityofthe335
Komodotorapidlyincreasebloodpressureandcardiacoutputforattacksonprey,extended336
periodsoflocomotionincludinginter-islandswimming,andmale-malecombatduringthe337
breedingseason.DirectmeasuresofcardiacfunctionhavenotbeenmadeinKomododragons,338
butinothervaranidlizards,alargeaerobicscopeduringexerciseisassociatedwithalarge339
factorialincreaseincardiacoutput57.Overall,thesecardiovasculargenessuggestaprofoundly340
differentcardiovascularandmetabolicprofilerelativetoothersquamates,endowingthe341
Komododragonwithuniquephysiologicalproperties.342
343
Wealsofoundevidenceforpositiveselectionacrossgenesthatregulatebloodclotting.344
Likeothermonitorlizards,thesalivaofKomododragonscontainsanticoagulants.Theextensive345
positiveselectiononthegenesencodingtheircoagulationsystemlikelyreflectsthatthereis346
selectivepressureforKomododragonstoevadetheanticoagulantandhypotensiveeffectsof347
thesalivaofconspecificrivalsforfood,territories,ormates.Whileallmonitorlizardstested348
containanticoagulantsintheirsaliva,theprecisemechanismbywhichtheyactvaries12.Itis349
likelythatmonitorlizardshaveevolveddifferenttypesofadaptationsthatreflectthediversity350
oftheiranticoagulants.Understandinghowthesesystemshaveevolvedhasthepotentialto351
furtherourunderstandingofthebiologyofthrombosis.352
353
Varanids,includingKomododragons,possessgenotypicsexdeterminationandshare354
ZZ/ZWsexchromosomeswithotheranguimorphanlizards14,18.Here,wewereabletodetail355
thecontentofZchromosomeofV.komodoensis.Thechromosomesequencingdataprovided356
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
significantinsightsintothecontentofV.komodoensisZchromosome.Allscaffoldsassignedto357
ZchromosomewerehomologoustoA.carolinensischromosome18(ACA18)andtochicken358
chromosome28,asconfirmedbycomparisonofbloodtranscriptomebetweensexes18.The359
samesyntenicblocksandgenesappeartobeimplicatedindifferentvertebratelineagesinsex360
determinationmechanisms58.Inparticular,theregionsofsexchromosomesthataresharedby361
thecommonancestorofvaranidsandseveralotherlineagesofanguimorphanlizardscontain362
theamh(anti-Müllerianhormone)gene18,whichplaysacrucialroleinthetestisdifferentiation363
pathway.Homologsoftheamhgenearealsostrongcandidatesforbeingthesex-determining364
genesinseverallineagesofteleostfishesandinmonotremes59–62.365
366
367
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368
MaterialsandMethods369
DNAisolationandprocessingforBionanoopticalmapping370
KomododragonwholebloodwasusedtoextracthighmolecularweightgenomicDNA371
forgenomemapping.Bloodwascentrifugedat2000gfor2minutes,plasmawasremoved,and372
thesamplewasstoredat4°C.2.5µlofbloodwasembeddedin100µlofagarosegelplugtogive373
~7µgDNA/plug,usingtheBioRadCHEFMammalianGenomicDNAPlugKit(Bio-Rad374
Laboratories,Hercules,CA,USA).PlugsweretreatedwithproteinaseKovernightat50°C.The375
plugswerethenwashed,melted,andthensolubilizedwithGELase(Epicentre,Madison,WI,376
USA).ThepurifiedDNAwassubjectedtofourhoursofdrop-dialysis.DNAconcentrationwas377
determinedusingQubit2.0Fluorometer(LifeTechnologies,Carlsbad,CA,USA),andthequality378
wasassessedwithpulsed-fieldgelelectrophoresis.379
ThehighmolecularweightDNAwaslabeledaccordingtocommercialprotocolsusing380
theIrysPrepReagentKit(BionanoGenomics,SanDiego,CA,USA).Specifically,300ngof381
purifiedgenomicDNAwasnickedwith7UnickingendonucleaseNb.BbvCI(NEB,Ipswich,MA,382
USA)at37°CfortwohoursinNEBBuffer2.ThenickedDNAwaslabeledwithafluorescent-383
dUTPnucleotideanalogusingTaqpolymerase(NEB)foronehourat72°C.Afterlabeling,the384
nickswererepairedwithTaqligase(NEB)inthepresenceofdNTPs.Thebackboneof385
fluorescentlylabeledDNAwasstainedwithDNAstain(BioNano).386
387
DNAprocessingfor10xGenomicslinkedreadsequencing388
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HighmolecularweightgenomicDNAextraction,sampleindexing,andgenerationof389
partitionbarcodedlibrarieswereperformedby10xGenomics(Pleasanton,CA,USA)according390
totheChromiumGenomeUserGuideandaspublishedpreviously64.391
392
Bionanomappingandassembly393
UsingtheBionanoIrysinstrument,automatedelectrophoresisofthelabeledDNA394
occurredinthenanochannelarrayofanIrysChip(BionanoGenomics),followedbyautomated395
imagingofthelinearizedDNA.TheDNAbackbone(outlinedbyYOYO-1staining)andlocations396
offluorescentlabelsalongeachmoleculeweredetectedusingtheIrysinstrument’ssoftware.397
ThelengthandsetoflabellocationsforeachDNAmoleculedefinesanindividualsingle-398
moleculemap.RawBionanosingle-moleculemapsweredenovoassembledintoconsensus399
mapsusingtheBionanoIrysSolveassemblypipeline(version5134)withdefaultsettings,with400
noisevaluescalculatedfromthe10xGenomicsSupernovaassembly.401
402
10xGenomicssequencingandassembly403
The10xGenomicsbarcodedlibrarywassequencedontheChromiummachine,andthe404
rawreadswereassembledusingthecompany’sSupernovasoftware(version1.0)withdefault405
parameters.OutputfastafilesofthephasedSupernovaassembliesweregeneratedin406
pseudohapformat.407
408
Mergingdatasetsintoasingleassembly409
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Sequencingandmappingdatatypesweremergedtogetherasfollows.First,Bionano410
assembledcontigsandthe10xGenomicsassemblywerecombinedusingBionano’shybrid411
assemblytoolwiththe-B2-N2options.SSPACE-LongRead(citehttps://doi.org/10.1186/1471-412
2105-15-211)wasusedinserieswithdefaultparameterstoscaffoldthehybridassemblyusing413
PacBioreads,Nanoporereads,andunincorporated10xGenomicsSupernovascaffolds/contigs,414
resultinginthefinalassembly.415
416
Assignmentofscaffoldstochromosomes417
IsolationofV.komodoensis(VKO)chromosome-specificDNApoolsaspreviously418
described14.Briefly,fibroblastcultivationofafemaleV.komodoensiswereobtainedfrom419
tissuesamplesofanearlyembryoofacaptiveindividual.Chromosomesobtainedbyfibroblast420
cultivationweresortedusingaMo-Flo(BeckmanCoulter)cellsorter.Fifteenchromosome421
poolsweresortedintotal.Chromosome-specificDNApoolswerethenamplifiedandlabelled422
bydegenerateoligonucleotideprimedPCR(DOP-PCR)andassignedtotheirrespective423
chromosomesbyhybridizationoflabelledprobestometaphases.V.komodoensischromosome424
poolsobtainedbyflowsortingwerenamedaccordingtochromosomes(e.g.majorityofDNAof425
VKO6/7belongtochromosomes6and7ofV.komodoensis).V.komodoensispoolsfor426
macrochromosomesareeachspecificforonesinglepairofchromosomes,exceptforVKO6/7427
andVKO8/7,whichcontainonespecificchromosomepaireach(pair6andpair8,respectively),428
plusathirdpairwhichoverlapsbetweenthetwoofthem(pair7).Formicrochromosomes,429
poolsVKO9/10,VKO17/18/19,VKO11/12/WandVKO17/18/Zcontainedmorethanone430
chromosomeeach,whiletherestarespecificforonesinglepairofmicrochromosomes.TheW431
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
andZchromosomesarecontainedinpoolsVKO11/12/WandVKO17/18/Z,respectively,432
togetherwithtwopairsofothermicrochromosomeseach.433
Chromosome-poolspecificgeneticmaterialwasamplifiedbyGenomePlex®Whole434
GenomeAmplification(WGA)Kit(Sigma)followingmanufacturerprotocols.DNAfromall15435
chromosomepoolswasusedtoprepareIlluminasequencinglibraries,whichwere436
independentlybarcodedandsequenced125bppaired-endinasingleIlluminaHiseq2500lane.437
Readsobtainedfromsequencingofflow-sorting-derivedchromosome-specificDNApoolswere438
processedwiththedopseqpipeline(https://github.com/lca-imcb/dopseq)17,65.Illumina439
adaptersandWGAprimersweretrimmedoffbycutadaptv1.1366.Then,pairsofreadswere440
alignedtothegenomeassemblyofV.komodoensisusingbwamem67.Readswerefilteredby441
MAPQ≥20andlength≥20bp,andalignedreadsweremergedintopositionsusingpybedtools442
0.7.1068,69.Referencegenomeregionswereassignedtospecificchromosomesbasedon443
distancebetweenpositions.Finally,severalstatisticswerecalculatedforeachscaffold.444
Calculatedparametersincluded:meanpairwisedistancebetweenpositionsonscaffold,mean445
numberofreadsperpositiononscaffold,numberofpositionsonscaffold,position446
representationratio(PRR)andp-valueofPRR.PRRofeachscaffoldwasusedtoevaluate447
enrichmentofgivenscaffoldonchromosomes.PRRwascalculatedasratioofpositionson448
scaffoldtopositionsingenomedividedbyratioofscaffoldlengthtogenomelength.PRRs>1449
correspondtoenrichment,whilePRRs<1correspondtodepletion.AsthePRRvalueis450
distributedlognormally,weuseitslogarithmicformforourcalculations.Tofilteroutonly451
statisticallysignificantPRRvaluesweusedthresholdsoflogPRR>0anditsp-value<=0.01.452
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ScaffoldswithlogPRR>0wereconsideredenrichedinthegivensample.Ifonescaffoldwas453
enrichedinseveralsampleswechosehighestPRRtoassignscaffoldastopsample.454
WealsoassignedhomologyofV.komodoensisgenometogenomesofAnoliscarolinensis455
(AnoCar2.0)andGallusgallus(galGal3)generatingalignmentbetweengenomeswithLAST70456
andsubsequentlyusingchainingandnettingtechnique71.ForLASTweuseddefaultscoring457
matrixandparametersof400forgapexistencecost,30forgapextensioncostand4500for458
minimumalignmentscore.ForaxtChainweusedsamedistancematrixanddefaultparameters459
forotherchain-netscripts.460
461
RNAsequencing462
RNAwasextractedfromhearttissueobtainedfromanadultmalespecimenthatdiedof463
naturalcauses.TrizolreagentwasusedtoextractRNAfollowingmanufacturer’sinstructions.464
RNAseqlibrarieswereproducedusingaNuGenRNAseqv2andUltralowv2kits,andsequenced465
onanIlluminaNextseq500.466
467
Genomeannotation468
RepeatMaskerwasusedtomaskrepetitiveelementsintheKomododragongenome469
usingthesquamatarepeatdatabaseasreference15.Aftermaskingrepetitiveelements,470
protein-codinggeneswereannotatedusingtheMAKERversion3.01.0272pipeline,combining471
proteinhomologyinformation,assembledtranscriptevidence,anddenovogenepredictions472
fromSNAPandAugustusversion3.3.173.Proteinhomologywasdeterminedbyaligning473
proteinsfrom15reptilespecies(TableS10)totheKomododragongenomeusingexonerate474
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
version2.2.074.RNA-seqdatawasalignedtotheKomodogenomewithSTARversion2.6.075475
andassembledinto900,722transcriptswithTrinityversion2.4.019.Proteindomainswere476
determinedusingInterProScanversion5.31.7076.GeneannotationsfromtheMAKERpipeline477
werefilteredbasedonthestrengthofevidenceforeachgene,thelengthofthepredicted478
protein,andthepresenceofproteindomains.Clustersoforthologousgenesacross15reptile479
speciesweredeterminedwithOrthoFinderv2.0.077.Atotalof284,107proteinswereclustered480
into16,546orthologousclusters.Intotal,96.4%ofKomodogenesweregroupedinto481
orthologousclusters.Forestimatingaspeciesphylogenyonly,proteinsequencesfromMus482
musculusandGallusgalluswereaddedtotheorthologousclusterswithOrthoFinder.tRNAs483
wereannotatedusingtRNAscan-SEversion1.3.178,andothernon-codingRNAswereannotated484
usingtheRfamdatabase79andtheInfernalsoftwaresuite80.485
486
Phylogeneticanalysis487
Atotalof2,752single-copyorthologousproteinsacross15reptilespecies,including488
Varanuskomodoensis,Shinisauruscrocodilurus,Ophisaurusgracilis,Anoliscarolinensis,Pogona489
vitticeps,Pythonmolorusbivittatus,Eublepharismacularius,Gekkojaponicus,Pelodiscus490
sinensis,Cheloniamydas,Chrysemyspictabellii,Alligatorsinensis,Alligatormississippiensis,491
Gavialisgangeticus,andCrocodylusporosus,alongwiththechickenGallusgallusandmouse492
Musmusculus,wereeachalignedusingPRANKv.17042781(TableS10).Alignedproteinswere493
concatenatedintoasupermatrix,andaspeciestreewasestimatedusingIQ-TREEversion494
1.6.7.182withmodelselectionacrosseachpartition83and10,000ultra-fastbootstrap495
replicates84.496
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497
Genefamilyevolutionanalysis498
GenefamilyexpansionandcontractionanalyseswereperformedwithCAFEv4.285for499
thesquamatereptilelineage,withaconstantgenebirthandgenedeathrateassumedacross500
allbranches.501
Vomeronasaltype2receptorswerefirstidentifiedinallspeciesbycontainingtheV2R502
domainInterProdomain(IPR004073)86.ToensurethatnoV2Rgenesweremissed,allproteins503
werealignedagainstasetofrepresentativeV2RgenesusingBLASTp87withane-valuecutoffof504
1e-6andabitscorecutoffof200orgreater.Anygenespassingthisthresholdwereaddedtothe505
setofputativeV2Rgenes.TransmembranedomainswereidentifiedineachputativeV2Rgene506
withTMHMMv2.088anddiscardediftheydidnotcontain7transmembranedomainsintheC-507
terminalregion.Beginningatthestartofthefirsttransmembranedomain,proteinswere508
alignedwithMAFFTv7.310(autoalignmentstrategy)89andtrimmedwithtrimAL(gappyout)90.509
AgenetreewasconstructedusingIQ-TREE82–84withtheJTT+modelofevolutionwithempirical510
basefrequenciesand10FreeRatemodelparameters,and10,000bootstrapreplicates.Genes511
werediscardediftheyfailedtheIQ-TREEcompositiontest.512
513
Positiveselectionanalysis514
Weanalyzed4,081genesthatwereuniversalandsingle-copyacrossallsquamate515
lineagestested(Varanuskomodoensis,Shinisauruscrocodilurus,Ophisaurusgracilis,Anolis516
carolinensis,Pogonavitticeps,Pythonmolorusbivittatus,Eublepharismacularius,andGekko517
japonicus)totestforpositiveselection(TableS8).Anadditional2,040genesthatwere518
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
universalandsingle-copyacrossasubsetofsquamatespecies(Varanuskomodoensis,Anolis519
carolinensis,Pythonmolurusbivittatus,andGekkojaponicus)werealsoanalyzed(TableS8).We520
excludedmulti-copygenesfromallpositiveselectionanalysestoavoidconfoundingfrom521
incorrectparalogyinference.ProteinswerealignedusingPRANK81andcodonalignmentswere522
generatedusingPAL2NAL91.523
Positiveselectionanalyseswereperformedwiththebranch-sitemodelaBSRELusingthe524
HYPHYframework92,93.Forthe4,081genesthatweresingle-copyacrossallsquamatelineages,525
thefullspeciesphylogenyofsquamateswasused.Forthe2,040genesthatwereuniversaland526
single-copyacrossasubsetofspecies,aprunedtreecontainingonlythosetaxawasused.We527
discardedgeneswithunreasonablyhighdN/dSvaluesacrossasmallproportionofsites,as528
thosewerefalsepositivesdrivenbylowqualitygeneannotationinoneormoretaxainthe529
alignment.WeusedacutoffofdN/dSoflessthan50across5%ormoreofsites,andap-value530
oflessthan0.05attheKomodonode.Eachgenewasfirsttestedforpositiveselectiononlyon531
theKomodobranch.GenesundergoingpositiveselectionintheKomodolineagewerethen532
testedforpositiveselectionatallnodesinthephylogeny.Thisresultedin201genesbeing533
underpositiveselectionintheKomodolineage(TableS9).534
535
536
Acknowledgements537
SpecialthanksfromB.G.B.toJohnRomanoforinspirationandhistoricalinformation.Weare538
gratefultostaffatZooAtlantaforcareofSlasherandRincaandhelpobtainingsamples,Jim539
PetherfromReptilandiazooinGranCanariaintheCanaryIslands,foradditionalsamples,and540
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
R.ChadwickandN.Carli(GladstoneGenomicsCore)forDNAandRNA-seqlibrarypreparation.541
WealsothankKristinaGiorda,RabeeaAbbas,DeannaChurch(10xGenomics)for10xGenomics542
ChromiumsequencingandSupernovaassembly.Thisworkwassupportedbyinstitutional543
fundingfromtheGladstoneInstitutestoB.G.BandK.S.P.;anNHLBIgranttoK.S.PandB.G.B544
(HL098179);theYoungerFamilygifttoB.G.B.;anNHGRIgranttoP.-Y.K.(R01HG005946);NIH545
traininggrants(T32AR007175)toA.C.Y.Mand(T32HL007731)toY.M.M.A.andM.R.were546
supportedbyGACR17-22141Y,M.R.wasadditionallysupportedbyCharlesUniversityprojects547
PRIMUS/SCI/46andResearchCentre(204069).548
549
Authorcontributions:A.L.L.didgenomeannotationandallcomparativegenomicsanalyses.550
Y.Y.Y.L.ledthesequencingandassemblyeffortswithY.M.andA.C.Y.M.A.I.sequencedisolated551
chromosomeswithM.RundersupervisionofL.K.,andassignedsequenceswithA.M.,I.K,and552
V.T.M.F.andV.O.contributedtogenomeassemblythegenomeinthelabofR.F.withC.C.553
A.K.H.ledtheinitialdevelopmentoftheproject.W.L.E.initiallyassembledthetranscriptomes554
andannotatedthegenome.O.A.R.providedfrozentissuesamples.J.M.collectedspecimen555
blood.M.M.andM.F.isolatedsamplesandobtainedPacBiosequenceinthelabofT.P.andC.C.556
E.S.performedPacBiosequencing.J.W.H.,J.M.,andC.C.provideddirectiononVaranid557
physiology. T.J.andC.C.provideddirectiononKomododragonecology.P.-Y.K.coordinatedthe558
genomicsefforts.K.S.P.directedcomparativegenomicanalysis.B.G.B.initiatedand559
coordinatedtheproject.A.L.,K.S.P.andB.G.B.wrotethepaperwithinputfromallauthors.560
561
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
Correspondence:[email protected](B.G.B.),562
[email protected](K.S.P.)563
564
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
Tables.565
Table1.GenomestatisticsoftheKomododragongenome.566
Assemblysize 1.51Gb(1,508,391,850bp)Numberofscaffolds 1,403Minimumscaffoldlength 10KbMaximumscaffoldlength 138MbN50 29Mb(29,129,838)Numberofprotein-codinggenes 18,462GCcontent 44.04%
567
Table2.ResultsofscaffoldassignmentstochromosomesofV.komodoensis.568
V.komodoensischromosome GGAhomology ACAhomology No.of
scaffolds
Totallengthofassignedscaffolds
(bp)
Chr1 Chr1,3,5,18,Z Chr1,2,3 94 245,019,529
Chr2 Chr1,3,5,7 Chr1,2,6 14 156,023,568
Chr3 Chr1,4 Chr3,5 11 115,571,927
Chr4 Chr1,2,5,27 Chr1,4,6 39 117,170,416
Chr5 Chr1 Chr3 6 75,951,376
Chr6,7,8 Chr2,6,8,9,20 Chr1,2,3,4 25 200,178,831
Chr9,10 Chr11,22,24 Chr7,8 8 69,008,218
Chr11,12 Chr4,10 Chr10,11 6 52,491,606
Chr13 Chr1,5,23 Chr9 9 19,625,567
Chr14 Chr14 Chr12 3 21,537,982
Chr15 Chr15 ChrX 4 14,821,201
Chr16 Chr17 Chr16 2 13,367,238
Chr17,18 Chr1,19,21 Chr1,9,15,17 10 17,262,365
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
Chr19 Chr1,3,25 Chr14 6 11,765,548
ChrZ Chr1,28 Chr18 6 10,642,498
GGAhomology:homologyofscaffoldstoG.galluschromosomes;ACAhomology:homologyof569
scaffoldstoA.carolinensischromosomes;Totallengthofassignedscaffolds(bp):sizeinbase570
pairsofthesumofallscaffoldsforeachchromosome.571
572573
574
575
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
Figurelegends.576Figure1.(A)Komododragons(left,Slasher;right,Rinca)sampledforDNAinthisstudy.Photos577
courtesyofAdamKThompson/ZooAtlanta.(B)Genomeassemblyworkflow.Twoseparatede578
novoassembliesweregeneratedwith10xgenomicsandBionanophysicalmappingdataand579
mergedintoanintermediatehybridassembly.LongreadsfromPacBioandOxfordNanopore580
MinIonwereusedtoscaffoldthehybridassemblyintoafinalversion.581
582
Figure2.Estimatedspeciesphylogenyof15non-avianreptilesspeciesand2additional583
vertebrates.Maximumlikelihoodphylogenywasconstructedfrom2,752single-copy584
orthologousproteins.Supportvaluesfrom10,000bootstrapreplicatesareshown.Allimages585
obtainedfromPhyloPic.org.586
587
Figure3.Type2vomeronasalreceptorshaveexpandedinKomododragonsandseveralother588
squamatereptiles.(A)Type2vomeronasalgenecountsinsquamatereptiles.(B)Unrooted589
genephylogenyof1,093vomeronasalType2receptortransmembranedomainsacross590
squamatereptiles.Thetopologyofthetreesupportsageneexpansionancestraltosquamates591
(i.e.,cladescontainingrepresentativesfromallspecies)aswellasmultiplespecies-specific592
expansionsthroughgeneduplicationevents(i.e.,cladescontainingmultiplegenesfromone593
species).Brancheswithbootstrapsupportlessthan60arecollapsed.Colorscorrespondto594
speciesin(A).Cladescontaininggenesfromasinglespeciesarecollapsed.595
596
Figure4.GeneclustersofType2vomeronasalreceptorsevolvedthroughgeneduplication.597
(A)GenesinaclusterofvomeronasalType2receptorgenesintheKomododragongenome598
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
containing14V2Rgenes.PinkgenesareV2Rgenesandgraygenesarenon-V2Rgenes.Gene599
labelscorrespondtolabelsin(B).(B)Unrootedphylogenyof151vomeronasalType2receptor600
genesinKomodo.Asmostofthegenesinthisgeneclustergrouptogetherinagenephylogeny601
ofallKomododragonV2Rgenes,itislikelythatthisclusterevolvedthroughgeneduplication602
events.Brancheswithbootstrapsupportlessthan80arecollapsed.Cladeswithoutgenesin603
thisV2Rgeneclusterarecollapsed.GenesintheV2Rclusterarecoloredpinkandlabeledasin604
(A).605
606
Figure5.MitochondrialgenesunderpositiveselectionintheKomododragon.Genesinthe607
Komododragongenomeunderpositiveselectionincludecomponentsoftheelectrontransport608
chain,regulatorsoftranscription,regulatorsoftranslation,andfattyacidbeta-oxidation.609
610
Supplementalfigurelegends.611
FigureS1.Percentidentitiesofsingle-copyorthologsbetweentheKomododragonandthe612
greenanoleandtheKomododragonandtheChinesecrocodilelizard.613
614
FigureS2.Positiveselectionongenesencodingstructuralproteinsintheelectrontransport615
chain.Darkgraygeneswerenottestedforpositiveselectionduetoeithermissingdatainone616
ormorespeciesordifficultyresolvingortholog/paralogrelationships.Pinkgeneshave617
signaturesofpositiveselection,andlightgraygenesdidnothavesignaturesofpositive618
selection.FiguremodifiedfromWikiPathways63.619
620
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
Supplementalfiledescriptions.621
TableS1.Genomestatisticsfornon-avianreptilesusedinthisstudy.622
TableS2.RepetitiveelementsintheKomododragongenome.623
TableS3.Readstatisticsforchromosomalanchoring.624
TableS4.ScaffoldassignmentandhomologiesofKomododragonscaffoldstogreenanoleand625
chickenchromosomes.626
TableS5.Numberofreads,scaffolds,andpositionsassignedtochromosomes.627
TableS6.NumberofV1R/V2Rgenesacrossnon-avianreptiles.628
TableS7.V2RgeneclustersintheKomododragongenome.629
TableS8.GenesassayedforpositiveselectionintheKomododragongenome.630
TableS9.PositivelyselectedgenesintheKomododragongenome.631
TableS10.Sourcesandversionsofgenomesusedforphylogeneticandcomparativemethods.632
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0
25
50
75
100
Anolis
carol
inens
is
(7,37
1 gen
es)
Shinisa
uraus
croc
odilur
us
(7,22
1 gen
es)
Species
Perc
ent i
dent
ity o
ver s
ingl
e−co
py o
rthol
ogs
Figure S1. Percent identities of single-copy orthologs between the Komodo dragon and the green anole and the Komodo dragon and the Chinese crocodile lizard.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint
C subunit
Uncoupling Protein
Complex II
Succinate-UbiquinoneOxidoreductase
Stalk
Cytochrome COxidase F1 ComplexIntermembrane
Space
NADH-UbiquinoneOxidoreductase
Complex IV
Matrix
Adenine NucleotideTranslocator
F0 Complex
binds ATP Synthase beta
Assembly
Complex IIIComplex I
Ubiquinol-Cytochrome CReductase
Complex VATP Synthase
SLC25A6ND3
UCP1
ATP5E SLC25A14
NDUFA3 NDUFA5
COX3
COX8A
ATP5F1
UQCR
ND6
COX7A2L
NDUFB5
ATP5A1
ND2
NAD+
FADH2
e-
ATP5J
UQCRFS1
NDUFB2
H2O
e-
ATP5G2
COX7A3
ATP5D
Assembly PWComplex 1
COX6A2
COX6C
ATP5C1
ATPIF1
NDUFS1
QP-C
NDUFB8
H+
H+
Cytochrome C
ATP6
COX2
COX5A
CYTB
NDUFB4
NDUFS3
COX6A1
H+
NDUFA8
NDUFV3
ATP5I
H+
NDUFC1
NDUFC2
ND5
NDUFB10
NDUFB6
SDHA
NDUFA9
NDUFS7
NDUFB9
NDUFV2
NDUFS8
SDHB ATP5B
UCRC
COX7B
UCP2NDUFB1
NDUFA10
SDHD
DAP13
H+
ATP5G1
NDUFB7
COX6B1
UQCRH
ATP5O
SURF1
COX7A1
NDUFA6
O2
COX17
H+
COX4I1
e-
COX15
NDUFS4
NDUFS6
SDHC
NADH
COX11
ND4L
SLC25A27
COX1
NDUFA7
ATP5J2NDUFS2
UCP3
ND1
UQCRB
Ubiquinone
ATP
COX5B
COX7A2
NDUFV1
Succinate
FAD
SCO1
ND4
TCA Cycle
UQCRC2
NDUFA4
ATP5S
NDUFA1
COX7C
SLC25A5
NDUFA2
ATP8
NDUFB3
H+
SLC25A4
ATP5G3
ATP5H
ATP5L
NDUFS5
NDUFAB1
UQCRC1
Positive selection
No selection
Not included in analysis
Legend
Figure S2. Positive selection on genes encoding structural proteins in the electron transport chain. Dark gray genes were not tested for positive selection due to either missing data in one or more species or difficulty resolving ortholog/paralog relationships. Pink genes have signatures of positive selection, and light gray genes did not have signatures of positive selection. Figure modified from WikiPathways 63.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted February 26, 2019. . https://doi.org/10.1101/551978doi: bioRxiv preprint