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

https://doi.org/10.1101/551978

http://creativecommons.org/licenses/by-nc-nd/4.0/

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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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.


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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.


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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.


https://doi.org/10.1101/551978


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).


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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.


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


Correspondence:[email protected](B.G.B.),562

[email protected](K.S.P.)563

564


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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


https://doi.org/10.1101/551978


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

References 633

1. Chapman,A.D.NumbersofLivingSpeciesinAustraliaandtheWorld.(Australian634

BiologicalResourcesStudy,2009).635

2. Collar,D.C.,Schulte,J.A.&Losos,J.B.Evolutionofextremebodysizedisparityin636

monitorlizards(Varanus).Evolution(N.Y).65,2664–2680(2011).637

3. Jensen,B.,Wang,T.,Christoffels,V.M.&Moorman,A.F.M.Evolutionanddevelopment638

ofthebuildingplanofthevertebrateheart.Biochim.Biophys.Acta-Mol.CellRes.1833,639

783–794(2013).640

4. Clemente,C.J.,Withers,P.C.&Thompson,G.G.Metabolicrateandendurancecapacity641

inAustralianvaranidlizards(Squamata:Varanidae:Varanus).Biol.J.Linn.Soc.97,664–642


https://doi.org/10.1101/551978


676(2009).643

5. Burggren,W.&Johansen,K.VentricularHaemodynamicsintheMonitorLizardVaranus644

Exanthematicus:PulmonaryandSystemicPressureSeparation.J.Exp.Biol.96,(1982).645

6. Ishimatsu,A.,Hicks,J.W.&Heisler,N.Analysisofintracardiacshuntinginthelizard,646

Varanusniloticus:anewmodelbasedonbloodoxygenlevelsandmicrosphere647

distribution.Respir.Physiol.71,83–100(1988).648

7. King,D.&Green,B.Goannas:TheBiologyofVaranidLizards.(UniversityofNewSouth649

Wales,1998).650

8. Auffenberg,W.TheBehavioralEcologyoftheKomodoMonitor.(UniversityPressesof651

Florida,1981).652

9. Green,B.,King,D.,Braysher,M.&Saim,A.Thermoregulation,waterturnoverand653

energeticsoffree-livingkomododragons,Varanuskomodoensis.Comp.Biochem.654

Physiol.PartAPhysiol.99,97–101(1991).655

10. Purwandana,D.etal.EcologicalallometriesandnicheusedynamicsacrossKomodo656

dragonontogeny.Sci.Nat.103,27(2016).657

11. Fry,B.G.etal.AcentralroleforvenominpredationbyVaranuskomodoensis(Komodo658

Dragon)andtheextinctgiantVaranus(Megalania)priscus.Proc.Natl.Acad.Sci.U.S.A.659

106,8969–8974(2009).660

12. Koludarov,I.etal.EntertheDragon:TheDynamicandMultifunctionalEvolutionof661

AnguimorphaLizardVenoms.Toxins9,(2017).662

13. JohnsonPokorná,M.etal.FirstDescriptionoftheKaryotypeandSexChromosomesin663

theKomodoDragon(Varanuskomodoensis).Cytogenet.GenomeRes.148,284–291664


https://doi.org/10.1101/551978


(2016).665

14. Iannucci,A.etal.IsolatingChromosomesoftheKomodoDragon:NewToolsfor666

ComparativeMappingandSequenceAssembly.Cytogenet.GenomeRes.157,42–50667

(2019).668

15. Smit,A.,Hubley,R.&Green,P.RepeatmaskerOpen-4.0.(2013).Availableat:669

http://www.repeatmasker.org.(Accessed:10thJanuary2015)670

16. Gao,J.etal.Sequencing,denovoassembling,andannotatingthegenomeofthe671

endangeredChinesecrocodilelizardShinisauruscrocodilurus.Gigascience6,1–6(2017).672

17. Kichigin,I.G.etal.EvolutionarydynamicsofAnolissexchromosomesrevealedby673

sequencingofflowsorting-derivedmicrochromosome-specificDNA.Mol.Genet.674

Genomics291,1955–1966(2016).675

18. Rovatsos,M.,Rehák,I.,Velenský,P.&Kratochvíl,L.Sharedancientsexchromosomesin676

varanids,beadedlizardsandalligatorlizards.Mol.Biol.Evol.(2019).677

doi:10.1093/molbev/msz024678

19. Haas,B.J.etal.DenovotranscriptsequencereconstructionfromRNA-sequsingthe679

Trinityplatformforreferencegenerationandanalysis.Nat.Protoc.8,1494–512(2013).680

20. Pyron,R.,Burbrink,F.T.&Wiens,J.J.Aphylogenyandrevisedclassificationof681

Squamata,including4161speciesoflizardsandsnakes.BMCEvol.Biol.13,93(2013).682

21. Welton,L.J.,Travers,S.L.,Siler,C.D.&Brown,R.M.Integrativetaxonomyand683

phylogeny-basedspeciesdelimitationofPhilippinewatermonitorlizards(Varanus684

salvatorComplex)withdescriptionsoftwonewcrypticspecies.Zootaxa3881,201685

(2014).686


https://doi.org/10.1101/551978


22. Silva,L.&Antunes,A.VomeronasalReceptorsinVertebratesandtheEvolutionof687

PheromoneDetection.Annu.Rev.Anim.Biosci.5,353–370(2017).688

23. Stoddart,D.M.TheEcologyofVertebrateOlfaction.(SpringerNetherlands,1980).689

24. Mason,R.T.&Parker,M.R.Socialbehaviorandpheromonalcommunicationinreptiles.690

J.Comp.Physiol.A196,729–749(2010).691

25. Green,R.E.etal.Threecrocodiliangenomesrevealancestralpatternsofevolution692

amongarchosaurs.Science(80-.).346,1254449–1254449(2014).693

26. Brykczynska,U.,Tzika,A.C.,Rodriguez,I.&Milinkovitch,M.C.Contrastedevolutionof694

thevomeronasalreceptorrepertoiresinmammalsandsquamatereptiles.GenomeBiol.695

Evol.5,389–401(2013).696

27. Yang,H.,Shi,P.,Zhang,Y.&Zhang,J.CompositionandevolutionoftheV2rvomeronasal697

receptorgenerepertoireinmiceandrats.Genomics86,306–315(2005).698

28. Riquelme,C.A.etal.FattyAcidsIdentifiedintheBurmesePythonPromoteBeneficial699

CardiacGrowth.Science(80-.).334,528LP-531(2011).700

29. Falkenberg,M.etal.MitochondrialtranscriptionfactorsB1andB2activatetranscription701

ofhumanmtDNA.Nat.Genet.31,289–294(2002).702

30. Cotney,J.,McKay,S.E.&Shadel,G.S.Elucidationofseparate,butcollaborative703

functionsoftherRNAmethyltransferase-relatedhumanmitochondrialtranscription704

factorsB1andB2inmitochondrialbiogenesisrevealsnewinsightintomaternally705

inheriteddeafness.Hum.Mol.Genet.18,2670–2682(2009).706

31. Cho,Y.,Hazen,B.C.,Russell,A.P.&Kralli,A.PeroxisomeProliferator-activatedReceptor707

γCoactivator1(PGC-1)-andEstrogen-relatedReceptor(ERR)-inducedRegulatorin708


https://doi.org/10.1101/551978


Muscle1(PERM1)IsaTissue-specificRegulatorofOxidativeCapacityinSkeletalMuscle709

Cells.J.Biol.Chem.288,25207–25218(2013).710

32. Cho,Y.etal.Perm1enhancesmitochondrialbiogenesis,oxidativecapacity,andfatigue711

resistanceinadultskeletalmuscle.FASEBJ.30,674–687(2016).712

33. Zhao,S.etal.RegulationofCellularMetabolismbyProteinLysineAcetylation.Science713

(80-.).327,1000–1004(2010).714

34. Brzezniak,L.K.,Bijata,M.,Szczesny,R.J.&Stepien,P.P.InvolvementofhumanELAC2715

geneproductin3’endprocessingofmitochondrialtRNAs.RNABiol.8,616–626(2011).716

35. Holzmann,J.etal.RNasePwithoutRNA:IdentificationandFunctionalReconstitutionof717

theHumanMitochondrialtRNAProcessingEnzyme.Cell135,462–474(2008).718

36. Lee,K.-W.&Bogenhagen,D.F.Assignmentof2ʹ-O-MethyltransferasestoModification719

SitesontheMammalianMitochondrialLargeSubunit16SRibosomalRNA(rRNA).J.Biol.720

Chem.289,24936–24942(2014).721

37. Forrester,S.J.etal.AngiotensinIISignalTransduction:AnUpdateonMechanismsof722

PhysiologyandPathophysiology.Physiol.Rev.98,1627–1738(2018).723

38. Kim,S.&Iwao,H.MolecularandCellularMechanismsofAngiotensinII-Mediated724

CardiovascularandRenalDiseases.Pharmacol.Rev.52,11LP-34(2000).725

39. WILSON,J.X.TheRenin-AngiotensinSysteminNonmammalianVertebrates.Endocr.Rev.726

5,45–61(1984).727

40. Fournier,D.,Luft,F.C.,Bader,M.,Ganten,D.&Andrade-Navarro,M.A.Emergenceand728

evolutionoftherenin–angiotensin–aldosteronesystem.J.Mol.Med.90,495–508729

(2012).730


https://doi.org/10.1101/551978


41. Mueller,C.A.,Eme,J.,Tate,K.B.&Crossley,D.A.Chroniccaptopriltreatmentreveals731

theroleofANGIIincardiovascularfunctionofembryonicAmericanalligators(Alligator732

mississippiensis).J.Comp.Physiol.B188,657–669(2018).733

42. Antl,M.etal.IRAGmediatesNO/cGMP-dependentinhibitionofplateletaggregationand734

thrombusformation.Blood109,552–559(2007).735

43. Puetz,J.&Boudreaux,M.K.Evaluationofthegeneencodingcalciumanddiacylglycerol736

regulatedguaninenucleotideexchangefactorI(CalDAG-GEFI)inhumanpatientswith737

congenitalqualitativeplateletdisorders.Platelets23,401–403(2012).738

44. Bezman,N.A.etal.RequirementsofSLP76tyrosinesinITAMandintegrinreceptor739

signalingandinplateletfunctioninvivo.J.Exp.Med.205,1775–88(2008).740

45. Israels,S.&McMillan-Ward,E.CD63modulatesspreadingandtyrosinephosphorylation741

ofplateletsonimmobilizedfibrinogen.Thromb.Haemost.93,311–318(2005).742

46. Cooper,D.N.,Millar,D.S.,Wacey,A.,Pemberton,S.&Tuddenham,E.G.Inheritedfactor743

Xdeficiency:moleculargeneticsandpathophysiology.Thromb.Haemost.78,161—172744

(1997).745

47. Takahashi,N.,Takahashi,Y.&Putnam,F.W.Primarystructureofbloodcoagulation746

factorXIIIa(fibrinoligase,transglutaminase)fromhumanplacenta.Proc.Natl.Acad.Sci.747

83,8019LP-8023(1986).748

48. Mosesson,M.W.Therolesoffibrinogenandfibrininhemostasisandthrombosis.Semin.749

Hematol.29,177—188(1992).750

49. Halpern,M.Nasalchemicalsensesinreptiles:structureandfunction.Pp423–523inGans751

C,CrewsD(eds)BiologyoftheReptilia,Vol.18,Brain.Horm.Behav.Chicago/ILUniv.752


https://doi.org/10.1101/551978


ChicagoPressGoogleSch.(1992).753

50. Martin,J.&Lopez,P.Chemoreception,symmetryandmatechoiceinlizards.Proc.R.Soc.754

BBiol.Sci.267,1265–1269(2000).755

51. Baeckens,S.,Martín,J.,García-Roa,R.&vanDamme,R.Sexualselectionandthe756

chemicalsignaldesignoflacertidlizards.Zool.J.Linn.Soc.183,445–457(2018).757

52. vanDamme,R.,Bauwens,D.,Thoen,C.,Vanderstighelen,D.&Verheyen,R.F.Responses758

ofNaiveLizardstoPredatorChemicalCues.J.Herpetol.29,38(1995).759

53. vanDamme,R.&Castilla,A.M.Chemosensorypredatorrecognitioninthelizard760

Podarcishispanica:effectsofpredationpressurerelaxation.J.Chem.Ecol.22,13–22761

(1996).762

54. Cooper,W.E.Correlatedevolutionofpreychemicaldiscriminationwithforaging,lingual763

morphologyandvomeronasalchemoreceptorabundanceinlizards.Behav.Ecol.764

Sociobiol.41,257–265(1997).765

55. Cooper,W.Tandemevolutionofdietandchemosensoryresponsesinsnakes.Amphibia-766

Reptilia29,393–398(2008).767

56. Hulbert,A.J.&Else,P.L.Evolutionofmammalianendothermicmetabolism:768

mitochondrialactivityandcellcomposition.Am.J.Physiol.Integr.Comp.Physiol.256,769

R63–R69(1989).770

57. Gleeson,T.T.,Mitchell,G.S.&Bennett,A.F.Cardiovascularresponsestogradedactivity771

inthelizardsVaranusandIguana.Am.J.Physiol.Integr.Comp.Physiol.239,R174–R179772

(1980).773

58. MarshallGraves,J.A.&Peichel,C.L.Arehomologiesinvertebratesexdetermination774


https://doi.org/10.1101/551978


duetosharedancestryortolimitedoptions?GenomeBiol.11,205(2010).775

59. Hattori,R.S.etal.AY-linkedanti-Müllerianhormoneduplicationtakesoveracriticalrole776

insexdetermination.Proc.Natl.Acad.Sci.109,2955LP-2959(2012).777

60. Cortez,D.etal.OriginsandfunctionalevolutionofYchromosomesacrossmammals.778

Nature508,488(2014).779

61. Bej,D.K.,Miyoshi,K.,Hattori,R.S.,Strüssmann,C.A.&Yamamoto,Y.ADuplicated,780

TruncatedamhGeneIsInvolvedinMaleSexDeterminationinanOldWorldSilverside.781

G3Genes|Genomes|Genetics7,2489–2495(2017).782

62. Ieda,R.etal.Identificationofthesex-determininglocusingrasspuffer(Takifugu783

niphobles)providesevidenceforsex-chromosometurnoverinasubsetofTakifugu784

species.PLoSOne13,e0190635(2018).785

63. Slenter,D.N.etal.WikiPathways:amultifacetedpathwaydatabasebridging786

metabolomicstootheromicsresearch.NucleicAcidsRes.46,D661–D667(2018).787

64. Weisenfeld,N.I.,Kumar,V.,Shah,P.,Church,D.M.&Jaffe,D.B.Directdeterminationof788

diploidgenomesequences.GenomeRes.27,757–767(2017).789

65. Makunin,A.I.etal.ContrastingoriginofBchromosomesintwocervids(Siberianroe790

deerandgreybrocketdeer)unravelledbychromosome-specificDNAsequencing.BMC791

Genomics17,618(2016).792

66. Martin,M.Cutadaptremovesadaptersequencesfromhigh-throughputsequencing793

reads.EMBnet.journal17,10(2011).794

67. Li,H.Aligningsequencereads,clonesequencesandassemblycontigswithBWA-MEM.795

(2013).796


https://doi.org/10.1101/551978


68. Quinlan,A.R.&Hall,I.M.BEDTools:aflexiblesuiteofutilitiesforcomparinggenomic797

features.Bioinformatics26,841–2(2010).798

69. Quinlan,A.R.,Pedersen,B.S.&Dale,R.K.Pybedtools:aflexiblePythonlibraryfor799

manipulatinggenomicdatasetsandannotations.Bioinformatics27,3423–3424(2011).800

70. Kielbasa,S.M.,Wan,R.,Sato,K.,Horton,P.&Frith,M.Adaptiveseedstamegenomic801

sequencecomparison.GenomeRes.(2011).doi:10.1101/gr.113985.110802

71. Kent,W.J.,Baertsch,R.,Hinrichs,A.,Miller,W.&Haussler,D.Evolution’scauldron:803

Duplication,deletion,andrearrangementinthemouseandhumangenomes.Proc.Natl.804

Acad.Sci.100,11484–11489(2003).805

72. Cantarel,B.L.etal.MAKER:aneasy-to-useannotationpipelinedesignedforemerging806

modelorganismgenomes.GenomeRes.18,188–96(2008).807

73. Stanke,M.&Morgenstern,B.AUGUSTUS:awebserverforgenepredictionineukaryotes808

thatallowsuser-definedconstraints.NucleicAcidsRes.33,W465-7(2005).809

74. Slater,G.&Birney,E.Automatedgenerationofheuristicsforbiologicalsequence810

comparison.BMCBioinformatics6,31(2005).811

75. Dobin,A.etal.STAR:ultrafastuniversalRNA-seqaligner.Bioinformatics29,15–21812

(2013).813

76. Jones,P.etal.InterProScan5:genome-scaleproteinfunctionclassification.814

Bioinformatics30,1236–1240(2014).815

77. Emms,D.M.&Kelly,S.OrthoFinder:solvingfundamentalbiasesinwholegenome816

comparisonsdramaticallyimprovesorthogroupinferenceaccuracy.GenomeBiol.16,157817

(2015).818


https://doi.org/10.1101/551978


78. Lowe,T.M.&Eddy,S.R.tRNAscan-SE:aprogramforimproveddetectionoftransferRNA819

genesingenomicsequence.NucleicAcidsRes.25,955–64(1997).820

79. Griffiths-Jones,S.,Bateman,A.,Marshall,M.,Khanna,A.&Eddy,S.R.Rfam:anRNA821

familydatabase.NucleicAcidsRes.31,439–41(2003).822

80. Nawrocki,E.P.&Eddy,S.R.Infernal1.1:100-foldfasterRNAhomologysearches.823

Bioinformatics29,2933–5(2013).824

81. Löytynoja,A.Phylogeny-awarealignmentwithPRANK.inMethodsinmolecularbiology825

(Clifton,N.J.)1079,155–170(2014).826

82. Nguyen,L.T.,Schmidt,H.A.,VonHaeseler,A.&Minh,B.Q.IQ-TREE:Afastandeffective827

stochasticalgorithmforestimatingmaximum-likelihoodphylogenies.Mol.Biol.Evol.32,828

268–274(2015).829

83. Kalyaanamoorthy,S.,Minh,B.Q.,Wong,T.K.F.,vonHaeseler,A.&Jermiin,L.S.830

ModelFinder:fastmodelselectionforaccuratephylogeneticestimates.Nat.Methods14,831

587–589(2017).832

84. Hoang,D.T.,Chernomor,O.,vonHaeseler,A.,Minh,B.Q.&Vinh,L.S.UFBoot2:833

ImprovingtheUltrafastBootstrapApproximation.Mol.Biol.Evol.35,518–522(2018).834

85. Han,M.V.,Thomas,G.W.C.,Lugo-Martinez,J.&Hahn,M.W.EstimatingGeneGainand835

LossRatesinthePresenceofErrorinGenomeAssemblyandAnnotationUsingCAFE3.836

Mol.Biol.Evol.30,1987–1997(2013).837

86. Mitchell,A.L.etal.InterProin2019:improvingcoverage,classificationandaccessto838

proteinsequenceannotations.NucleicAcidsRes.(2018).doi:10.1093/nar/gky1100839

87. Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.&Lipman,D.J.Basiclocalalignment840


https://doi.org/10.1101/551978


searchtool.J.Mol.Biol.215,403–410(1990).841

88. Krogh,A.,Larsson,B.,vonHeijne,G.&Sonnhammer,E.L..Predictingtransmembrane842

proteintopologywithahiddenmarkovmodel:applicationtocomplete843

genomes11EditedbyF.Cohen.J.Mol.Biol.305,567–580(2001).844

89. Katoh,K.&Standley,D.M.MAFFTMultipleSequenceAlignmentSoftwareVersion7:845

ImprovementsinPerformanceandUsability.Mol.Biol.Evol.30,772–780(2013).846

90. Capella-Gutiérrez,S.,Silla-Martínez,J.M.&Gabaldón,T.trimAl:Atoolforautomated847

alignmenttrimminginlarge-scalephylogeneticanalyses.Bioinformatics25,1972–1973848

(2009).849

91. Suyama,M.,Torrents,D.&Bork,P.PAL2NAL:Robustconversionofproteinsequence850

alignmentsintothecorrespondingcodonalignments.NucleicAcidsRes.34,(2006).851

92. Smith,M.D.etal.Lessismore:anadaptivebranch-siterandomeffectsmodelfor852

efficientdetectionofepisodicdiversifyingselection.Mol.Biol.Evol.32,1342–53(2015).853

93. Pond,S.L.K.,Frost,S.D.W.&Muse,S.V.HyPhy:hypothesistestingusingphylogenies.854

Bioinformatics21,676–679(2005).855

856

857


https://doi.org/10.1101/551978


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.


https://doi.org/10.1101/551978


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.


https://doi.org/10.1101/551978


a high-resolution, chromosome-assigned komodo dragon ... · 71 lizards are all present in the...

Documents