Temperature-dependent mutational robustness can explain
faster molecular evolution at warm temperatures, affecting
speciation rate and global patterns of species diversity
Mikael Puurtinen
1,2*
1
1,2
1,3
1,2
1,4
, Merja Elo , Matti Jalasvuori , Aapo Kahilainen , Tarmo Ketola , Janne S. Kotiaho ,
1
Mikko Mönkkönen , Olli T. Pentikäinen
1,5
Correspondence to: [email protected]
Accepted for publication in Ecography, doi: 10.1111/ecog.01948
Abstract
Distribution of species across the Earth shows strong latitudinal and altitudinal gradients with the number of
species decreasing with declining temperatures. While these patterns have been recognized for well over a
century, the mechanisms generating and maintaining them have remained elusive. Here, we propose a
mechanistic explanation for temperature-dependent rates of molecular evolution that can influence speciation
rates and global biodiversity gradients. Our hypothesis is based on the effects of temperature and
temperature-adaptation on stability of proteins and other catalytic biomolecules. First, due to the nature of
physical forces between biomolecules and water, stability of biomolecules is maximal around +20°C and
decreases as temperature either decreases or increases. Second, organisms that have adapted to cold
temperatures have evolved especially flexible (but unstable) proteins to facilitate catalytic reactions in cold,
where molecular movements slow down. Both these effects should result in mutations being on average more
detrimental at cold temperatures (i.e. lower mutational robustness in cold). At high temperatures,
destabilizing water-biomolecule interactions, and the need to maintain structures that withstand heat
denaturation, should decrease mutational robustness similarly. Decreased mutational robustness at extreme
temperatures will slow down molecular evolution, as a larger fraction of new mutations will be removed by
selection. Lower mutational robustness may also select for reduced mutation rates, further slowing down the
rate of molecular evolution. As speciation requires the evolution of epistatic incompatibilities that prevent
gene flow among incipient species, slow rate of molecular evolution at extreme temperatures will directly slow
down the rate at which new species arise. The proposed mechanism can thus explain why molecular evolution
is faster at warm temperatures, contributing to higher speciation rate and elevated species richness in
environments characterized by stable and warm temperatures.
1
Department of Biological and Environmental Science, 40014 University of Jyvaskyla, Finland.
2
Centre of Excellence in Biological Interactions, 40014 University of Jyvaskyla, Finland.
3
Current address: Metapopulation Research Centre, 00014 University of Helsinki, Finland
4
Natural History Museum, 40014 University of Jyvaskyla, Finland.
5
Nanoscience Center, 40014 University of Jyvaskyla, Finland.
1
Introduction
Theglobaldistributionofspeciesrichnessisfarfromrandomoruniform.Instead,speciesrichnessshowsa
numberofgeographicalgradientsacrosstheEarth.Perhapsthemoststrikingofthesepatternsisthe
decreasingnumberofspeciesfromtheequatortowardthepoles,thelatitudinaldiversitygradient(LDG).
LDGshavebeendocumentedforawidevarietyoftaxainbothaquaticandterrestrialenvironments
(Hillebrand2004a).Oververylongtime-scales,theLDGhasbeenfoundtobesteeperduringperiods
characterizedbycolderandmoreseasonalclimates,suggestingthatclimatehasanimportantrolein
determiningdistributionofspeciesdiversityovertheglobe(Yasuharaetal.2012,Mannionetal.2014).
AlthoughalargenumberofhypotheseshavebeenputforwardtoexplainLDGs(Rohde1992,Willigetal.
2003,Mittelbachetal.2007),theideathatgreaterspeedofevolutionanddiversificationinlower(warmer)
latitudesmayexplaincurrentLDGshasgatheredempiricalsupportoverthepastfewdecades(Gillmanand
Wright2014).This“evolutionaryspeedhypothesis”suggeststhattemperatureaffectstherateofspeciation
byinfluencingmutationratesand/oraveragegenerationtimes(Rohde1992,EvansandGaston2005,Gillman
andWright2014).
Thereisampleandstrongevidencethatevolutionaryspeed,measuredasrateofmolecularevolution,isfaster
atwarmertemperatures(GillmanandWright2014).Positiveassociationwithvariablesrelatedtothe
functionaltemperatureoforganismsandtherateofmolecularevolutionhasbeenreportedacrossmetazoa
(Gilloolyetal.2005),inmarineprotists(AllenandGillooly2006),infish(Estabrooketal.2007,Wrightetal.
2011),inamphibians(Wrightetal.2010),inaquaticturtles(Lourencoetal.2013),infloweringplants(Davies
etal.2004),andintrees(Wrightetal.2006,Gillmanetal.2010).Associationsbetweenratesofmolecular
evolutionandnetdiversification(speciationminusextinction)havebeenreportedfrombirdsandreptiles(Eo
andDeWoody2010,Lanfearetal.2010)andfromfloweringplants(BarracloughandSavolainen2001),but
notfrommammals(Goldieetal.2011).Arecentreviewsummarizingpaleoecologicalandphylogenetic
studiesconcludedthat“thereisstrongevidencethatabroadrangeoflifeformshaveexperiencedfasternet
ratesofdiversificationatlowerlatitudes”(GillmanandWright2014).
Inarecentstudy,JetzandFine(2012)modelledglobalterrestrialvertebraterichnessacrossbioregionsusing
environmentaldatafrompast55millionyears.Forectotherms,temperaturealoneexplainedmoreofthe
variationincurrentrichnessthananyoftheothervariables(productivity,area,ortheircombination
integratedoverthe55millionyears).Forbothecto–andendotherms,thebestmodelsalwaysincluded
temperature.Thisstudystressestheindependenteffectoftemperatureonterrestrialvertebraterichness,
especiallyforectotherms.Alsostudiesfrommarinesystemssuggestthattemperature,insteadofe.g.
productivity,isthemostimportantfactorexplainingbothcurrent(Royetal.1998,Rutherfordetal.1999,
Romboutsetal.2009,Romboutsetal.2010,Tittensoretal.2010),butseeMcCallumetal.(2015)),and
historicalpatternsofspeciesrichness(Huntetal.2005,Yasuharaetal.2012).Thestrongandconsistent
2
marineLDGsarealsonoteworthy,astheycannoteasilybeexplainedbyarea,timeforspeciation,spatial
heterogeneity,orwateravailabilitythathavebeenproposedasmechanismsunderlyingterrestrialLDGs
(Hillebrand2004a,Hillebrand2004b).
However,themechanismsbywhichtemperatureacceleratesgeneticevolutionanddiversificationremain
elusive(EvansandGaston2005).Previously,modelsbasedontheeffectsofbodysizeandtemperatureon
metabolicratehavebeenproposedtoexplainmutationratesandglobaldiversitypatterns.Themetabolic
theoryofecology(MTE;Brownetal.2004)predictstheeffectsofbodysizeandtemperatureonmetabolism
throughconsiderationsofvasculardistributionnetworksandbiochemicalkinetics.Allenetal.(2006)
combinedMTEwiththeoryofpopulationgeneticstopredicttheeffectoftemperatureonratesofgenetic
divergenceamongpopulationsandspeciationincommunitiesbyspecifyingpopulationsize,communitysize,
andmutationrateastemperature-dependentparameters.ThetheoryhasbeenfurtherelaboratedbyStegen
andco-workers(Stegenetal.2009,Stegenetal.2012)toincorporatetheeffectsoftemperatureonecological
interactionsanddynamicsofresourcesupply.ThevalidityofassumptionsunderlyingMTEisnevertheless
stronglycontested(Priceetal.2012,Storch2012,Glazier2014).
Akeyassumptioninmetabolictheoriesforbiodiversityistemperature-dependenceofmutationrate,which
hasbeensuggestedtobecausedbyelevatedproductionofDNA-damagingoxygenradicalswithhigher
metabolism(MartinandPalumbi1993).However,thereisnoevidenceforadirectlinkbetweenmetabolic
rateandmolecularevolution(MooersandHarvey1994,Hoffmannetal.2004,Lanfearetal.2007),anda
directtestoftheroleofoxidativestressinincreasingheritablemutationsalsofailedtofindsupportforthis
mechanism(Joyner-Matosetal.2011).Furthermore,itisnotclearhowhighsomaticmetabolicratecould
influencegerm-linemutations(Lanfearetal.2007,Galtieretal.2009).Theproductionofoxygenradicalsby
metabolismisalsoexpectedtomostlyaffectmitochondrialDNA,whereasnuclearDNAshouldbemuchless
affected(MartinandPalumbi1993,Hoffmannetal.2004).Thedirecteffectofmetabolicrateonmutations
maythusbeoflimitedimportancetooverallgenomicmutationrates.Indeed,arecentreviewonmolecular
evolutionandLDGconcludedthat“metabolicratemayinformlittleonratesofmutationormolecular
evolutionandwearenoclosertounderstandingthedriverofvariationintherateofmolecularevolutionand
theformationofthelatitudinalbiodiversitygradient”(Dowleetal.2013).
MutationsoftenoccurintheprocessofDNAreplication,andfactorsthatincreasetherateofgermlinecell
division,especiallyshortergenerationtimes,couldthusexplainfasterratesofmolecularevolutionperunit
timeinwarmertemperatures(Rohde1992).Shortergenerationtimeshavebeenassociatedwithfaster
molecularevolutioninbirds(MooersandHarvey1994),mammals(Bromhametal.1996,Welchetal.2008)
andinvertebrates(Thomasetal.2010).However,itispossiblethatselectionagainstsomaticmutationsin
long-livedorganisms,andnotgenerationtimeperse,isthecrucialfactoraffectingmolecularevolution
(Nabholzetal.2008,Galtieretal.2009,Lanfearetal.2013,Bromhametal.2015).Althoughthereisevidence
forastatisticalassociationbetweengenerationtimeandmolecularevolution,dataontheassociationbetween
3
ambient(orphysiological)temperatureandgenerationtimeisscarce(McCoyandGillooly2008showthat
highertemperaturesareassociatedwithshorterlifespans),makingitdifficulttoevaluatethegeneral
importanceofthegenerationtimehypothesisontheassociationbetweentemperatureandratesofmolecular
evolution.Ontheotherhand,temperaturehasbeenlinkedtotherateofmolecularevolutioneveninthe
absenceofgenerationtimedifferencesine.g.hummingbirds(Bleiweiss1998)andtropicalplants(Wrightet
al.2006).
Therearealsootherfactorsthatmayaffectsubstitutionrates,butwhoserelationshipwithtemperatureor
latitudeisnotwellknown.Suchfactorsincludethedegreeofspermcompetition(Wong2014),organismsize
(Welchetal.2008,e.g.Bromhametal.2015),andpopulationsize(seeDowleetal.2013).Disentanglingthe
effectsofseveral,oftenintercorrelated,factorsonmolecularevolutioncanbedonebyincorporatingmultiple
factorstothesamemodel(seee.g.MartinandPalumbi1993,MooersandHarvey1994,Welchetal.2008,
Wong2014).Itisalsoimportanttobearinmindthatnotallgenesormutationsareexpectedtobeequally
affectedbyvariousfactors.Forexample,spermcompetitionintensityisnotexpectedtoinfluence
mitochondrialDNAevolution,sincemitochondriaareonlyinheritedfromfemales.
Temperature and mutational robustness
Wesuggestthattheeffectsoftemperatureonmutationalrobustnessofproteinsandothercatalytic
biomolecules(e.g.ribozymes)mayprovideamechanisticexplanationfortheslowerrateofmolecular
evolutioninenvironmentswhereambienttemperaturesarecold(highlatitudes,highaltitudes,deepoceans).
Inthispaper,wefocusmainlyontheeffectsofcoldtemperaturesonmutationalrobustnessandmolecular
evolution.Ourhypothesishoweversuggeststhatsimilarpatternsshouldalsobeevidentathightemperatures,
andwewillcommentevidenceforslowdownofmolecularevolutionathightemperatures,whereappropriate.
Temperaturemayaffectmutationalrobustnessintwoways.First,regardlessofthestructuraldetailsof
proteins,proteinthermodynamicstabilityatneutralpHismaximalaroundroomtemperature(Fig.1A).This
isbecausethehydrophobiceffect,themainforcedrivingproteinfolding,ismaximalatthistemperature
(Privalov1990,Privalov1997,Kumaretal.2002).Thedecreaseinstabilityatcoldertemperaturescouldbe
explainedthroughformationsofsolvent-separated-configurationswherethenonpolarproteincoreis
hydratedbyathinlayerofwater(Privalov1990,Dias2012).Averyrecentstudysuggestthatinlivecells,
cold-denaturationofproteinscanbeaseriousconcernatrealisticphysiologicaltemperatures(Danielssonet
al.2015).
Second,andperhapsmoreimportant,proteinsoforganismsthathaveevolvedtofunctionincold
environmentshaveevolvedtobethermodynamicallylessstable(Fig.1B).Itisawell-establishedempirical
factthatthereisatightassociationbetweenthetemperaturetowhichanorganismsisadaptedtoandthe
denaturationtemperatureofproteins(seee.g.Fig.1inSomero1995).Theloweredstabilityofcold-adapted
4
proteinsisduetostructuralpropertieslikedecreasedcorehydrophobicity,increasedsurfacehydrophobicity,
increasedmovementofinternalgroups,andreducednumberofinterdomainandintersubunitinteractions
(Feller2003,SiddiquiandCavicchioli2006).Thedecreaseinthermodynamicstabilityismostlikelya
consequenceofselectionforincreasedmechanicalflexibilitynecessaryformaintainingactivityoftheprotein
inthecoldenvironmentwheremolecularmotionsslowdown(FellerandGerday2003,DePristoetal.2005,
SiddiquiandCavicchioli2006).Ithasevenbeenarguedthatcold-adaptedproteinsmayhavereachedastate
thatisclosetothelowestpossiblestability,i.e.thattheycannotbecomelessstablewithoutlosingthenative
andactiveconformation(FellerandGerday2003).Importantly,coldadaptationdoesnotseemtoshiftthe
temperatureofmaximalstability,butreducesthermodynamicstabilityatalltemperatures(Cipollaetal.
2012)(seeFig.1B).
Figure1.Stylizedeffectsoftemperatureonproteinstability.Theintensityoftheorangecolourindicatesthe
probabilitythatarandommutationwillrendertheproteinnon-functional,andthemutationwillthusbe
removedbyselection(markedwithx).(A)Thestabilitymaximumofmoststudiedproteinsisaround+20°C.A
destabilizingmutationmaynotimpairproteinfoldingandstabilityat+20°C,butatalower(orhigher)
temperaturethesamemutationmaybedetrimental,andberemovedbyselection.Arecentstudy(Danielsson
etal.2015)foundthatinlivecells,proteinstabilitydecreasesmuchmoredramaticallyatlowtemperatures
thandepictedinthisfigure,suggestingthatcold-denaturationisaseriousconcerninrealisticphysiological
temperatures.(B)Incold-adaptedorganisms(psychrophiles),proteinsareunstableasaconsequenceof
selectionformaintainingflexibilityessentialforcatalysingreactions.Thelowstabilityofcold-adapted
enzymesishypothesizedtoresultinverylargeproportionofmutationsbeingharmfulandbeingremovedby
selection.
Thermostabilityisimportantformolecularevolutionbecausethermolabileproteinshavelowermutational
robustness.Thegreatmajorityofamino-acidchangingmutationsaredestabilizing(TokurikiandTawfik
2009).Mutationsarelikelytorenderathermolabileproteinnon-functionalbecausethemutatedproteinwill
notfoldtoitsnativeform,orbecauseloweredstabilityreducestheconcentrationofdissolvedproteindueto
increasedaggregationorproteolysis(Sanchez-Ruiz2010).Misfoldingproteinsmayevenhavetoxiceffects,
5
furtherincreasingthedeleteriousconsequencesofdestabilizingmutations(Geiler-Samerotteetal.2011).A
recentcomputationalstudyconcludedthatmorethermodynamicallystableproteinsaremorerobustagainst
randomamino-acidreplacements(Hormoz2013).Thermostabilityhasalsobeenfoundtosubstantially
increasethelikelihoodofproteinsmaintainingfunctionalityaftermajorrestructuring(Besenmatteretal.
2007),andthelikelihoodofevolvingneworimprovedfunctions(Bloometal.2006).Thermostabilityhas
furtherbeenfoundtodecreasetheharmfuleffectofmutagenexposureinanRNAvirus(Domingo-Calapetal.
2010).Atthelevelofamino-acidsubstitutions,lessdestabilizingsubstitutionsaremorelikelytobefixedin
evolvinglineages(Godoy-Ruizetal.2004,Godoy-Ruizetal.2006),supportingthenotionthatstabilityeffects
ofmutationsareimportantindeterminingtherateofproteinsequenceevolution.
Temperature effects on mutation and substitution rates
Inthissection,weoutlinetheeffectsoftemperatureonmutationandsubstitutionrates.Thelogicofthe
argumentispresentedwithaflow-chartinFigure2,andinFigure3wegiveacalculatedexampleofthe
relationshipbetweentemperatureandkeypropertiesofmutationsandsubstitutions,basedontheformulas
derivedbelow.
Insexualorganisms,mutationrateisthoughttobeprimarilydeterminedbythebalancebetweenreductionin
theharmfuleffectsofnewmutationsandthecostsofmorestringentproof-reading(Dawson1998,
Sniegowskietal.2000).Dawson(1998)derivedthesolutionforevolutionarystablemutationratewhen
mutationsthatdecreasethefitnessofanindividualbyafraction(1–s)occuratinfinitenumberofunlinked
diallelicloci,andwhenmodifiersthatdecreasemutationratedirectlydecreaseindividualfitness.Thesolution
givenbyDawson(1998)fortheevolutionarystablemutationrate(U*)is
∗
=
( )−
1+
,
(1)
whereα(U)describestherelationshipbetweenthemutationrateUcodedbythemodifier,andthedirectcost
(proportionalreductioninfitness)ofthemodifiertoindividualfitness.Aplausibleexplicitfunctionforα(U)is
( ) = 1−
(2)
,
wherekisascalingfactordeterminingtherateatwhichthecostsofreducingthemutationratedepress
individualfitness.Withthisfunction,wecanwritethefunctionforU*as
∗
=
1−
−
1+
,
(3)
wherethefunctiontobemaximizedisthefitnessofthemodifierallele,w:
6
=1−
−
1+
(4)
Theevolutionarilystablemutationrateinfitness-affectinglociU*canbefoundbydifferentiatingwwith
respecttoU
=
−
,andsolving
= 0withrespecttoU,whichyields
∗
=
ln
+ (5)
.
Thesolution(eq.5)impliesthatmutationrateshouldevolvetolowerlevelwhennewmutationsaremore
harmful(i.e.,whensincreases).Ourhypothesissuggeststhatmutationsaremoreharmfulincold
environmentsandinorganismswithlowfunctionaltemperatures,duetoboththedirecteffectoftemperature
tomoleculestabilityandtheevolvedlowerthermostabilityofcold-adaptedproteins,andthiswillresultin
reducedmutationrateincoldtemperatures.Inhightemperatures,theneedtomaintainproteinstructures
thatareabletowithstandheatdenaturationshouldalsomakemutationsmoreharmfulthaninbenign
temperatures.Toillustratetheargument,letusassumethattheharmfulnessofmutations(s)isafunctionof
temperature(T,in°C),withaminimum(m)aroundroomtemperature(25°C;thisvalueisarbitrarilychosen
toyieldapatternroughlymatchingempiricaldata).Notethattherelationshipformalizedinfunction6does
notapplytoanysingleprotein,butisaheuristicrelationshipintendedtoportraythecombinedeffectsof
temperatureadaptationandprotein-solventinteractionsonmutationaleffects.Wepositthatthefurther
temperaturedeviatesfromroomtemperature,themoreharmfulmutationsbecome.
=
(25 − )
+
(6)
,
whereaisaconstantscalingfactor(Fig.3A).Substitutingeq.6toeq.5andsimplifying,wegetthe
evolutionarystablemutationrateasafunctionoftemperature(Fig.3A):
∗
=
[ (1 + ) + (25 − ) ]
ln
+ (25 − )
.
(7)
Notingthatthepersitemutationrateµisproportionalthegenome-widemutationrateU,andthattherateof
neutralsubstitutionsonlydependsonmutationrate(HartlandClark1997),therateofneutralsubstitutions
(e.g.synonymousmutationsthatdonotchangetheamino-acidinaprotein,dS)issimplyproportionaltothe
mutationrate
∝
∝
∗
andcanbemodelledas
= ∗
,
(8)
7
wherepisascalingfactortotranslatethegenomicmutationratetoper-locusmutationrate.Wecanthus
makethequalitativepredictionthattherateofneutralsubstitutionswillbehighestattemperaturesnearthe
roomtemperature,anddecreaseasthetemperatureapproaches0°C,orreacheshightemperatures(Fig.3B).
Therateofsubstitutionswithfitnesseffects(e.g.mostnon-synonymousmutationsthataltertheamino-acidin
aprotein,dN)ismoredifficulttopredict,asitdependsonthedistributionofmutationeffectsandonthe
effectivepopulationsize(Lanfearetal.2014).Accountingforthesefactorsisbeyondthescopeofthecurrent
treatment.However,asthegreatmajorityofmutationswithfitnesseffectsareharmful(Eyre-Walkerand
Keightley2007),andasthelikelihoodoffixationdecreasesrapidlyastheharmfulnessofmutationsincreases
(CrowandKimura1970),forcurrentpurposeswecanmodelratesubstitutionsatfitness-affectinglocias
=
(9)
wherebisascalingfactordeterminingtherateofdeclineinfixationprobabilitywithincreasings,andthus
relatestotheeffectivepopulationsize(largerbmeansmoreefficientselection,correspondingtolarger
effectivepopulationsize)(Lanfearetal.2014).Thisformalizationpredictsthattherateofsubstitutionswith
fitnesseffectsdecreasesrapidlyatextremetemperatures,wheremutationsaremoreharmful(Fig.3B).
Theideathattheharmfulnessofmutationsdependsontemperature,andisreflectedinmutationand
substitutionrates,hasbeenputforwardbeforeinthecontextofslowmolecularevolutioninbirdsand
thermophiles.Prageretal.(1974)notedthatbirdsseemtohaveaslowerrateofmolecularevolutionthan
othervertebrates,andsuggestedthatthismightbeexplainedbythehighbodytemperatureofbirds.Since
then,severalstudieshaveconfirmedthatmolecularevolutionisslowerinbirdsthanmammals,butfaster
thaninectothermicvertebrates.StanleyandHarrison(1999),studyingcytochromebevolution,showedthat
selectionagainstnonsynonymousmutationsisstrongerinbirdsthaninmammals.Thesefindingsgivestrong
supportforthehypothesisthathighbodytemperaturesinbirdsimposestrictrequirementsforproteinsand
leadtoreducedtoleranceforaminoacidreplacements.
Inthermophiles,anecdotalevidencethatmanymissensemutations(mutationsthatchangetheamino-acidin
aprotein)arewelltoleratedinstandardgrowthtemperaturesbutbecomelethalatelevatedtemperatures
leadDrake(2009)toformulatethe“hypothesisofdangerousmissense”:theaveragemissensemutation
harmsthermophilesmorethanmesophiles.Inaccordancewiththishypothesis,Friedmanetal.(2004)found
thattheratioofnon-synonymoustosynonymousmutations(dN/dS)waslowerinthermophilicthanin
mesophilicmicrobes,andattributedthisfindingtostrongerpurifyingselectionagainstamino-acidchanging
mutationsathightemperatures.Drake(2009)foundthattherateofspontaneousmutationislowerin
thermophilesthaninmesophiles,andsuggestedthatthelowermutationrateisanadaptationtothemore
harmfuleffectsofmutationsinthermophiles.However,asmanystudiesdonotincludethermophilestothe
data(e.g.Gilloolyetal.2005,Gilloolyetal.2007),anddonotconsidermodelswhereincreasingtemperature
8
couldslowmolecularevolutiondownathigh(above40°C)temperatures,itisnotsurprisingthatthe
phenomenonisnotwidelyacknowledged.
Ourhypothesissuggeststhatlikehightemperatures,alsolowtemperaturesleadtoreducedmutational
robustness(Fig.3C),potentiallyexplainingwhyratesofmolecularevolutionandspeciationareslowerinin
coldenvironments(e.g.athighlatitudesandaltitudes.Weareawareoftwodatasetsthatcaninformaboutthe
effectofcoldertemperaturesonstrengthofselectionagainstnon-synonymousmutations.Gilloolyetal.
(2007)showedthattemperaturespeedsupbothsynonymousandnon-synonymousevolutionoveradiverse
setofanimaltaxa(rangingfrompsychrophilestomesophiles).Althoughnotreportedintheiroriginal
manuscript,therelationshipbetweentemperatureanddN/dScanbeeasilycarriedoutfromdatapublished
withthemanuscript.Intriguingly,datasuggeststhatselectionagainstamino-acidchangingmutationsin
cytochromebisindeedstrongerincoldertemperatures(Pearsoncorrelationbetweentemperatureand
dN/dS;r=0.426,n=48,P=0.003).Fortheotherstudiedgene(nicotinamideadeninedinucleotide),therewas
anon-significantrelationshiptotheoppositedirection(r=-0.311,n=36,P=0.065).Wrightetal.(2011)
studiedcytochromebevolutioninmarinefishes,anddidnotfindasignificantassociationbetween
temperatureanddN/dS.Moreempiricalresearchisclearlyneededtoassesstheeffectofcoldtemperatures
onselectionagainstnon-synonymousmutations,andconsequentselectionforreducedmutationrates.
However,itmustbenotedthatsynonymouschangesmaynotallbeneutral(Shabalinaetal.2013)ascertain
codonscanservemultiplefunctionsby,forexample,codingforanaminoacidwhilesimultaneouslyactingasa
bindingsitefortranscriptionalregulators(Stergachisetal.2013). Furthermore,organismscanprefersome
codonsoverothers.Theusageofsynonymouscodonsappearstofollowtheproportionsofavailabletransfer
RNAmoleculesinthecell,andsuboptimalcodonusageislikelytoselectformutationsthatre-establishthe
codon-tRNAbalance(Qianetal.2012).IftheconcentrationsoftRNAsareknown,themutationratetowards
balancecouldbemeasuredbycomparingdifferenttypesofrecentlyacquiredgenes.Forsomegenes,thisrate
maybenecessarytobetakenintoaccountwhendN/dSisdetermined.
9
Figure2.Theeffectsoftemperatureonmutationandspeciationrates.Atextremetemperatures(loworhigh),
mutationswithfitnesseffectsaremoredetrimental,whichdirectlyslowsdownevolutionatsitesthataffect
fitness(e.g.non-synonymoussubstitutions,dN).Becausemutationsareonaveragemoredetrimentalat
extremetemperatures,selectionfavoursreducedmutationrate,andslowsdownevolutionatsiteswithout
fitnessaffectsaswell(e.g.synonymoussubstitutions,dS).Slowermutationratefurtherslowsdownevolution
atsitesthataffectfitness.Slowevolutionofprotein-codingsequencesmeansthatgeneticincompatibilities
betweenisolatedpopulationsevolveataslowerrate,reducingtherateatwhichnewspeciesform.
10
Figure3.Effectsoftemperatureonmutationandsubstitutionrates.A.Wehypothesizethatmutationswillbe
moredetrimentalinbothlowandhightemperatures(redline,leftaxis),andthiswillselectforreduced
mutationratesatextremetemperatures(blueline,rightaxis;eq.7).B.Thesynonymoussubstitutionrate(dS;
continuousline)mirrorsthemutationrate(eq.8),butthenon-synonymoussubstitutionrate(dN;dashed
line)isdepressedatextremetemperatures,duetothemoreintensepurifyingselection(eq.9).C.Theratioof
substitutionratesatcodingandnon-codinglocishowsstrongeffectsoftemperature,beingpositiveatthecold
temperaturesandnegativeathightemperatures.Valuesusedinthecalculations:k=0.5;a=8×106;m=0.05;
p=10-7;b=50.
11
Operative temperatures in ecto- and endotherms
Theoperativetemperaturesoforganismsareofcentralimportancetoourhypothesis.Forterrestrial
ectothermsthatcanseekshadetoavoiddirectsunlight,maximaloperativetemperaturesshowlittle
latitudinalgradientfromtheequatortolatitudesupto50degrees,beingaround25 °Cto30°C.However,
minimaloperativetemperaturesdeclinefromaround22 °Cneartheequatorto0°Corbelowathigher
latitudes(seeFigure4inSundayetal.2014).Athigherlatitudes,proteinsthereforeneedtooperateandbe
stableinbothcoldandwarmenvironments.Wesuggestthatwithintherangeofoperationaltemperatures
encounteredbyorganismsincurrentglobalclimate(excludingexposedorday-activeorganismsinhot
deserts),minimaloperationaltemperaturesexertthestrongestselectionagainstnon-synonymousmutations
andexplaintheslowerrateofmolecularevolutioninectothermsathighaltitudesandlatitudes
Anumberofstudieshavefoundslowerratesofmolecularevolutionatcoldambienttemperaturesalsoin
endotherms.Bleiweiss(1998) showedthattherateofDNAevolutioninhummingbirdsdecreaseswith
altitude.Morerecentstudieshavereportedslowercytochromebevolutionathigherlatitudesandaltitudesin
mammals(Gillmanetal.2009)andinbirds(Gillmanetal.2012).Atfirstsight,thesefindingswouldseemto
suggestthatsomeotherfactorthantemperaturemustdrivetherateofmolecularevolution,sinceendotherms
cancontroltheirbodytemperature.However,manybirdsandmammalsreducetheirbodytemperatureclose
toambienttemperaturestoconserveenergyduringadverse(cold)conditionsbygoingintotorpor.Low
torporbodytemperatureswerespeculatedasonepossiblecausefortheobservedslowdownofmolecular
evolutionbyBleiweiss(1998)andGillmanetal.(2009).Wesuggestthatcoldtorporselectsforcold-active
proteinsthatfacilitateheatingupthebodyfromtorportoactivetemperature;seee.g.Williamsetal.(2011)
forrapidchangesinthecorebodytemperatureofahibernatinggroundsquirrel.Hence,likepsychrophiles,
endothermsthatregularlygointocoldtorpormayhaveevolvedthermolabileproteinstoenhancecatalytic
activityincold,andthishascoincidentallyincreasedmutationalsensitivity.Inmammals,temporalvariation
inbodytemperaturesincreaseswithincreasinglatitudeanddecreasingaveragetemperature(Boylesetal.
2013),supportingthesuggestionthatendothermfunctionaltemperaturescanfollowenvironmental
temperatureclines.
Althoughwesuggestthatminimalratherthanmaximaloperationaltemperaturesaremoreimportantin
limitingratesofmolecularevolutionwithintheconditionsofcurrentclimate(excludinghotdeserts),the
situationmaybeverydifferentifglobalclimatewasmuchwarmer.Elevatedtemperaturesexertstronger
boundariestoorganismalsurvivalthanlowtemperatures,possiblybecauseofthedestabilizingeffectsofheat
onproteins(Araujoetal.2013).Ifglobaltemperaturesweretoincreasesignificantly,wecouldthusexpect
diversitytodeclineinwarmestregions,bothviaextinctionandpossiblyalsoviaslowdowninmolecular
evolution.Indeed,duringtheextremegreenhouseconditionsca.250Mya,equatorialregionsweredevoidof
mostterrestrialandmarinetaxapresentinotherpartsoftheworldatthattime(Sunetal.2012)
12
Genetics of speciation
Completionofspeciationrequirestheevolutionofepistaticgeneticincompatibilitiesthatpreventgeneflow
betweenincipientspeciesbycausinghybridinviabilityorinfertility.Suchincompatibilitiesarisemosteasily
inpopulationsthatarereproductivelyisolated.AsoriginallyexplainedbyBateson,Dobzhansky,andMuller,
mutationsthatareadaptiveorneutralinthepopulationtheyoriginatedincanbefunctionallyincompatible
whenbroughttogetherincross-populationhybrids(Presgraves2010a).Recently,molecularstudieshave
beguntorevealtheexactidentitiesandfunctionsofsomesuchhybridincompatibilitygenes.Thegreat
majorityoftheidentifiedlociareprotein-codinggenes(Presgraves2010b,Presgraves2010a).Iftherateof
amino-acidsubstitutionsislowerincoldtemperatures,aswehypothesize,coldtemperatureswillslowdown
thespeciationrate.Speciationratewillslowdownirrespectivewhetherthelociunderlyingincompatibility
evolveneutrally,orwhethertheyareunderpositiveselection,e.g.duetointragenomicorintersexualconflicts
(Presgraves2010a,CrespiandNosil2013),orduetoenvironmentalselectivepressures.Slowermutationrate
willalsotranslatetolowerstandingepistaticgeneticvariation,meaningthatthereislessvariationthatcan
potentiallyevolvetohybridincompatibilitiesbetweenisolatedpopulations(Cutter2012,Corbett-Detigetal.
2013).
Assessing the validity of different hypotheses
Wehaveproposedamechanismthatcanexplainthestrongandconsistentassociationsbetweentemperature,
molecularevolution,diversification,andglobalpatternsofspeciesdiversity.Oursuggestedmechanismis
basedongeneralbiophysicalpropertiesofproteinsandotherbiomolecules(likeribozymes),andhence
appliestoallorganismsregardlessofthetypeofenvironmentortrophicposition.Currentlythemostpopular
hypothesisforexplainingtheassociationbetweentemperatureandmolecularevolutionistheMetabolic
TheoryofEcology(MTE;Brownetal.2004,Gilloolyetal.2005,AllenandGillooly2006,Gilloolyetal.2007),
whichproposesthatmass-specificmetabolicratesarelinearlyandpositivelyassociatedwithratesof
molecularevolution.Table1listsanumberofpredictionsthatdifferbetweenMTEandourtemperaturedependentmutationalrobustnesshypothesis,allowingforcomparisonofthetwohypotheses.Ourhypothesis
isbasedontheuniqueassumptionthathomologousproteinsfromorganismsfromcold(orhot)temperatures
willbemoresensitivetorandomamino-acidsubstitutionsatoperativetemperaturesthanproteinsfrom
mesophilicorganisms.Testingthisassumptionwillbecrucialforassessingthevalidityofthemechanism
proposedinthisarticle.
13
Table1.Differingpredictionsforpatternsbetweentemperatureandmolecularevolutionbetweenthe
metabolictheoryofecologyandthetemperature-dependentmutationalrobustnesshypothesispresentedin
thisarticle.Seemaintextfordatabearingonthesepredictionsandfurtherdiscussion.
Metabolictheoryofecology
(MTE)
Relationshipbetween
temperatureandper
generationmutationratein
ectotherms
Relationshipbetween
operativetemperatureand
dN/dS(lowerdN/dSindicates
strongerpurifyingselection)
Relationshipbetween
operativetemperatureandrate
ofsynonymousmutations(per
timeunit)
Relationshipbetween
environmentaltemperature
andrateofmolecularevolution
inendotherms
Norelationship(Gilloolyetal.
2005,Gilloolyetal.2007)
Norelationship(Gilloolyetal.
2007)
Increasing,practicallylinear
(aftercontrollingforbody
mass-0.25).
Fororganismofequalsize,
molecularevolutionshouldbe
fasterincoldenvironments
sinceactivemetabolicrate
increaseswithdecreasing
temperature(Andersonand
Jetz2005,Clarkeetal.2010).
Temperature-dependent
mutationalrobustness
hypothesis
Hump-shaped(increasingfrom
psycrophilestomesophiles;
decreasingfrommesophilesto
thermophiles)
Hump-shapedfrom
psycrophilestothermophiles.
Hump-shapedfrom
psycrophilestothermophiles
(aftercontrollingforpossible
effectofgenerationtime).
Slowerincoldenvironments
(forspecieswithcoldtorpor),
sincecold-activeenzymesare
lessmutationallyrobustand
thusevolvemoreslowly.
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17
Acknowledgements–WethankAdamAlgar,FolmerBokma,RichardField,andJean-MichelRobergefor
contributingtotheworkshoponenergyandbiodiversityinKotiahomansionin2011.Fundinghasbeen
providedbyAcademyofFinland(forMPandME)andbyKoneFoundation(forMP,MEandJSK).Wethankthe
threeanonymousrefereesforconstructivecommentsonearlierversionofthemanuscript.
18