Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral

22 Pages·2017·4.14 MB·English

SHORTREPORTS
Loss of RXFP2 and INSL3 genes in Afrotheria
shows that testicular descent is the ancestral
condition in placental mammals
ViragSharma1,2,3,ThomasLehmann4,HeikoStuckas5,LianeFunke1,MichaelHiller1,2,3*
1 MaxPlanckInstituteofMolecularCellBiologyandGenetics,Dresden,Germany,2 MaxPlanckInstitutefor
thePhysicsofComplexSystems,Dresden,Germany,3 CenterforSystemsBiologyDresden,Germany,
4 SenckenbergResearchInstituteandNaturalHistoryMuseumFrankfurt,FrankfurtamMain,Germany,
5 MuseumofZoology,SenckenbergDresden,Germany
a1111111111
a1111111111 *[email protected]
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a1111111111 Abstract
Descentoftestesfromapositionnearthekidneysintothelowerabdomenorintothescro-
tumisanimportantdevelopmentalprocessthatoccursinallplacentalmammals,withthe
OPENACCESS exceptionoffiveafrotherianlineages.Sincesoft-tissuestructuresliketestesarenotpre-
servedinthefossilrecordandsincekeypartsoftheplacentalmammalphylogenyremain
Citation:SharmaV,LehmannT,StuckasH,Funke
L,HillerM(2018)LossofRXFP2andINSL3genes controversial,ithasbeendebatedwhethertesticulardescentistheancestralorderivedcon-
inAfrotheriashowsthattesticulardescentisthe ditioninplacentalmammals.Toresolvethisdebate,weusedgenomicdataof71mamma-
ancestralconditioninplacentalmammals.PLoS
lianspeciesandanalyzedtheevolutionoftwokeygenes(relaxin/insulin-likefamilypeptide
Biol16(6):e2005293.https://doi.org/10.1371/
receptor2[RXFP2]andinsulin-like3[INSL3])thatinducethedevelopmentoftheguberna-
journal.pbio.2005293
culum,theligamentthatiscrucialfortesticulardescent.WeshowthatbothRXFP2and
AcademicEditor:LaurenceHurst,Universityof
INSL3arelostornonfunctionalexclusivelyinfourafrotherians(tenrec,capeelephant
Bath,UnitedKingdomofGreatBritainandNorthern
Ireland shrew,capegoldenmole,andmanatee)thatcompletelylacktesticulardescent.Thepres-
enceofremnantsofoncefunctionalorthologsofbothgenesintheseafrotherianspecies
Received:January1,2018
showsthatthesegenelosseshappenedafterthesplitfromtheplacentalmammalancestor.
Accepted:May24,2018
These“molecularvestiges”providestrongevidencethattesticulardescentistheancestral
Published:June28,2018
condition,irrespectiveofpersistingphylogeneticdiscrepancies.Furthermore,theabsence
Copyright:©2018Sharmaetal.Thisisanopen ofsharedgene-inactivatingmutationsandourestimatesthatthelossofRXFP2happened
accessarticledistributedunderthetermsofthe
atdifferenttimepointsstronglysuggestthattesticulardescentwaslostindependentlyin
CreativeCommonsAttributionLicense,which
Afrotheria.Ourresultsprovideamolecularmechanismthatexplainsthelossoftesticular
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal descentinafrotheriansand,moregenerally,highlighthowmolecularvestigescanprovide
authorandsourcearecredited. insightsintotheevolutionofsoft-tissuecharacters.
DataAvailabilityStatement:Allgenome
alignmentsareavailableathttps://bds.mpi-cbg.de/
hillerlab/testicondy/.Sangersequencesare
availablefromGenBank(accessionnumbers
Authorsummary
MH347268-MH347272).Alldataarealsoavailable
onrequest.
Whilefossilsofwhaleswithlegsdemonstratethatthesespeciesevolvedfromleggedances-
Funding:MaxPlanckSociety.ReceivedbyMH. tors,theancestralstateofnonfossilizingsoft-tissuestructurescanonlybeindirectly
Thefunderhadnoroleinstudydesign,data inferred.Thisdifficultyisalsoconfoundedbyuncertaintiesinthephylogeneticrelation-
collectionandanalysis,decisiontopublish,or
shipsbetweentheanimalsconcerned.Aprimeexampleisthecaseoftesticulardescent,a
preparationofthemanuscript.GermanResearch
PLOSBiology|https://doi.org/10.1371/journal.pbio.2005293 June28,2018 1/22
LossoftesticulardescentinAfrotheria
Foundation(grantnumberHI1423/3-1).Received
byMH.Thefunderhadnoroleinstudydesign, developmentalprocessthatdeterminesthefinalpositionoftestes,whichoccursinmost
datacollectionandanalysis,decisiontopublish,or placentalmammalsbutisabsentfromseveralafrotherianlineages.Here,wediscovered
preparationofthemanuscript.LeibnizAssociation
thatafrotherianspossessremnantsofgenesknowntoberequiredfortesticulardescent.
(grantnumberSAW-2016-SGN-2).Receivedby
These“molecularvestiges”showthattesticulardescentwasalreadypresentintheplacen-
TL,HS,MH.Thefunderhadnoroleinstudy
talancestorandwassubsequentlylostinAfrotheria.Ourstudyhighlightsthepotentialof
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. molecularvestigesinresolvingcontradictoryancestralstatesofsoft-tissuecharacters.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Abbreviations:CESAR,CodingExon-Structure Introduction
AwareRealigner;INSL3,insulin-like3;Mya,million
yearsago;RXFP2,relaxin/insulin-likefamily Inplacentalmammals—theeutheriancrowngroupconsistingofthecladesAfrotheria,Xenar-
peptidereceptor2;SRA,SequenceReadArchive. thra,andBoreoeutheria[1]—optimaltesticularfunctionrequiresatemperaturethatislower
thanthebodytemperature.Toachievethis,thetestesarelocatedoutsideoftheabdominalcav-
ityinascrotuminmanyspeciessuchasprimates,mostrodents,lagomorphs,mostcarnivores,
andmostterrestrialartiodactyls[2,3].Alternatively,testesarelocatedinthelowerabdomen
indolphins,trueseals,pangolins,andothermammals.Inthesespecies,testicularcoolingis
achievedbyvascularcountercurrentheatexchangersystems,asobservedindolphin[4];direct
coolingwithbloodfromthehindlimbs,asobservedinseals[5];ortesticularcoolingmaynot
benecessary,asthesespecieshavelowerbodytemperatures[2,6,7].
Thepositionofthetestesinthelowerabdomenorinthescrotumistheresultofadevelop-
mentaldescentprocess(S1Fig).Duringmammaliandevelopment,testesinitiallyformata
positionnearthekidneysintheembryo.Testiculardescentintothescrotumoccursintwo
phases:firstfromtheabdomentotheinguinalcanalandsecondthroughtheinguinalcanal
intothescrotum[8,9].Thefirsttransabdominalphaseisgovernedbythegrowthandreorga-
nizationofthegubernaculum,aligamentthatconnectsthelowerpoleofthetestesandinner
ringofthefutureinguinalcanal[8–10].Migrationofthetestesiscausedbytheswellingofthe
distalgubernaculum,whichanchorsthetestistotheinguinalcanal,whiletheabdominalcavity
enlarges.Thesecondinguinoscrotalphaseisdependentonandrogensignalingandrequires
theelongationofthegubernaculum,whichmigratesintothescrotum[8–10].Theinvolved
signalingandmechanicsmaketesticulardescentadifficultandcomplexdevelopmentalpro-
cess.Failureinanyofthedescentphasesresultsinapathologicalconditioncalledcryptorchi-
dism(absenceoftestesfromthescrotum),whichisacongenitalbirthdefectobservedatan
appreciablefrequencyinhumanmales(2%–4%atbirth[11])andotheranimals(upto10%in
maledogs[12],2%inmalecats[13],2%–8%inmalehorses[14]).
Almostallplacentalmammalsexhibiteitherpartialdescent(onlythetransabdominal
phase),whichresultsinascrotaltesteslocatedinthelowerabdomen,orcompletedescent
(transabdominalandinguinoscrotalphase),whichresultsinscrotaltestes[2,3].Anotable
exceptionisAfrotheria,inwhichfiveofthesixmainlineages(representedherebythelesser
hedgehogtenrec,capegoldenmole,capeelephantshrew,manatee,elephant,androckhyrax)
donotshowanytesticulardescentandhavetestespositionedattheirinitialabdominalposi-
tionnearthekidneys[2,3,15–17].Thislackofanytesticulardescentistermedtesticondy.
Theaardvarkistheonlyafrotherianexhibitingdescendedbutascrotaltestes[2,3,18].Asche-
maticillustrationofthedifferentpositionoftestesinmammalsisshowninS1Fig.
SinceAfrotheriarepresentoneofthethreemaincladesofplacentalmammals(together
withXenarthraandBoreoeutheria),twodifferentevolutionaryscenarioscouldexplaintesti-
condyinseveralafrotherianlineages.First,iftesticondyistheancestralconditioninplacental
mammals,thentesticulardescentwasgainedtwoorthreetimes(dependingonthephylogeny)
inXenarthra,Boreoeutheria,andtheaardvarklineage.Second,iftesticulardescentisthe
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LossoftesticulardescentinAfrotheria
ancestralconditioninplacentalmammals,thentesticulardescentwaslostonceormoreoften
(againdependingonthephylogeny)infiveofthesixafrotherianlineages.Sincesoft-tissue
structuresliketestesandthetransientgubernaculumligamentaretypicallynotpreservedin
thefossilrecord,theevolutionofsuchsoft-tissuestructurescanonlybeinferredbyanalytical
methodssuchasparsimony,extantphylogeneticbracketing,ormaximumlikelihood[19–22],
allofwhichrelyonthegivenphylogenetictree.Consequently,resolvingwhethertesticondyor
testiculardescentistheancestralconditioninplacentalmammalsrequiresaccurateknowledge
oftheunderlyingphylogeny.
Unfortunately,althoughintegrativeapproachesusingbothmorphologicalandmolecular
charactershavebroughtmajoradvancesinourunderstandingofmammalianphylogeny[23–
26],thereisstillnofinalconsensusontherelationshipsbetween(andsometimeswithin)the
maincladesofplacentalmammals.Inparticular,theplacentalrootandbranchingpatternof
thecladesAfrotheria,Xenarthra,andBoreoeutheriaarestilldebated[26–28](S2Fig),andan
analysisofraregenomiceventsraisedtheconcretepossibilityofanear-simultaneoussplit
[29].Furthermore,thephylogenywithinAfrotheriaisnotwellresolved,becauseofconflicting
evidenceforthepositionoftheaardvark(theonlynontesticondafrotherianlineage)andthe
relationshipsbetweenmanatees,elephants,andhyraxes[30–34](S3Fig).
Giventhesephylogeneticuncertainties,itisprobablynotsurprisingthattwodifferentstud-
iesreachedoppositeconclusionsaboutwhethertesticondyistheancestralorderivedstatefor
placentalmammalsandforAfrotheria.WerdelinandNilsonne[2]inferredthattesticular
descentinplacentalmammalsandAfrotheriaistheancestralcondition(testiculardescentwas
subsequentlylost).However,theirresultswerebasedonaphylogenyinwhichAfrotheriawere
nestedwithinBoreoeutheria,whichisnotsupportedbycurrentphylogenies.Morerecently,
Kleisnerandcolleagues[3]reexaminedtheevolutionoftesticulardescentinthecontextof
currentphylogeniesandcametotheoppositeconclusionthattesticondyinplacentalmam-
malsandAfrotheriaistheancestralphenotypiccharacter.
Here,wesoughttoresolvethisdebatewhethertesticondyortesticulardescentistheances-
tralconditioninplacentalmammalsandinAfrotheriabyusingmolecularevidence.First,we
reasonedthatiftesticulardescentisancestral,thentesticondafrotherianlineagesmayhave
lostkeygeneticinformationthatisnecessaryfortesticulardescent.Suchalossofgeneticinfor-
mationmaybedetectablebycomparativegenomicsanalysis.Second,wereasonedthatiftes-
ticulardescentisancestralandifaardvarksarenestedwithinAfrotheria,thentesticondy
wouldhaveevolvedindependentlyseveraltimes.Thisisexpectedtoleaveasignatureofinde-
pendentlossofthegeneticinformationthatisnecessaryfortesticulardescent.Byanalyzing
theevolutionoftwokeygenes(relaxin/insulin-likefamilypeptidereceptor2[RXFP2]and
insulin-like3[INSL3])thatarerequiredforgubernaculumdevelopmentandfunctionin71
placentalmammals,wefoundthatbothgeneshaveloss-of-functionmutationsonlyinseveral
testicondafrotherianspecies.Theabsenceofsharedinactivatingmutationsandourageesti-
matesforthelossofRXFP2furthersuggestthattesticondyevolvedindependentlyinafrother-
ianlineagesatdifferenttimepoints.Together,theseresultsprovidenotonlyamolecular
mechanismthatexplainsthelossoftesticulardescentinafrotherianlineagesbutalsoshows
thattesticulardescentistheancestralstateforplacentalmammalsandAfrotheria.
Results
Comparativeanalysisofthegubernaculum-inducingRXFP2andINSL3
genesin71placentalmammals
Todetermineiftesticondyistheancestralorderivedconditionforplacentalmammalsandfor
Afrotheria,weexaminedtwokeygenesthatarenecessaryandsufficientforthedevelopment
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LossoftesticulardescentinAfrotheria
ofthegubernaculum:INSL3andRXFP2.INSL3encodesarelaxin-likehormonethatissecreted
byLeydigcellsofthetestesandbindsspecificallytothetransmembranereceptorencodedby
RXFP2,whichishighlyexpressedingubernacularcells[35–39].TheINSL3-RXFP2ligand-
receptorpairpromotesgubernacularcellproliferationandstimulatestheswellingreaction
[40–42].Bothgenesarenecessaryforgubernacularfunction,asknockoutofRXFP2[37,39]or
INSL3[40,43,44]inmiceresultsintheabsenceofthegubernaculumandnotesticular
descent,whichinturnleadstospermatogenesisdefectsandmaleinfertility.Despitethefact
thatRXFP2isalsoexpressedinpostmeioticspermatogeniccells,surgicallycorrectingtheposi-
tionofundescendedtestesinglobalINSL3knockoutmiceoraknockoutofRXFP2thatis
restrictedtomalespermcellsresultsinnormalspermatogenesisandfertility[39,43],suggest-
ingthatbothgenesaredispensableforspermatogenesisandgermcellsurvivalinadultmale
mice.
ToinvestigatetheevolutionofRXFP2andINSL3inplacentalmammals,wemadeuseof
existinggenomealignmentsbetweenhumansand68othermammals[45].Inaddition,wefur-
thercomputedagenomealignmentbetweenhumanandthemostrecentgenomeassemblies
oftherockhyraxandHoffmann’stwo-toedsloth(Materialsandmethods).Inspectingthe
genomiclocithatcorrespondtohumanRXFP2andINSL3allowedustoexaminebothgenes
inall70mammals,evenintheabsenceofgeneannotationsformostofthesespecies.
LossoftheRXFP2andINSL3genesinfourtesticondafrotherianlineages
ToinvestigateiftesticondAfrotherialostthegeneticinformationnecessaryfortesticular
descent,wefirstexaminedthecodingregionofRXFP2andINSL3insevenafrotherianswith
availablegenomes(aardvark,lesserhedgehogtenrec,capegoldenmole,capeelephantshrew,
manatee,elephant,andhyrax).Ourgenomealignmentsrevealedthatfourtesticondlineages
(tenrec,capegoldenmole,capeelephantshrew,andmanatee)haveseveralmutationsin
RXFP2thatinactivateitsreadingframe.Thesegene-inactivatingmutationscreatepremature
stopcodons,shiftthereadingframe,disruptthesplicesitedinucleotides,anddeleteentire
exons(Fig1A).Furthermore,threeoutofthesefourspecies(tenrec,capeelephantshrew,
manatee)alsohaveinactivatingmutationsintheINSL3gene(Fig2A).Sincethesemutations
affectseveralexonsanddestroyfunctionalproteindomainsinINSL3(A-andB-chain,Fig
2A),itishighlyunlikelythattheremnantsofthesegenesencodeafunctionalprotein.Impor-
tantly,reciprocal-bestBLASThitsandconservedgeneorderclearlyshowthattheseremnants
are“molecularvestiges”thatcorrespondtotheRXFP2andINSL3genes(S4Fig).Inanalogyto
vestigialorgans,thesemolecularvestigesimplythepresenceofoncefunctionalRXFP2and
INSL3orthologsthatweresubsequentlylostinseveralafrotheriansduringevolution.
Toconfirmthattheseinactivatingmutationsarerealanddonotrepresentgenomeassem-
blyoralignmenterrors,weusedamultistepvalidationapproach.Sincegenomealignmentsdo
nottakereadingframeandsplicesiteinformationintoaccount,wefirstsoughttoruleoutthe
possibilitythatinactivatingmutationsareaconsequenceofalignmentambiguities.Tothis
end,werealignedallcodingexonswiththeCodingExon-StructureAwareRealigner
(CESAR),anexonalignmentmethodthatproducesanalignmentwithconsensussplicesites
andanintactreadingframewheneverpossible[49,50].CESARconfirmedthatallaffected
exonsexhibitinactivatingmutations(Figs1and2A).Second,tovalidatethatthesemutations
arenotsequencingorassemblyerrors,weinvestigatedrawsequencingreadsfromthe
SequenceReadArchive(SRA)[51].ForbothRXFP2andINSL3,wefoundthatallgenomic
locicontaininganinactivatingmutationaresupportedbyatleast10sequencingreads,while
notasinglereadalignstoaputativesequence,inwhichtheinactivatingmutationwasreversed
toitsancestralstate.Wefurtherconfirmedthepresenceoftwoframeshiftingmutationsin
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LossoftesticulardescentinAfrotheria
Fig1.Gene-inactivatingmutationsinRXFP2infourafrotherianspecies.(A)Theexon-intronstructureofthecodingregionoftheRXFP2geneis
shownasboxes(exons,drawntoscale)andlines(introns,notdrawntoscale).Averticalredline/arrowheadindicatesaframeshiftingdeletion/
insertion,withthenumberofdeleted/insertedbasesgivenabove.Stopcodonmutationsareshownasablackverticalline.Splicesitemutationsare
indicatedbythemutateddinucleotide.Ablueverticallineindicatesaframe-preservingdeletion.Redboxesareexonsthatareeitherdeletedor
accumulatednumerousmutationsthatdestroyanysequencesimilarity.Allinactivatingmutationswerevalidatedbyunassembledgenome
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LossoftesticulardescentinAfrotheria
sequencingreadsstoredintheSRA.Elephant,rockhyrax,andaardvarkhaveanintactRXFP2geneandarenotshown.Afilledstarindicates
mutationsthatweconfirmedbyPCRandSangersequencinginthelesserhedgehogtenrec;theexon17frameshiftwasalsofoundinthegreater
hedgehogtenrec(S5AandS5BFig).AnopenstarindicatesmutationsthatweconfirmedbyPCRandsequencinginthedugong,thesisterspeciesof
themanatee(S5CandS5DFig).(B-I)ExamplesofinactivatingmutationsandtheirvalidationbyunassembledSRAreads.RXFP2,relaxin/insulin-
likefamilypeptidereceptor2;SRA,SequenceReadArchive.
https://doi.org/10.1371/journal.pbio.2005293.g001
Fig2.Gene-inactivatingmutationsandcriticalaminoacidmutationsinINSL3.(A)FunctionaldomainsoftheINSL3proteinandtheexon-intronstructureofthe
INSL3gene.InactivatingmutationsareasinFig1,frame-preservinginsertions/deletionsareshownasbluelines/arrowheads.Exonsbutnotintronsaredrawntoscale.
Elephant,rockhyrax,andaardvarkhaveanintactINSL3andarenotshown.(B)Whilethecapegoldenmoledoesnotexhibitanygene-inactivatingmutations(A),an
INSL3proteinalignmentoftheA-andB-chainshowsmutations(redbackground)ataminoacidsthatarecriticalforstructureandfunctionofthematurehormone
(graybackground)[46–48].DisulfidebondsbetweenCysresiduesareindicatedbybluelines.Residuesthatareaffectedbyframeshiftingdeletionsintheunderlying
tenrecorcapeelephantshrewnucleotidesequenceareindicatedbyasterisks.Forthesetwospecies,weignoredtheseframeshiftsandusedtheancestralreadingframe.
Speciesinredfontaretesticond.Notethatelephantandrockhyraxhavenomutationsatanyofthecriticalsites.INSL3,insulin-like3.
https://doi.org/10.1371/journal.pbio.2005293.g002
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LossoftesticulardescentinAfrotheria
RXFP2inthelesserhedgehogtenrecbyPCRandSangersequencing(S5AandS5BFig).Over-
all,thisshowsthattheinactivatingmutationsshowninFigs1and2Aarenotsequencing
errorsorartefactsarisingfromgenomeassemblyoralignmentissues.Finally,weinvestigated
whetherhithertoundetectedfunctionalcopiesofRXFP2orINSL3existinafrotheriansthat
mayhavearisenbylineage-specificduplications.Byperformingultra-sensitivegenomealign-
ments,weonlydetectedasingleorthologouslocusforRXFP2andINSL3.Inaddition,we
foundalignmentstoRXFP1,aparalogofRXFP2thatexistsinallplacentalmammals(S6Fig),
showingthatthesealignmentparametersaresufficientlysensitivetoevendetectmoreancient
geneduplications.Together,thisexcludesthepossibilitythatafrotherianspossessanother
functionalduplicatedcopyofRXFP2orINSL3.
IfRXFP2andINSL3aretrulylost,wefurtherexpectthattheyevolveneutrallyintheline-
ageswithinactivatingmutations.Indeed,usingRELAX[52],wefoundthatRXFP2evolves
underrelaxedselectioninallfourgene-lossspecies(adjustedPvalues<3.2e−5,S1Table).For
INSL3,nosignificantevidenceforrelaxedselectionwasfound,likelybecauselargedeletionsin
thisshort131-residueproteinintenrec,capeelephantshrew,andmanatee(Fig2A)severely
reducedalignmentlength.Therefore,weinspectedthetwoproteindomainsthatarenecessary
forthefunctionofthematureINSL3hormone.Similartoinsulin,thepreprohormoneINSL3
isprocessedintoanA-andB-chainpeptide.TheA-andB-chainthenformsaheterodimer
thatisstabilizedbytwodisulfidebondsbetweentheA-andB-chainsandonedisulfidebond
withintheA-chain[47].Wefoundthattenrec,capeelephantshrew,andmanateehavedele-
tionsthatoverlaptheA-andB-chainandaffectresiduesthatarecriticalforINSL3structure
andfunction(Fig2B).Together,ourresultsconclusivelyshowthattheremnantsofRXFP2
andINSL3cannotencodefunctionalproteinsinseveraltesticondafrotherianlineages.
SinceINSL3lacksclearinactivatingmutationsinthecapegoldenmole,weexaminedthe
residuesthatareimportantforINSL3structureandfunction.WefoundthattheCysatposi-
tion10intheA-chainthatformsadisulfidebondwithCysatposition15[47]ismutatedtoa
Tyrinthecapegoldenmole(Fig2B).Furthermore,theLysatposition8intheB-chain(Fig
2B),aresiduethatisimportantforreceptoractivation[48],isdeletedinthisspecies.Thissug-
geststhat,whileINSL3stillhasanintactreadingframeinthecapegoldenmole,itaccumulated
mutationsthatmostlikelyrendertheencodedproteinnonfunctional.
RXFP2andINSL3areintactinelephantandrockhyrax
Interestingly,bothRXFP2andINSL3lackanygene-inactivatingmutationsintheelephantand
therockhyrax,twoafrotheriansthatarealsotesticond[15,17].Whiletheelephanthasa2-bp
deletioninthelastexonofRXFP2,thismerelytruncatestheC-terminusby25residuesandis
notanindicationofloss(seesectionRXFP2andINSL3areintactinallnontesticondplacental
mammalsandS7Fig).Furthermore,RELAXestimatesaKa/Ksvalueof0.31and0.33forele-
phantandrockhyrax,respectively,whichisslightlybutnotsignificantlyhigherthantheKa/
Ksvalueof0.27observedforothermammals.Thus,thereisnosignificantevidenceforrelaxed
selectioninthesetwolineages.Wealsoscannedbothgenesforaminoacidmutationsthat
wereonlyobservedinhumancryptorchidismpatients(V18M,P49S,W69R,P93L,R102C,
R102H,R105H,N110KinINSL3andT222PinRXFP2[8]).WhereaselephantINSL3exhibits
theR102Hmutation,thismutationisobservedinmanyothernontesticondmammals,and
celllineexperimentshaveshownthatthismutationdoesnotaffectINSL3activity[38].Simi-
larly,elephantRXFP2hasaT222A(ThrtoAla)mutationatapositionwhereamutationfrom
ThrtoProrendersRXFP2nonfunctional[37,53].However,theT222Amutationthatispres-
entinelephantisalsoobservedinthenontesticondaardvarkandpangolin,andexperiments
haveshownthatmutatingthisThrtoAladoesnotaffectRXFP2function[53].Therockhyrax
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LossoftesticulardescentinAfrotheria
doesnotexhibitanyofthemutationsobservedinhumancryptorchidismpatients.Basedon
theseevidences,elephantandrockhyraxRXFP2andINSL3mayencodefunctionalproteins.
RXFP2andINSL3areintactinallnontesticondplacentalmammals
Sofar,ouranalysissuggeststhatthefunctionofRXFP2andINSL3isonlycompromisedinsev-
eraltesticondafrotherians.Therefore,weexaminedbothgenesintheaardvark,theonly
afrotherianexhibitingpartialtesticulardescent[2,18],andfoundthatRXFP2andINSL3are
intactandevolveunderselection(S1Table)andthatINSL3lacksanymutationsofcritical
aminoacids(Fig2B).TofurtherinvestigatetherelationbetweenlossofRXFP2andINSL3and
testicondy,weexaminedbothgenesin64othernontesticondmammals.Whilethegenome
alignmentshowedafewputativeinactivatingmutationsinsomespecies,adetailedmanual
inspectionrevealedthattheseareassemblyerrors(S8Fig),assemblygaps(S9Fig),andalign-
mentambiguities(S10Fig).ForRXFP2,wefurtherfoundthattheN-terminusis17amino
acidslongerinhuman,chimpanzee,bonoboandgorilla(S11Fig)andthatseveralspecieshave
smalltruncationsandelongationsoftheC-terminuswithoutaffectingthetransmembrane
domainsofthereceptor(S7Fig).SinceRXFP2doesnotevolveunderrelaxedselectioninany
ofthese64mammals(S1Table),theselengthvariationsarenotanindicationofgenelossbut
supportpreviousobservationsthatN-andC-terminiofproteinsareevolutionarilylesscon-
strained[49,54].Together,thisshowsthatbothgenesareintactandunderselectioninall
othernontesticondplacentalmammals.
Datingthegenelosseventsindicatesthattesticondyevolvedindependently
inAfrotheria
Interestingly,weobservednoinactivatingmutationsinRXFP2andINSL3thatareshared
amonganytesticondafrotherianspecies,suggestingthatthelossofthesegeneshappened
independently,afterthesespeciessplitfromtheircommonancestors.SinceRXFP2andINSL3
areexpectedtoevolveneutrallyafterthelossoftesticulardescent,anestimateofhowlong
thesegeneshavebeenevolvingneutrallyprovidesanestimateforwhentesticondyoccurred.
Tothisend,forthefourbranchesinthephylogenetictreeleadingtothefourgene-lossspecies,
weestimatedtheportionofthebranchwherethegeneevolvedunderselectionandtheportion
whereitevolvedneutrally,asdescribedin[55,56].SincelargepartsofINSL3aredeletedin
tenrec,capeelephantshrewandmanatee(Fig2A)andsinceexon1overlapsassemblygapsin
severalotherspecies(Fig3),wefocusedonRXFP2,forwhicheachgene-lossspeciesprovides
atleast594bpinthecodonalignment,toobtainrobustestimates.
AsshowninFig4andS2Table,weestimatethateachlineagelosttheRXFP2geneatdiffer-
enttimepoints.Consistentwiththelargenumberofinactivatingmutations,RXFP2appearsto
belostfirstinthecapeelephantshrewaround66–83millionyearsago(Mya).Forthelesser
hedgehogtenrec,weestimatethatRXFP2losshappenedaround50–59Mya.Consistentwith
thisestimate,wefoundthatthesamegene-inactivatingmutationinRXFP2exon17isshared
withitssisterspeciesgreaterhedgehogtenrec(S5BFig),suggestingthatRXFP2lossalready
occurredintheancestorofbothtenrecspeciesthatlived7–14Mya(Fig4).ThelossofRXFP2
inmanateeisestimatedtohavehappenedaround43–51Myaandthuslikelypredatesthesplit
ofthemanateeanddugonglineage26–53Mya.Totestthis,weusedPCRandsequencing
experimentsandfoundthatthedugongsharestwostopcodonmutationsindifferentexons
withthemanatee(S5CandS5DFig),confirmingthatRXFP2losspredatesthesplitofmana-
teesanddugongs.Thelossinthecapegoldenmolelikelyhappenedmorerecently(23–28
Mya),consistentwithourobservationsthatINSL3didnotyetaccumulateagene-inactivating
mutation.Whiletheabsoluteestimatesofgene-losstimesaretentative(asfossil-basedtime
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LossoftesticulardescentinAfrotheria
Fig3.RXFP2andINSL3areonlylostinseveraltesticondafrotherianlineages.Theexon-intronstructuresofRXFP2(18codingexons)and
INSL3(2codingexons)areshown.Ayellowrectangleindicatesanintactexon.Exonswithinactivatingmutations(stopcodonorsplicesite
mutations,frameshifts)areindicatedinorange;completelydeletedorhighlydivergedexonsareindicatedinred.Grayexonscouldnotbe
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LossoftesticulardescentinAfrotheria
examinedbecausetherespectivegenomeassemblyisincompleteatthislocus(assemblygap).WecouldnotexaminetheRXFP2inthe
fragmentedpangolingenome,becausedifferentpartsofRXFP2locusaligntoatleast5differentshortscaffolds.Neitherexonsnorintronsare
drawntoscale.INSL3,insulin-like3;RXFP2,relaxin/insulin-likefamilypeptidereceptor2.
https://doi.org/10.1371/journal.pbio.2005293.g003
calibrationsofthespeciesdivergencetimesarelacking),thesetimeintervalsaresubstantially
differentfromeachother(Fig4).Togetherwiththeabsenceofsharedinactivatingmutations,
thisstronglysuggeststhattesticondyevolvedindependentlyinAfrotheria.
Discussion
Theevolutionoftesticondyandwhethertesticulardescentistheancestral[2]orderivedstate
[3]forplacentalmammalsandforAfrotheriahasbeencontroversial,despiteagreementinthe
phenotypiccharacterassignment.Thedifferentconclusionsaremainlyduetopersistingdif-
ferencesinthephylogeny,whichaffectancestralcharacterreconstruction.Toresolvethis
debate,weinvestigatedtheevolutionofRXFP2andINSL3,twogenesencodingahormone
receptorpairthatisrequiredforthedevelopmentofthetestes-descendinggubernaculumliga-
ment.WefoundremnantsofoncefunctionalorthologsofRXFP2andINSL3asmolecularves-
tigesinfourtesticondafrotherianlineages.Togetherwiththepresenceoforthologsofboth
genesinotherAfrotheriaandotherplacentalmammals,thisshowsthatthesegeneswerelost
Fig4.EvolutionofRXFP2andestimatedtimeintervalsduringwhichRXFP2cameunderneutralevolution.Redboxesrepresent
alowerandupperboundforanestimatedtimeintervalduringwhichRXFP2startedtoevolveneutrally(Materialsandmethods,S2
Table),whichshowsthatthegenewaslostindependentlyatdifferenttimepoints.Grayboxesrepresentanestimatedintervalfor
speciesdivergencetimestakenfromTimeTree[57](seealsoS4Table).Speciesinbluefontaretesticond.Thephylogeneticposition
oftenrecs,goldenmoles,elephantshrewsiswellsupportedbymorphologicalandmolecularcharacters[23,24,26].Uncertain
phylogeneticrelationshipsareshownasapolytomy.NotethatwhilethelossofRXFP2intheelephantshrewisestimatedtohave
happenedatthebaseofthelineage,thislossisclearlyindependentfromthemuchlaterlossinthetenrecandinthegoldenmole
lineage.EvidenceforthelossofRXFP2inthedugongandthegreaterhedgehogtenrecwasobtainedbyPCRandsequencing
experiments(S5Fig).RXFP2,relaxin/insulin-likefamilypeptidereceptor2.
https://doi.org/10.1371/journal.pbio.2005293.g004
PLOSBiology|https://doi.org/10.1371/journal.pbio.2005293 June28,2018 10/22