Genomic Legacy of the African Cheetah, Acinonyx jubatus

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Biology Faculty Articles Department of Biological Sciences
12-10-2015
Genomic Legacy of the African Cheetah, Acinonyx
jubatus
Pavel Dobrynin
St. Petersburg State University - Russia
Shiping Liu
BGI-Shenzhen - China; Sun Yat-sen University - China
Gaik Tamazian
St. Petersburg State University - Russia
Zijun Xiong
BGI-Shenzhen - China
Andrey A. Yurchenko
St. Petersburg State University - Russia
See next page for additional authors
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NSUWorks Citation
Dobrynin, Pavel; Shiping Liu; Gaik Tamazian; Zijun Xiong; Andrey A. Yurchenko; Ksenia Krasheninnikova; Sergey Kliver; A.
Schmidt-Kunzel; Klaus-Peter Koepfli; Warren E. Johnson; Lukas F. K. Kuderna; Raquel Garcia-Perez; Marc de Manuel; Ricardo
Godinez; Aleksey Komissarov; Alexey Makunin; Vladimir Brukhin; Weilin Qiu; Long Zhou; Fang Li; Jian Yi; Carlos A. Driscoll;
Agostinho Antunes; T. K. Oleksyk; Eduardo Eizirik; Polina Perelman; Melody E. Roelke; David E. Wildt; Mark Diekhans; Tomas
Marques-Bonet; Laurie Marker; Jong Bhak; Jun Wang; Guojie Zhang; and Stephen J. O'Brien. 2015. "Genomic Legacy of the African
Cheetah, Acinonyx jubatus."Genome Biology16, (277): 1-19.https://nsuworks.nova.edu/cnso_bio_facarticles/733
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Authors
Pavel Dobrynin, Shiping Liu, Gaik Tamazian, Zijun Xiong, Andrey A. Yurchenko, Ksenia Krasheninnikova,
Sergey Kliver, A. Schmidt-Kunzel, Klaus-Peter Koepfli, Warren E. Johnson, Lukas F. K. Kuderna, Raquel
Garcia-Perez, Marc de Manuel, Ricardo Godinez, Aleksey Komissarov, Alexey Makunin, Vladimir Brukhin,
Weilin Qiu, Long Zhou, Fang Li, Jian Yi, Carlos A. Driscoll, Agostinho Antunes, T. K. Oleksyk, Eduardo
Eizirik, Polina Perelman, Melody E. Roelke, David E. Wildt, Mark Diekhans, Tomas Marques-Bonet, Laurie
Marker, Jong Bhak, Jun Wang, Guojie Zhang, and Stephen J. O'Brien
This article is available at NSUWorks:https://nsuworks.nova.edu/cnso_bio_facarticles/733
Genomic legacy of the African cheetah,
Acinonyx jubatus
Dobrynin etal.
Dobryninetal.GenomeBiology (2015) 16:277
DOI10.1186/s13059-015-0837-4
Dobryninetal.GenomeBiology (2015) 16:277
DOI10.1186/s13059-015-0837-4
RESEARCH OpenAccess
Genomic legacy of the African cheetah,
Acinonyx jubatus
Pavel Dobrynin1†,Shiping Liu2,25†,Gaik Tamazian1,ZijunXiong2,AndreyA.Yurchenko1,
KseniaKrasheninnikova1,SergeyKliver1,AnneSchmidt-Küntzel15,Klaus-PeterKoepfli1,3,
WarrenJohnson3,LukasF.K.Kuderna4,RaquelGarcía-Pérez4,MarcdeManuel4,RicardoGodinez5,
AlekseyKomissarov1,AlexeyMakunin1,11,VladimirBrukhin1,WeilinQiu2,LongZhou2,FangLi2,JianYi2,
CarlosDriscoll6,AgostinhoAntunes7,8,TarasK.Oleksyk9,EduardoEizirik10,PolinaPerelman11,12,
MelodyRoelke13,DavidWildt3,MarkDiekhans14,TomasMarques-Bonet4,24,25,LaurieMarker16,
JongBhak17,JunWang18,19,20,21,GuojieZhang2,26andStephenJ.O’Brien1,22*
Abstract
Background: Patternsofgeneticandgenomicvarianceareinformativeininferringpopulationhistoryforhuman,
modelspeciesandendangeredpopulations.
Results: Herethegenomesequenceofwild-bornAfricancheetahsrevealsextremegenomicdepletioninSNV
incidence,SNVdensity,SNVsofcodinggenes,MHCclassIandIIgenes,andmitochondrialDNASNVs.Cheetah
genomesareonaverage95%homozygouscomparedtothegenomesoftheoutbreddomesticcat(24.08%
homozygous),VirungaMountainGorilla(78.12%),inbredAbyssiniancat(62.63%),Tasmaniandevil,domesticdogand
othermammalianspecies.Demographicestimatorsimputetwoancestralpopulationbottlenecks:one>100,000years
agocoincidentwithcheetahmigrationsoutoftheAmericasandintoEurasiaandAfrica,andasecond11,084–12,589
yearsagoinAfricacoincidentwithlatePleistocenelargemammalextinctions.MHCclassIgenelossanddramatic
reductioninfunctionaldiversityofMHCgeneswouldexplainwhycheetahsablateskingraftrejectionamong
unrelatedindividuals.Significantexcessofnon-synonymousmutationsinAKAP4(p<0.02),agenemediating
spermatozoondevelopment,indicatescheetahfixationoffivefunction-damagingaminoacidvariantsdistinctfrom
AKAP4homologuesofotherFelidaeormammals;AKAP4dysfunctionmaycausethecheetah’sextremelyhigh(>80%)
pleiomorphicsperm.
Conclusions: Thestudyprovidesanunprecedentedgenomicperspectivefortherarecheetah,withpotential
relevancetothespecies’naturalhistory,physiologicaladaptationsanduniquereproductivedisposition.
Keywords: Geneticdiversity,Conservationbiology,Populationbiology
Background haveelongatedlegs,slimaerodynamicskullsandenlarged
The African cheetah—the world’s fastest land animal— adrenal glands, liver and heart, plus semi-retractable
is a paradigm of physical prowess that displays numer- claws that grip the earth like football cleats as they race
ous physiological adaptations allowing for magnificent after prey at >100 km/hour. Cheetahs have captured
high-speed sprints across the African plains. Cheetahs the imagination of artists, writers, regal potentates and
wildlifeloversforcenturies.Initiallydescendedfromearly
*Correspondence:[email protected] Pliocene precursors related to American pumas, their
†Equalcontributors fossil record extends across the Americas, Europe and
1TheodosiusDobzhanskyCenterforGenomeBioinformatics,SaintPetersburg
AsiauntilthelatePleistocene(∼10,000–12,000yearsago)
StateUniversity,41ASredniyAvenue,199004St.Petersburg,Russia
22OceanographicCenter,NovaSoutheasternUniversityFtLauderdale,8000 when an abrupt extinction after the last glacial retreat
N.OceanDrive,33004FtLauderdale,Florida,USA extirpated∼40speciesoflargemammals,includingchee-
Fulllistofauthorinformationisavailableattheendofthearticle
tahsandpumasfromNorthAmerica[1–5].
©2015Dobryninetal.OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktothe
CreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Dobryninetal.GenomeBiology (2015) 16:277 Page2of19
Modern cheetahs range across eastern and south- assemblies with annotated genomic feature details
ern Africa (a small number are in Iran, a relict of (Table 1) are publicly posted in the GARfield browser
the Asiatic cheetah subspecies [6]) and are considered (http://garfield.dobzhanskycenter.org)andthehubforthe
highly endangered by wildlife authorities and govern- UCSCGenomeBrowser(http://genome.ucsc.edu).
ments. As a species, cheetahs show a dramatic reduc- Three additional cheetahs from Tanzania and three
tion in overall genetic variation revealed by multiple fromNamibiaweresequencedatlowcoverage(5–6-fold;
genomic markers, including an ability to accept recip- 500bpinsertsize;Additionalfile1:FigureS4;Additional
rocal skin grafts from unrelated cheetahs [7–9]. Their file 2: Table S2) and 1,820,419 variable nucleotide sites
genetic depletion correlates with elevated juvenile mor- were identified and compared to SNV variation in other
tality, extreme abnormalities in sperm development, dif- speciesofFelidaeandmammals(Figs.1and2;Additional
ficulties until recently in achieving sustainable captive file2:TablesS15–S24).Weassessedtheextentandpattern
breeding,andincreasedvulnerabilitytoinfectiousdisease ofgenomicdiversityusingsevendifferentmeasures,each
outbreaks[10–13].Cheetahstodayremainaconservation of which affirmed the remarkable reduction in the chee-
icon and a symbol for the cost of genetic impoverish- tah’sgenicandgenomicvariability.First,cheetahsdisplay
ment caused by demographic reduction, close inbreed- thelowestoverallgenome-wideSNVincidenceamong11
ing and near extinction in small free-ranging natural species including the human, domestic cat, gorilla, lion
populations. Genetic loss in modern cheetahs has been and Tasmanian devil, and 90 % less than a feral domes-
debated,validatedandresearchedonmultiplelevels,and tic cat (Boris from St. Petersburg; Fig. 1a) [19]. Second,
is believed to derive from one or more severe popula- genomes were parsed into 50-kbp windows, which were
tionbottlenecksthatoccurredovertimeandspaceduring used to estimate SNV density; in total, 46,787 windows
the Pleistocene epoch [7, 14–18]. That precipitous drop comprised2.337Gbor99.12%ofthetotallengthofthe
in number and genetic diversity, aggravated by behav- genome.Themajorityofwindowsshowed8–15-foldless
ioral reinforcement of immense range boundaries, led variationincheetahsthaninthehuman,domesticcator
tothegeneticallydepletedcheetahpopulationssurviving wildcat (Fig. 1b). The only sampled species or popula-
today. tionwithcomparableorlowergenomicvariationthanthe
Here we present a detailed annotation and analysis of cheetahwastheGirForestlionsfromAsia,apopulation
the assembled whole-genome sequence of African chee- known to have undergone extreme genetic homogeniza-
tah that affirms the genome-wide reduction of cheetah tioninitsrecenthistory[23–27].
diversityandidentifiesgeneadaptationsthatoccurredin Third, cheetah coding genes showed dramatic genetic
thecheetah’sevolutionarylineage. diminutionasgreatas50-fold(∼98%)relativetodomes-
ticcatorwildcatgenomevariation(Fig.1c).Theextreme
Results reduction in coding gene variants would explain the ini-
DNA from a male Namibian cheetah, Chewbaaka, was tial discovery of the cheetah’s depauperate genetic vari-
parsed into seven mate-pair libraries and sequenced ation three decades ago with studies using allozymes,
to high (75-fold) coverage on Illumina HiSeq2000 and cellular protein electrophoretic variants and gene-based
assembled de novo (Additional file 1: Figures S1–S3; restriction fragment length polymorphism (RFLP) [7–9].
Additionalfile2:TablesS1,S3–S5).Cheetahgenomescaf- Fourth, cheetahs show on average 10–15-fold longer
folds(2332scaffolds;N50contig:28.2kbp,N50scaffold: homozygous stretches relative to the feral domestic cat
3.1 Mb) were aligned to the reference Felis catus 6.2 cat genome; on average 93 % of each cheetah’s genome was
genomeassembly(hereaftercalledFca-6.2)anchoredwith homozygous (Fig. 1d; Additional file 1: Figure S8). Fifth,
linkage and radiation hybrid maps [19, 20] as well as to cheetah genomes show far less heterozygous SNV sites,
thegenomesofthelion(Pantheraleo),tiger(P.tigris)and 0.019–0.021 %, reduced to 50–61 % of the incidence
domesticdog(Canisfamiliaris)usingamultiplesequence in tigers, 30 % of humans and 15 % of domestic cats
alignmentestimatedwiththeProgressiveCactussoftware [19,28](Additionalfile2:TablesS20andS21).Sixth,com-
[21].Featuresofthecheetahgenomewereannotatedfrom pletemitochondrialgenomesofcheetahsimilarlyshowon
the alignments including 20,343 protein-coding genes, average 90 % reduction in SNVs relative to other species
repeat families (∼39.5 % of the genome) and single (Additionalfile2:TableS25).
nucleotide variants (SNVs) (Table 1; Additional file 2: Seventh, we also investigated in detail the cheetahs’
TablesS6–S11andS15).Comparativeanalysisofcat(Felis major histocompatibility complex (MHC), a cluster of
catus),cheetah,lionandtigergenomesusingtheGRIMM ∼280 immune-related genes, given their functional role
and GRIMM Synteny tools [22] identified 220 break- and the remarkable observation that cheetahs accepted
pointsincluding19–121exchangesamongdifferentfelids reciprocal skinallograftsfromunrelated individualsasif
(Additional file 1: Figures S5 and S6; Additional file 2: theywereimmunological“self”[9].Anassistedassembly
TablesS13andS14).ThealignedcheetahandcatFca-6.2 of 20 cheetah MHC sequence scaffolds on the domestic
Dobryninetal.GenomeBiology (2015) 16:277 Page3of19
Table1Assemblyandannotationofthecheetahgenome
Number Feature Size Source
Genomesequenceandassembly 7cheetahs
1 A.jubatusraineyii(Tanzania) 3cheetahs 75×reference TableS2
2 A.jubatusjubatus(Namibia) 4cheetahs 5×resequencing TableS2
3 SOAPdeNovoassembly TablesS1,S4
4 AssistedassemblywithdomesticcatFca-6.2 Fca-6.2frameworkanchors: TableS5
a.RadiationHybridmap 3000markers
b.Linkagemap 60,000SNVs
5 Estimatedgenomesize(assemblyand17-mer) 2.375–2.395Gb TableS3
6 N50contigs 28.2kbp TableS4
7 N50scaffolds 3.1Mb TableS4
8 AverageGCcontent 0.475 FigureS3
Annotation
9 Codinggenes 20,343genes 601.2Mb TableS10
10 Non-codingRNA200,045loci 17Mb TableS11
a.43,878microRNA 4.41Mb TableS11
b.1,605smallnuclearRNA 186kbp TableS11
c.154,031transportRNA 12.7Mb TableS11
d.531ribosomalRNA 85kbp TableS11
11 Singlenucleotidevariants(SNVs) 1,820,419loci TablesS15–S20
12 Repetitiveelements Interspersedrepeats 746Mb TablesS6,S7
39.48%ofcheetahgenome Tandemrepeats 51.2Mb TableS8
Complextandemrepeats
2.04Mb TableS9
3,126loci
Microsatellites
23.47Mb TableS8
487,898loci
FiguresS5,S6
13 Genomicrearrangementsofcheetahvsdomesticcat 93Mb
TablesS13,S14
14 Nuclearmitochondrialsegments 105.6kbp TableS12
15 Positivelyselectedgenes 946genes DatasheetS5
16 GARfieldGenomeBrowser http://garfield.dobzhanskycenter.org
catBAClibraryMHCassembly(totalsize8.3Mb)[29,30] and non-synonymous) in the MHC immune genes from
resolved 278 genes from extended class II, class II, class the cheetah (from Namibia and Tanzania), domestic cat,
I and extended class I regions. Although most regions wildcat, human and dog [19, 20, 32]. We found a 95–
werewellcovered,completehomologuesofcertainclass 98 % reduction in both populations of cheetahs and
I MHC genes (FLA-I F, H and M) were not detected also for Cinnamon (a highly inbred Abyssinian cat who
(Additional file 1: Figures S9 and S10; Additional file 2: supplied the reference domestic cat genome Fca-6.2)
Table S26). When we compared the structural organiza- [19,20]relativetoabundantSNVsinanoutbreddomes-
tion and gene order of the MHC with other species, the tic cat (Boris), human and dog MHC regions (Fig. 2).
cheetahanddomesticcatwerehighlysimilar,butdifferent TheMHC-SNVreductionsintheinbredcatandcheetah
fromthedogandhuman.CheetahandcatMHCsinclude involve both synonymous and non-synonymous amino
three functional vomeronasal receptor genes (important acid-altering substitutions. These numerous function-
for pheromone recognition [31]) in the extended class I altering variants reflect a history of pathogen-based
region (these genes are absent in the human, nonhuman frequency-dependent selection driving MHC diversity
primates and dog). The cat and cheetah also displayed higheracrossmammals(Additionalfile2:TableS26)[33].
expansionofcertainolfactoryreceptorgenes(0.9Mband Patternsofwhole-genomesequencevariationwereused
30genes)withintheextendedclassIregion[20].Wecom- to model and infer the population history of cheetahs
paredthenumberofdetectedSNVvariants(synonymous from eastern and southern Africa (from Tanzania and
Dobryninetal.GenomeBiology (2015) 16:277 Page4of19
Fig.1Estimatesofgenomediversityinthecheetahgenomerelativetoothermammalgenomes.aSNVrateinmammals.SNVrateforeach
individualwasestimatedusingallvariantpositions,withrepetitiveregionsnotfiltered.bSNVdensityincheetahs,fourotherfelidsandhuman
baseduponestimatesin50-kbpslidingwindows.Ofthese,38,661fragmentshadlengthslessthanthespecifiedwindowsizeandthuswere
excludedfromfurtheranalysis;mostofthosefragmentsarecontigswithlengthlessthan500bp,andthus46,787windowsoftotallength2.337Gb
werebuiltandanalyzed.cNumberofSNVsinprotein-codinggenesinfelidgenomes.dThecheetahgenomeiscomposedof93%homozygous
stretches.ThegenomeofBoris,anoutbredferaldomesticcatlivinginSt.Petersburg(top)iscomparedtoCinnamon,ahighlyinbredAbyssiniancat
(Fca-6.2referencefordomesticcatgenomesequence[19,20],middle)andacheetah(Chewbacca,bottom)asdescribedhere.Approximately15,000
regionsof100MbacrossthegenomeforeachspecieswereassessedforSNVs.Regionsofhighvariability(>40SNVs/100kbp)arecoloredred;
highlyhomozygousregions(≤40SNVs/100kbp)arecoloredgreen.ThefirstsevenchromosomehomologuesofthegenomesofBoris,Cinnamon
andChewbaccaaredisplayedfordirectcomparison.Themedianlengthsofhomozygositystretchesincheetahs(sevenindividuals),Africanlions
(fiveindividuals),SiberianandBengaltigers,andthedomesticcatarepresentedinAdditionalfile1:FigureS7
Namibia,respectively)usingthediffusionapproximation subdivides into two bottlenecked derivative populations,
to the allele frequency spectrum (AFS) implemented in showed the best fit based on low bootstrap variance
the DaDi software tool [34]. The DaDi approximation and high maximum likelihood (LL = −43,587) (see
comparestheexpectedallelefrequencyandtheobserved “Materials and methods”; Additional file 1: Figure S12;
AFS over the parameter value space by computing a Additional file 2: Table S27), as illustrated in Fig. 3.
composite-likelihoodscoreforthebestofdistinctivebut The DaDi modeling results imply a >100,000-year-old
plausibleevolutionaryscenarios.Thescenariosweresim- founder event for cheetahs, perhaps a consequence of
ulated with the AFS data and the results were used their long Pleistocene migration history from North
to calculate the likelihoods of best fit for each model America across the Beringian land bridge to Asia,
(see Fig. 3 legend and “Materials and methods” for the then south to Africa, punctuated by regular popula-
decision algorithm pathway that identified the optimal tion reduction as well as limiting gene flow through
model). territory protection. Alternatively, Barnett et al. [35]
Model 4 (also denoted by 2D ISB), a two-dimensional have postulated, based on a study of ancient DNA
(2D) model of an expanding ancestral population that ofMiracinonyxtrumani(Americancheetahs),thattoday’s
Dobryninetal.GenomeBiology (2015) 16:277 Page5of19
Fig.2ComparisonofMHCregionstructurebetweencheetahanddomesticcats.Leftside:TwochromosomeB2segmentscontainingdomesticcat
MHCgenesorderedonBAClibraries[29,30].Rightside:CheetahscaffoldsrelatedtoMHCregion.Orderofscaffoldsisbasedontheresultsofsynteny
analysis(lightbluefill).IndividualgenesaredenotedbydotsandcoloredaccordingtotheirMHCclass:lightblueforextendedclassII,blueforclassII,
greenforclassIII,orangeforclassI,redforolfactoryreceptorsandpurpleforhistones.GeneticdiversityintheMHCregionwasestimatedby
calculatingSNVcountsinnon-overlapping50-kbpwindows.Thesecountsarevisualizedbycoloredlinesintheplot;forcats:greenforwildcat,redfor
BorisandpurpleforCinnamon;forcheetahs:redforTanzaniaandorangeforNamibia
AfricancheetahsoriginatedfromAsia,whichwouldindi- the Namibian population compared to the Tanzanian
cate that the 10,000-year-old founder effect coincided population, implying historic gene flow from Namibian
with an Asia to Africa cheetah dispersal around that to Tanzanian predecessors estimated at >11,084–12,589
time. years ago in Africa (Fig. 3; Additional file 1: Figure S12;
More recent late Pleistocene bottlenecks for eastern Additional file 2: Table S28). A parallel analysis using
and southern African populations would further deplete the pairwise sequentially Markovian coalescent (PSMC)
variation in both populations [2, 7, 9]. The AFS mod- algorithm for estimating demographic history lent sup-
eling indicated a notable excess in derived alleles in port to the inference of decreasing cheetah population
Dobryninetal.GenomeBiology (2015) 16:277 Page6of19
Fig.3DemographichistoryanalysisofAfricancheetah.aDemographichistoryoftwocheetahpopulations(southerninNamibiaandeasternin
Tanzania)basedonDaDianalyses.FourdistinctivebutplausiblemodelscenariosweresimulatedbytheDaDianalysiswiththeAFSdata.Model4fits
thedatabest;see“Materialsandmethods”forourdecisionalgorithmpathwaythatidentifiedmodel4asbest.bFirstandsecondgraphsrepresent
marginalspectraforapairofpopulations.Thethirdgraphshowsresidualsbetweenthemodelandtheobserveddata.Redorblueresidualsindicate
thatthemodelpredictstoomanyortoofewallelesinagivencell,respectively.Thefourthgraphshowsgoodness-of-fittestsbasedonthe
likelihoodandPearson’sstatistic,withbothindicatingthatourmodelisareasonable,thoughincomplete,descriptionofthedata
size in the last 100,000 years (Additional file 1: [37]. We aligned these genes using the parallel tool
FigureS11). ParaAT [38] and using PAML to search for genes with
Modern cheetahs display multiple physiological corre- an accelerated rate of non-synonymous to synonymous
lates of inbreeding depression in both captive and free- substitution(Dn/Ds)accumulationinthecheetahlineage
ranging populations. Compared to other Felidae species, [37]. Overall, cheetahs displayed a far more accelerated
cheetahsshowconstitutiveimpairmentsinreproduction, accumulation of non-synonymous mutations relative to
including low fecundity in captivity, an average of 80 % otherspecies(Fig.4).Weidentified92cheetahgeneswith
malformed spermatozoa per ejaculate and an elevated statistically significant elevated Dn/Ds ratios; for these,
incidence of acrosomal defects, as has been observed we identified the type and frequency of damaging muta-
in other inbred natural populations [9, 11, 12, 36]. To tions.Eighteengeneshaddamagingcommonorinvariant
explore genes that might have mediated the cheetah’s constitutive damaging mutations previously implicated
reproductive issues, we first identified 964 human genes in spermatogenesis, azoospermia, oligospermia, gonadal
with gene ontology (GO) terms related to reproduc- dysfunction and oogenesis (Additional file 2: Tables S29
tion, encoding 1730 RNA transcripts. The list was nar- and S30; Additional file 3: Datasheet S6). Of these, one
rowed to 656 genes that had a 1 : 1 ortholog match gene (AKAP4) showed an accelerated accumulation of
among the cheetah, cat, tiger, dog and human based on damaging deletions or missense mutations among sam-
BLAST and syntenic orthology using Proteinortho/PoFF pled cheetahs based upon the Polyphen2 database. An
Dobryninetal.GenomeBiology (2015) 16:277 Page7of19
Fig.4ComparisonofDn/Dsdistributionsforreproduction-relatedandallcheetahgenes.aDistributionsofbranch-specificvaluesofDn/Dsfor
reproductivesystemgenes.Dn/Dsratioswerecalculatedforfivespecies(dog,human,cat,tigerandcheetah)basedon500bootstrapreplications
andthefree-ratiomodelinPAML[37].bDistributionsofbranch-specificDn/Dsvaluesforfourspecies(dog,cat,tigerandcheetah)andancestral
reconstructedFelidaebranch.Dn/Dsratiosforbranchesbasedon200bootstrapreplicationsof10Mbprotein-codingsequences