THEVITALQUESTION

WHYISLIFETHEWAYITIS?

NICKLANE

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ForAnaMyinspirationandcompanionOnthismagicaljourney

CONTENTS

Introduction:Whyislifethewayitis?

PARTI:TheProblem1Whatislife?2Whatisliving?

PARTII:TheOriginofLife3Energyatlife’sorigin4Theemergenceofcells

PARTIII:Complexity5Theoriginofcomplexcells6Sexandtheoriginsofdeath

PARTIV:Predictions7Thepowerandtheglory

Epilogue:Fromthedeep

Glossary

AcknowledgementsBibliography

ListofillustrationsIndex

THEVITALQUESTION

T

INTRODUCTION:WHYISLIFETHEWAYITIS?

hereisablackholeattheheartofbiology.Bluntlyput,wedonotknowwhylifeisthewayitis.All complex life on earth shares a common ancestor, a cell that arose from simple bacterial

progenitors on just one occasion in 4 billion years. Was this a freak accident, or did other‘experiments’ in the evolution of complexity fail?We don’t know.We do know that this commonancestorwasalreadyaverycomplexcell.Ithadmoreorlessthesamesophisticationasoneofyourcells,and itpassed thisgreatcomplexityonnot just toyouandmebut toall itsdescendants, fromtreestobees.Ichallengeyoutolookatoneofyourowncellsdownamicroscopeanddistinguishitfromthecellsofamushroom.Theyarepracticallyidentical.Idon’tlivemuchlikeamushroom,sowhyaremycellssosimilar?It’snotjustthattheylookalike.Allcomplexlifesharesanastonishingcatalogue of elaborate traits, from sex to cell suicide to senescence, none of which is seen in acomparableforminbacteria.Thereisnoagreementaboutwhysomanyuniquetraitsaccumulatedinthatsingleancestor,orwhynoneofthemshowsanysignofevolvingindependentlyinbacteria.Why,ifallof thesetraitsarosebynaturalselection, inwhicheachstepofferssomesmalladvantage,didequivalenttraitsnotariseonotheroccasionsinvariousbacterialgroups?

Thesequestionshighlightthepeculiarevolutionarytrajectoryoflifeonearth.Lifearosearoundhalfabillionyearsaftertheearth’sformation,perhaps4billionyearsago,butthengotstuckatthebacteriallevelofcomplexityformorethan2billionyears,halftheageofourplanet.Indeed,bacteriahaveremainedsimpleintheirmorphology(butnottheirbiochemistry)throughout4billionyears.Instark contrast, all morphologically complex organisms – all plants, animals, fungi, seaweeds andsingle-celled ‘protists’ such as amoeba – descend from that singular ancestor about 1.5–2 billionyearsago.Thisancestorwas recognisablya ‘modern’cell,withanexquisite internal structureandunprecedentedmoleculardynamism,alldrivenbysophisticatednanomachinesencodedbythousandsof new genes that are largely unknown in bacteria. There are no surviving evolutionaryintermediates,no‘missinglinks’togiveanyindicationofhoworwhythesecomplextraitsarose,justanunexplainedvoidbetweenthemorphologicalsimplicityofbacteriaandtheawesomecomplexityofeverythingelse.Anevolutionaryblackhole.

We spend billions of dollars a year on biomedical research, teasing out the answers tounimaginablycomplexquestionsaboutwhywegetill.Weknowinenormousdetailhowgenesandproteinsrelatetoeachother,howregulatorynetworksfeedbackintoeachother.Webuildelaboratemathematicalmodelsanddesigncomputersimulationstoplayoutourprojections.Yetwedon’tknowhowthepartsevolved!Howcanwehopetounderstanddiseaseifwehavenoideawhycellsworkthewaytheydo?Wecan’tunderstandsocietyifweknownothingofitshistory;norcanweunderstand

the workings of the cell if we don’t know how it evolved. This isn’t just a matter of practicalimportance.Thesearehumanquestionsaboutwhywearehere.Whatlawsgaverisetotheuniverse,thestars, thesun, theearth,andlife itself?Will thesamelawsbeget lifeelsewherein theuniverse?Wouldalienlifebeanythinglikeus?Suchmetaphysicalquestionslieat theheartofwhatmakesushuman.Some350yearsafterthediscoveryofcells,westilldon’tknowwhylifeonearthisthewayitis.

Youmightnothavenoticed thatwedon’tknow.It’snotyourfault.Textbooksand journalsarefullofinformation,butoftenfailtoaddressthese‘childlike’questions.Theinternetswampsuswithallmannerofindiscriminatefacts,mixedwithvaryingproportionsofnonsense.Butit’snotmerelyacaseofinformationoverload.Fewbiologistsaremorethandimlyawareoftheblackholeattheheartoftheirsubject.Mostworkonotherquestions.Thegreatmajoritystudylargeorganisms,particulargroupsofplantsoranimals.Relativelyfewworkonmicrobes,andevenfewerontheearlyevolutionofcells.There’salsoaconcernaboutcreationistsandintelligentdesign–toadmitwedon’tknowalltheanswersrisksopeningthedoortonaysayers,whodenythatwehaveanymeaningfulknowledgeofevolution.Ofcoursewedo.Weknowanawfullot.Hypothesesontheoriginsoflifeandtheearlyevolutionofcellsmustexplainanencyclopediaoffacts,conformtoastraitjacketofknowledge,aswell as predict unexpected relationships that can be tested empirically.We understand a great dealaboutnaturalselectionandsomeofthemorerandomprocessesthatsculptgenomes.Allthesefactsareconsistentwiththeevolutionofcells.Butthissamestraitjacketoffactsispreciselywhatraisestheproblem.Wedon’tknowwhylifetookthepeculiarcoursethatitdid.

Scientistsarecuriouspeople,andifthisproblemwereasstarkasI’msuggesting,itwouldbewellknown.Thefactis–it’sfarfromobvious.Thevariouscompetinganswersareesoteric,andallbutobscurethequestion.Thenthere’stheproblemthatcluescomefrommanydisparatedisciplines,frombiochemistry, geology, phylogenetics, ecology, chemistry and cosmology. Few can claim realexpertiseinallthosefields.Andnowweareinthemidstofagenomicrevolution.Wehavethousandsof completegenome sequences, codes that stretchovermillionsor billionsof digits, all toooftencontainingconflictingsignalsfromthedeeppast. Interpretingthesedatademandsrigorouslogical,computationalandstatisticalknow-how;anybiologicalunderstandingisabonus.Andsothecloudshave been swirling around with arguments. Each time a gap opens up, it reveals an increasinglysurreal landscape. The old comforts have been evaporating. We’re now faced with a stark newpicture, and it’s both real and troubling.And from a researcher ’s standpoint, hoping to find somesignificantnewproblemtosolve,it’sflatoutthrilling!Thebiggestquestionsinbiologyareyettobesolved.Thisbookismyownattempttomakeastart.

Howdobacteriarelatetocomplexlife?Therootsofthequestiondaterightbacktothediscoveryofmicrobes by the Dutchmicroscopist Antony van Leeuwenhoek in the 1670s. Hismenagerie of‘littleanimals’ thrivingunder themicroscope tooksomebelieving,butwassoonconfirmedby theequallyingeniousRobertHooke.Leeuwenhoekalsodiscoveredbacteria,andwroteabouttheminafamouspaperof1677:theywere‘incrediblysmall;nay,sosmall,inmysight,thatIjudgedthatevenif100oftheseveryweeanimalslaystretchedoutoneagainstanother,theycouldnotreachthelengthofagrainofcoursesand;andifthisbetrue,thentenhundredthousandoftheselivingcreaturescouldscarceequalthebulkofacoursegrainofsand’.ManydoubtedthatLeeuwenhoekcouldpossiblyhaveseenbacteriausinghissimplesingle-lensmicroscopes,thoughitisnowincontrovertiblethathedidso.Twopointsstandout.Hefoundbacteriaeverywhere–inrainwaterandthesea,notjustonhisownteeth.Andheintuitivelymadesomedistinctionbetweenthese‘veryweeanimals’andthe‘gygantickmonsters’–microscopicprotists!–withtheirenthrallingbehaviourand‘littlefeet’(cilia).Heevennoticed thatsome largercellswerecomposedofanumberof little ‘globules’,whichhecomparedwithbacteria(thoughnotinthoseterms).Amongtheselittleglobules,Leeuwenhoekalmostcertainly

saw the cell nucleus, the repositoryof thegenes in all complexcells.And there thematter lay forseveral centuries. The famous classifierCarl Linnaeus, 50 years after Leeuwenhoek’s discoveries,hadjustlumpedallmicrobesintothegenusChaos(formless)ofthephylumVermes(worms).Inthenineteenth century Ernst Haeckel, the great German evolutionist and contemporary of Darwin,formalised the deep distinction again, separating bacteria from other microbes. But in conceptualterms,therewaslittleadvanceuntilthemiddleofthetwentiethcentury.

The unification of biochemistry brought matters to a head. The sheer metabolic virtuosity ofbacteriahadmadethemseemuncategorisable.Theycangrowonanything,fromconcretetobatteryacid togases. If these totallydifferentwaysof lifehadnothing incommon,howcouldbacteriabeclassified?And ifnotclassified,howcouldweunderstand them?Justas theperiodic tablebroughtcoherence to chemistry, so biochemistry brought an order to the evolution of cells. AnotherDutchman,AlbertKluyver,showedthatsimilarbiochemicalprocessesunderpinnedtheextraordinarydiversity of life. Processes as distinct as respiration, fermentation and photosynthesis all shared acommon basis, a conceptual integrity which attested that all life had descended from a commonancestor. What was true of bacteria, he said, was also true of elephants. At the level of theirbiochemistry,thebarrierbetweenbacteriaandcomplexcellsbarelyexists.Bacteriaareenormouslymore versatile, but the basic processes that keep them alive are similar. Kluyver ’s own studentCornelisvanNiel,togetherwithRogerStanier,perhapscameclosesttoappreciatingthedifference:bacteria,likeatoms,couldnotbebrokendownanyfurther,theysaid:bacteriaarethesmallestunitoffunction.Manybacteriacanrespireoxygeninthesamewaythatwedo,forexample,butittakesthewholebacteriumtodoso.Unlikeourowncells,therearenointernalpartsdedicatedtorespiration.Bacteriadivideinhalfastheygrow,butinfunctiontheyareindivisible.

Andthencamethefirstofthreemajorrevolutionsthathavewrackedourviewoflifeinthepasthalfcentury.ThisfirstwasinstigatedbyLynnMargulisinthesummeroflove,1967.Complexcellsdid not evolve by ‘standard’ natural selection,Margulis argued, but in an orgy of cooperation, inwhichcellsengagedwitheachothersoclosely that theyevengot insideeachother.Symbiosis isalong-terminteractionbetweentwoormorespecies,usuallysomesortoftradeforwaresorservices.In thecaseofmicrobes, thosewaresare thesubstancesof life, thesubstratesofmetabolism,whichpowerthelivesofcells.Margulistalkedaboutendosymbiosis–thesametypesoftrade,butnowsointimatethatsomecollaboratingcellsphysicallyliveinsidetheirhostcelllikethetraderswhosoldfrom within the temple. These ideas trace their roots to the early twentieth century, and arereminiscentofplate tectonics. It ‘looks’as ifAfricaandSouthAmericawereonce joined together,andlaterpulledapart,but thischildlikenotionwas longridiculedasabsurd.Likewise,someof thestructures inside complex cells look like bacteria, and even give the impression of growing anddividingindependently.Perhapstheexplanationreallywasassimpleasthat–theyarebacteria!

Like tectonics these ideas were ahead of their time, and it was not until the era of molecularbiology in the 1960s that it was possible to present a strong case. This Margulis did for twospecialisedstructuresinsidecells–themitochondria,seatsofrespiration,inwhichfoodisburnedinoxygentoprovidetheenergyneededforliving,andthechloroplasts,theenginesofphotosynthesisinplants, which convert solar power into chemical energy. Both of these ‘organelles’ (literallyminiature organs) retain tiny specialised genomes of their own, each onewith a handful of genesencodingatmosta fewdozenproteins involved in themechanicsof respirationorphotosynthesis.The exact sequences of these genes ultimately gave the game away – plainly, mitochondria andchloroplastsdoderivefrombacteria.ButnoticeIsay‘derive’.Theyarenolongerbacteria,anddon’thaveanyrealindependence,asthevastmajorityofthegenesneededfortheirexistence(atleast1,500ofthem)arefoundinthenucleus,thegenetic‘controlcentre’ofthecell.

Marguliswasrightaboutthemitochondriaandchloroplasts;bythe1980s,fewdoubtersremained.

Butherenterprisewasmuchgreater:forMargulis,theentirecomplexcell,nowgenerallyknownastheeukaryotic cell (from theGreekmeaning ‘truenucleus’)wasapatchworkof symbioses. Inhereyes,manyotherpartsofthecomplexcell,notablythecilia(Leeuwenhoek’s‘littlefeet’),alsoderivedfrombacteria(spirochetesinthecaseofcilia).Therehadbeenalongsuccessionofmergers,whichMargulisnowformalisedasthe‘serialendosymbiosistheory’.Notjustindividualcellsbutthewholeworldwasavastcollaborativenetworkofbacteria– ‘Gaia’,an idea thatshepioneeredwithJamesLovelock.While theconceptofGaiahasenjoyeda renaissance in themore formalguiseof ‘earthsystems science’ in recent years (stripping Lovelock’s original teleology), the idea that complex‘eukaryotic’cellsareanensembleofbacteriahasfarlesstosupportit.Mostofthestructuresofthecelldonotlookasiftheyderivedfrombacteria,andthere’snothinginthegenestosuggestthattheydo. So Margulis was right about some things and almost certainly wrong about others. But hercrusadingspirit,forcefulfemininity,dismissalofDarwiniancompetitionandtendencytobelieveinconspiracy theories,meant thatwhen she died prematurely from a stroke in 2011, her legacywasdecidedlymixed.Afeministheroineforsomeandloosecannonforothers,muchofthislegacywassadlyfarremovedfromscience.

Revolutionnumbertwowasthephylogeneticrevolution–theancestryofgenes.Thepossibilityhad been anticipated by Francis Crick as early as 1958. With characteristic aplomb, he wrote:‘Biologists should realise that before longwe shall have a subjectwhichmight be called “proteintaxonomy”– thestudyofaminoacidsequencesofproteinsofanorganismand thecomparisonofthembetweenspecies.Itcanbearguedthatthesesequencesarethemostdelicateexpressionpossibleof thephenotypeofanorganismand thatvastamountsofevolutionary informationmaybehiddenaway within them.’ And lo, it came to pass. Biology is now very much about the informationconcealed in the sequences of proteins and genes.We no longer compare the sequences of aminoacids directly, but the sequences of letters in DNA (which encodes proteins), giving even greatersensitivity. Yet for all his vision, neither Crick nor anyone else began to imagine the secrets thatactuallyemergedfromthegenes.

The scarred revolutionary was Carl Woese. In work beginning quietly in the 1960s and notbearing fruit until a decade later, Woese selected a single gene to compare between species.Obviously,thegenehadtobepresentinallspecies.What’smore,ithadtoservethesamepurpose.That purpose had to be so fundamental, so important to the cell, that even slight changes in itsfunctionwouldbepenalisedbynaturalselection.Ifmostchangesareeliminated,whatremainsmustberelativelyunchanging–evolvingextremelyslowly,andchanginglittleovervastperiodsoftime.That’snecessaryifwewanttocomparethedifferencesthataccumulatebetweenspeciesoverliterallybillions of years, to build a great tree of life, going back to the beginning. Thatwas the scale ofWoese’sambition.Bearingalltheserequirementsinmind,heturnedtoabasicpropertyofallcells,theabilitytomakeproteins.

Proteins are assembled on remarkable nanomachines found in all cells, called ribosomes.Excepting the iconic double helix ofDNA, nothing ismore symbolic of the informational age ofbiology than the ribosome. Its structure also epitomises a contradiction that is hard for the humanmindtofathom–scale.Theribosomeisunimaginablytiny.Cellsarealreadymicroscopic.Wehadnoinkling of their existence formost of human history.Ribosomes are orders ofmagnitude smallerstill. You have 13million of them in a single cell from your liver. But ribosomes are not onlyincomprehensibly small; on the scale of atoms, they are massive, sophisticated super-structures.They’re composed of scores of substantial subunits,movingmachine parts that actwith farmoreprecision thananautomated factory line.That’snot anexaggeration.Theydraw in the ‘tickertape’code-scriptthatencodesaprotein,andtranslateitssequenceprecisely,letterbyletter,intotheproteinitself.Todoso,theyrecruitallthebuildingblocks(aminoacids)needed,andlinkthemtogetherinto

a long chain, their order specified by the code-script.Ribosomes have an error rate of about oneletterin10,000,farlowerthanthedefectrateinourownhigh-qualitymanufacturingprocesses.Andthey operate at a rate of about 10 amino acids per second, building whole proteins with chainscomprisinghundredsofaminoacidsinlessthanaminute.Woesechoseoneofthesubunitsfromtheribosome, a singlemachine part, so to speak, and compared its sequence across different species,frombacteriasuchasE.colitoyeasttohumans.

His findings were a revelation, and turned our world view on its head. He could distinguishbetweenthebacteriaandcomplexeukaryoteswithoutanydifficulty,layingoutthebranchingtreeofgeneticrelatednesswithinandbetweeneachofthesemagisterialgroups.Theonlysurpriseinthiswashow littledifference there isbetweenplantsandanimalsand fungi, thegroups thatmostbiologistshavespentmostoftheirlivesstudying.Whatnobodyanticipatedwastheexistenceofathirddomainoflife.Someofthesesimplecellshadbeenknownforcenturies,butweremistakenforbacteria.Theylooklikebacteria.Exactlylikebacteria:equallytiny,andequallylackingindiscerniblestructure.ButthedifferenceintheirribosomeswaslikethesmileoftheCheshirecat,betrayingthepresenceofadifferent sort of absence.Thisnewgroupmighthave lacked the complexityof eukaryotes, but thegenesandproteinsthattheydidhavewereshockinglydifferentfromthoseofbacteria.Thissecondgroupof simple cellsbecameknownas thearchaea, on thehunch that they’re evenolder than thebacteria,whichisprobablynottrue;modernviewshaveitthattheyareequallyold.Butatthearcaneleveloftheirgenesandbiochemistry,thegulfbetweenbacteriaandarchaeaisasgreatasthatbetweenbacteriaandeukaryotes(us).Almostliterally.InWoese’sfamous‘threedomains’treeoflife,archaeaandeukaryotesare‘sistergroups’,sharingarelativelyrecentancestor.

Insomerespects,thearchaeaandeukaryotesdoindeedhavealotincommon,especiallyintermsof information flow (the way that they read off their genes and convert them into proteins). Inessence,archaeahaveafewsophisticatedmolecularmachinesresemblingthoseofeukaryotes,ifwithfewer parts – the seeds of eukaryotic complexity. Woese refused to countenance any deepmorphologicalgulfbetweenbacteriaandeukaryotes,butproposedthreeequivalentdomains,eachofwhich had explored vast realms of evolutionary space, none ofwhich could be given precedence.Mostforcefully,herejectedtheoldterm‘prokaryote’(meaningliterally‘beforethenucleus’,whichcouldbeapplied tobotharchaeaandbacteria)as therewasnothing inhis tree tosuggestageneticbasisforthatdistinction.Onthecontrary,hepicturedallthreedomainsasreachingrightbackintothedeepestpast, sharingamysteriouscommonancestor, fromwhich theyhadsomehow‘crystallised’.Towardstheendofhis lifeWoesebecamealmostmysticalabout thoseearlieststagesofevolution,callingforamoreholisticviewoflife.That’sironic,giventhattherevolutionhewroughtwasbasedonawholly reductionistanalysisofa singlegene.There isnodoubt that thebacteria,archaeaandeukaryotesaregenuinelydistinctgroupsand thatWoese’s revolutionwas real;buthisprescriptionforholism,takingwholeorganismsandfullgenomesintoaccount,isrightnowusheringinthethirdcellularrevolution–anditoverturnsWoese’sown.

This third revolution isn’t over yet. It’s a littlemore subtle in reasoning, but packs the biggestpunchofall.Itisrootedinthefirsttworevolutions,andspecificallyinthequestion:howdothetworelate?Woese’s tree depicts the divergence of one fundamental gene in the three domains of life.Margulis, in contrast, has genes from different species converging together in the mergers andacquisitionsofendosymbiosis.Depictedasatree,thisisthefusion,notthebifurcation,ofbranches–theoppositeofWoese.Theycan’tbothberight!Neitherdotheybothhavetobetotallywrong.Thetruth, as so often in science, lies somewhere between the two. But don’t think that makes it acompromise.Theanswerthat’semergingismoreexcitingthaneitheralternative.

We know that mitochondria and chloroplasts were indeed derived from bacteria byendosymbiosis, but that the other parts of complex cells probably evolved by conventionalmeans.

The question is:when, exactly?Chloroplasts are found only in algae and plants, henceweremostlikelyacquiredinanancestorof thosegroupsalone.Thatputs themasarelativelylateacquisition.Mitochondria,incontrast,arefoundinalleukaryotes(there’saback-storytherethatwe’llexamineinChapter1)andsomusthavebeenanearlieracquisition.Buthowearly?Putanotherway,whatkindofcell picked upmitochondria? The standard textbook view is that it was quite a sophisticated cell,somethinglikeanamoeba,apredatorthatcouldcrawlaround,changeshapeandengulfothercellsbyaprocessknownasphagocytosis.Inotherwords,mitochondriawereacquiredbyacellthatwasnotsofarfrombeingafullyfledged,card-carryingeukaryote.Wenowknowthat’swrong.Overthelastfewyears,comparisonsof largenumbersofgenes inmorerepresentativesamplesofspecieshavecometotheunequivocalconclusionthatthehostcellwasinfactanarchaeon–acellfromthedomainArchaea.Allarchaeaareprokaryotes.Bydefinition, theydon’thaveanucleusorsexoranyof theother traits of complex life, includingphagocytosis. In termsof itsmorphological complexity, thehost cell must have had next to nothing. Then, somehow, it acquired the bacteria that went on tobecomemitochondria.Only thendid itevolveall thosecomplex traits. Ifso, thesingularoriginofcomplexlifemighthavedependedontheacquisitionofmitochondria.Theysomehowtriggeredit.

This radical proposition – complex life arose from a singular endosymbiosis between anarchaeon host cell and the bacteria that became mitochondria – was predicted by the brilliantlyintuitive and free-thinking evolutionary biologist Bill Martin, in 1998, on the basis of theextraordinarymosaic of genes in eukaryotic cells, amosaic largely uncovered byMartin himself.Take a single biochemical pathway, say fermentation.Archaea do it oneway, and bacteria quite adifferentway;thegenesinvolvedaredistinct.Eukaryoteshavetakenafewgenesfrombacteria,andafew others from archaea, and woven them together into a tightly knit composite pathway. Thisintricate fusion of genes doesn’t merely apply to fermentation, but to almost all biochemicalprocessesincomplexcells.Itisanoutrageousstateofaffairs!

Martinthoughtallthisthroughingreatdetail.Whydidthehostcellpickupsomanygenesfromitsownendosymbionts,andwhydiditintegratethemsotightlyintoitsownfabric,replacingmanyofitsexistinggenesintheprocess?Hisanswer,withMiklósMüller,iscalledthehydrogenhypothesis.MartinandMüllerargued that thehostcellwasanarchaeon,capableofgrowing from twosimplegases,hydrogenandcarbondioxide.Theendosymbiont (the futuremitochondrion)wasaversatilebacterium(perfectlynormalforbacteria),whichprovideditshostcellwiththehydrogenitneededtogrow.Thedetailsofthisrelationship,workedoutstepbysteponalogicalbasis,explainwhyacellthatstartedoutlivingfromsimplegaseswouldendupscavengingorganics(food)tosupplyitsownendosymbionts.But that’snot the importantpoint forushere.Thesalientpoint is:Martinpredictedthatcomplexlifearosethroughasingularendosymbiosisbetweentwocellsonly.Hepredictedthatthehostcellwasanarchaeon,lackingthebaroquecomplexityofeukaryoticcells.Hepredictedthatthereneverwasanintermediate,simpleeukaryoticcell,whichlackedmitochondria; theacquisitionofmitochondriaandtheoriginofcomplexlifewasoneandthesameevent.Andhepredictedthatallthe elaborate traits of complex cells, from the nucleus to sex to phagocytosis, evolved after theacquisition ofmitochondria, in the context of that unique endosymbiosis. This is one of the finestinsightsinevolutionarybiology,anddeservestobemuchbetterknown.Itwouldbe,wereitnotsoeasily confoundedwith the serial endo-symbiosis theory (whichwe’ll seemakesnoneof the samepredictions).Alloftheseexplicitpredictionshavebeenborneoutinfullbygenomicresearchoverthepasttwodecades.It’samonumenttothepowerofbiochemicallogic.IftherewereaNobelPrizeinBiology,nobodywouldbeamoredeservingrecipientthanBillMartin.

Andsowehavecomefullcircle.Weknowanawful lot,butwestilldon’tknowwhylife is theway it is.We know that complex cells arose on just one occasion in 4 billion years of evolution,throughasingularendosymbiosisbetweenanarchaeonandabacterium(Figure1).Weknowthatthe

traits of complex life arose in the aftermath of this union; but we still do not know why thoseparticulartraitsaroseineukaryotes,whileshowingnosignsofevolvingineitherbacteriaorarchaea.We don’t know what forces constrain bacteria and archaea – why they remain morphologicallysimple,despitebeingsodifferentintheirbiochemistry,sovariedintheirgenes,soversatileintheirability to extract a living from gases and rocks.Whatwe do have is a radical new framework inwhichtoapproachtheproblem.

Figure1AtreeoflifeshowingthechimericoriginofcomplexcellsAcompositetreereflectingwholegenomes,asdepictedbyBillMartinin1998,showingthethreedomainsofBacteria,ArchaeaandEukaryotes.Eukaryoteshaveachimericorigin,inwhichgenesfromanarchaealhostcellandabacterialendosymbiontcoalesce,withthearchaealhostcellultimatelyevolvingintothemorphologicallycomplexeukaryoticcell,andtheendosymbiontsintomitochondria.Onegroupofeukaryoteslateracquiredasecondbacterialendosymbiont,whichbecamethechloroplastsofalgaeandplants.

I believe the clue lies in the bizarremechanism of biological energy generation in cells. Thisstrangemechanismexertspervasivebutlittleappreciatedphysicalconstraintsoncells.Essentiallyalllivingcellspowerthemselves throughtheflowofprotons(positivelychargedhydrogenatoms), inwhatamountstoakindofelectricity–proticity–withprotonsinplaceofelectrons.Theenergywegain from burning food in respiration is used to pump protons across a membrane, forming areservoirononesideofthemembrane.Theflowofprotonsbackfromthisreservoircanbeusedtopowerworkinthesamewayasaturbineinahydroelectricdam.Theuseofcross-membraneprotongradients to power cellswas utterly unanticipated. First proposed in 1961 and developed over theensuingthreedecadesbyoneofthemostoriginalscientistsofthetwentiethcentury,PeterMitchell,thisconceptionhasbeencalledthemostcounterintuitiveideainbiologysinceDarwin,andtheonlyonethatcompareswiththeideasofEinstein,HeisenbergandSchrödingerinphysics.Atthelevelofproteins, we now know how proton power works in detail.We also know that the use of proton

gradientsisuniversalacrosslifeonearth–protonpowerisasmuchanintegralpartofalllifeastheuniversal genetic code. Yet we know next to nothing about how or why this counterintuitivemechanismofenergyharnessingfirstevolved.Soitseemstometherearetwobigunknownsattheveryheartofbiologytoday:whylifeevolvedintheperplexingwayitdid,andwhycellsarepoweredinsuchapeculiarfashion.

Thisbookisanattempttoanswerthesequestions,whichIbelievearetightlyentwined.Ihopetopersuadeyouthatenergyiscentraltoevolution,thatwecanonlyunderstandthepropertiesoflifeifwebringenergyintotheequation.Iwanttoshowyouthatthisrelationshipbetweenenergyandlifegoesrightbacktothebeginning–thatthefundamentalpropertiesoflifenecessarilyemergedfromthedisequilibriumofarestlessplanet.Iwanttoshowyouthattheoriginoflifewasdrivenbyenergyflux, thatprotongradientswerecentral to theemergenceofcells,andthat theiruseconstrainedthestructureofbothbacteriaandarchaea.Iwanttodemonstratethattheseconstraintsdominatedthelaterevolution of cells, keeping the bacteria and archaea forever simple in morphology, despite theirbiochemicalvirtuosity. Iwant toprove thata rareevent,anendosymbiosis inwhichone bacteriumgotinsideanarchaeon,brokethoseconstraints,enablingtheevolutionofvastlymorecomplexcells.Iwant to show you that thiswas not easy – that the intimate relationship between cells living oneinside another explains why morphologically complex organisms only arose once. I hope to domore, to persuade you that this intimate relationship actually predicts some of the properties ofcomplexcells.These traits include thenucleus,sex, twosexes,andeven thedistinctionbetween theimmortal germline and the mortal body – the origins of a finite lifespan and geneticallypredetermineddeath.Finally,Iwanttoconvinceyouthatthinkingintheseenergetictermsallowsusto predict aspects of our own biology, notably a deep evolutionary trade-off between fertility andfitness in youth, on the one hand, and ageing and disease on the other. I’d like to think that theseinsightsmighthelpustoimproveourownhealth,oratleasttounderstanditbetter.

It can be frowned upon to act as an advocate in science, but there is a fine tradition of doingexactly that in biology, going back toDarwin himself; he calledTheOrigin of Species ‘one longargument’.Abook is still the bestway to layout a visionof how factsmight relate to eachotheracross the whole fabric of science – a hypothesis that makes sense of the shape of things. PeterMedawardescribedahypothesisasan imaginative leap into theunknown.Once the leap is taken,ahypothesisbecomesanattempttotellastorythatisunderstandableinhumanterms.Tobescience,thehypothesismustmakepredictionsthataretestable.There’snogreaterinsultinsciencethantosaythatanargumentis‘notevenwrong’,thatitisinvulnerabletodisproof.Inthisbook,then,Iwilllayoutahypothesis–tellacoherentstory–thatconnectsenergyandevolution.IwilldosoinenoughdetailthatIcanbeprovedwrong,whilewritingasaccessiblyandasexcitinglyasIcan.Thisstoryisbasedinpartonmyownresearch(you’llfindtheoriginalpapersinFurtherReading)andinpartonthatofothers. I have collaboratedmost fruitfully with BillMartin in Düsseldorf, who I’ve found has anuncanny knack of being right, andAndrew Pomiankowski, amathematicallyminded evolutionarygeneticistandbestofcolleaguesatUniversityCollegeLondon;andwithseveralextremelyablePhDstudents. Ithasbeenaprivilegeandanenormouspleasure,andweareonlyat thebeginningofanimmensejourney.

Ihavetriedtokeepthisbookshortandtothepoint,tocutdownondigressionsandinterestingbutunrelatedstories.Thebookisanargument,asspareordetailedasitneedstobe.Itisnotlackinginmetaphorand(Ihope)entertainingdetails;thatisvitaltobringabookgroundedinbiochemistrytolifeforthegeneralreader.Fewofuscaneasilyvisualisethealiensubmicroscopiclandscapeofgiantinteractingmolecules,theverystuffoflife.Butthepointisthescienceitself,andthathasfashionedmywriting.Tocallaspadeaspadeisagoodold-fashionedvirtue.It’ssuccinct,andbringsusstraighttothepoint;youwouldsoonbecomeirritatedifIinsistedonremindingyoueveryfewpagesthata

spadeisadiggingimplementusedforburyingpeople.Whileit’slesshelpfultocallamitochondrionamitochondrion,it’slikewisecumbersometokeepwriting“Alllarge,complexcellssuchasourowncontainminiaturepowerhouses,whichderivedlongagofromfree-livingbacteria,andwhichtodayprovideessentiallyallourenergyneeds.”Icouldwriteinstead:“Alleukaryoteshavemitochondria.”Thisisclearerandpacksagreaterpunch.Whenyouarecomfortablewithafewterms,theyconveymoreinformation,andsosuccinctly that in thiscase theyimmediatelybegaquestion:howdid thatcomeabout?Thatleadsstraighttotheedgeoftheunknown,tothemostinterestingscience.SoI’vetriedtoavoidunnecessaryjargon,andhaveincludedoccasionalremindersofthemeaningofterms;butbeyondthatIhopeyouwillgainfamiliaritywithrecurringterms.Asafailsafe,I’vealsoincludedashortglossaryofthemaintermsattheend.Withtheoccasionaldoublecheck,Ihopethisbookwillbewhollyaccessibletoanyonewhoisinterested.

And I sincerely hope you will be interested! For all its strangeness, this brave new world isgenuinely exciting: the ideas, the possibilities, the dawning understanding of our place in this vastuniverse. I will outline the contours of a new and largely uncharted landscape, a perspective thatstretchesfromtheveryoriginoflifetoourownhealthandmortality.Thiscolossalspanisthankfullyunitedbyafewsimpleideasthatrelatetoprotongradientsacrossmembranes.Formethebestbooksinbiology,eversinceDarwin,havebeenarguments.Thisbookaspirestofollowinthat tradition.Iwill argue that energyhasconstrained theevolutionof lifeonearth; that the same forcesought toapplyelsewhereintheuniverse;andthatasynthesisofenergyandevolutioncouldbethebasisforamore predictive biology, helping us understand why life is the way it is, not only on earth, butwhereveritmightexistintheuniverse.

PARTI

THEPROBLEM

U

1

WHATISLIFE?

nblinkingdayandnight,theradiotelescopesscrutinisetheskies.Forty-twoofthemarescatteredinalooseclusteracrossthescrubbysierraofnorthernCalifornia.Theirwhitebowlsresemble

blank faces, all focused hopefully in unison on somepoint beyond the horizon, as if thiswere anassemblypointforalieninvaderstryingtogohome.Theincongruityisapt.ThetelescopesbelongtoSETI,thesearchforextraterrestrialintelligence,anorganisationthathasbeenscanningtheheavensforsignsoflifeforhalfacentury,tonoavail.Eventheprotagonistsarenottoooptimisticaboutitschancesofsuccess;butwhenfundingdriedupafewyearsago,adirectappealtothepublicsoonhadthe Allen Telescope Array operational again. To my mind the venture is a poignant symbol ofhumanity’s uncertain sense of our place in the universe, and indeed the fragility of science itself:sciencefictiontechnologysoinscrutablethatithintsatomniscience,trainedonadreamsonaivethatitisbarelygroundedinscienceatall,thatwearenotalone.

Evenif thearrayneverdetects life, it isstillvaluable.Itmaynotbepossible to lookthewrongwaythroughthesetelescopes,yetthatistheirrealpower.Whatexactlyarewelookingforoutthere?Shouldlifeelsewhereintheuniversebesosimilartousthatittoousesradiowaves?Dowethinkthatlifeelsewhereshouldbecarbonbased?Woulditneedwater?Oxygen?Thesearenotreallyquestionsaboutthemake-upoflifesomewhereelseintheuniverse:theyareaboutlifeonearth,aboutwhylifeis the way we know it. These telescopes are mirrors, reflecting their questions back at earthlybiologists. The problem is that science is all about predictions. The most pressing questions inphysics are aboutwhy the laws of physics are as they are:what fundamental principles predict theknownpropertiesoftheuniverse?Biologyislesspredictive,andhasnolawstocomparewiththoseofphysics,butevensothepredictivepowerofevolutionarybiologyisembarrassinglybad.Weknowagreatdealaboutthemolecularmechanismsofevolutionandaboutthehistoryoflifeonourplanet,butfarlessaboutwhichpartsofthishistoryarechance–trajectoriesthatcouldhaveplayedoutquitedifferentlyonotherplanets–andwhichbitsaredictatedbyphysicallawsorconstraints.

That isnot forany lackofeffort.This terrain is theplaygroundof retiredNobel laureatesandothertoweringfiguresinbiology;yetforalltheirlearningandintellect,theycannotbegintoagreeamongthemselves.Fortyyearsago,atthedawnofmolecularbiology,theFrenchbiologistJacquesMonodwrotehisfamousbookChanceandNecessity,whicharguesbleaklythattheoriginoflifeonearthwasafreakaccident,andthatwearealoneinanemptyuniverse.Thefinallinesofhisbookareclosetopoetry,anamalgamofscienceandmetaphysics:

The ancient covenant is in pieces;man knows at last that he is alone in the universe’s unfeeling immensity, out ofwhich heemergedonlybychance.Hisdestinyisnowherespelledout,norishisduty.Thekingdomaboveorthedarknessbelow:itisforhimtochoose.

Sincethen,othershavearguedtheopposite:thatlifeisaninevitableoutcomeofcosmicchemistry.Itwillarisequickly,almosteverywhere.Oncelifeisthrivingonaplanet,whathappensnext?Again,thereisnoconsensus.Engineeringconstraintsmayforcelifedownconvergentpathwaystosimilarplaces,regardlessofwhereitstarts.Givengravity,animalsthatflyarelikelytobelightweight,andpossesssomethingakintowings.Inamoregeneralsense,itmaybenecessaryforlifetobecellular,composedofsmallunitsthatkeeptheirinsidesdifferentfromtheoutsideworld.Ifsuchconstraintsare dominant, life elsewheremay closely resemble life on earth.Conversely, perhaps contingencyrules–themake-upoflifedependsontherandomsurvivorsofglobalaccidentssuchastheasteroidimpactthatwipedoutthedinosaurs.WindbacktheclocktoCambriantimes,halfabillionyearsago,whenanimalsfirstexplodedintothefossilrecord,andletitplayforwardsagain.Wouldthatparallelworldbesimilartoourown?Perhapsthehillswouldbecrawlingwithgiantterrestrialoctopuses.

Oneofthereasonsforpointingtelescopesouttospaceisthathereonearthwearedealingwithasamplesizeofone.Fromastatisticalpointofview,wecan’tsaywhat, ifanything,constrained theevolutionoflifeonearth.Butifthatwerereallytrue,therewouldbenobasisforthisbook,oranyothers. The laws of physics apply throughout the universe, as do the properties and abundance ofelements,hencetheplausiblechemistry.Lifeonearthhasmanystrangepropertiesthathavetaxedthemindsofthefinestbiologistsforcenturies–traitslikesexandageing.Ifwecouldpredictfromfirstprinciples–fromthechemicalmake-upoftheuniverse–whysuchtraitsarose,whylifeisthewayitis,thenwewouldhaveaccesstotheworldofstatisticalprobabilityagain.Lifeonearthisnotreallyasampleofone,butforpracticalpurposesitisaninfinitevarietyoforganismsevolvingoverinfinitetime.Yetevolutionarytheorydoesnotpredict,fromfirstprinciples,whylifeonearthtookthecoursethatitdid.IdonotmeanbythisthatIthinkevolutionarytheoryiswrong–itisnot–butsimplythatitisnotpredictive.Myargumentinthisbookisthatthereareinfactstrongconstraintsonevolution–energeticconstraints–whichdomakeitpossible topredictsomeof themostfundamental traitsoflife from first principles. Before we can address these constraints, we must consider whyevolutionary biology is not predictive, and why these energetic constraints have passed largelyunnoticed; indeed,whywehavehardlyevennoticed there isaproblem.Ithasonlybecomestarklyapparentinthepastfewyears,andonlytothosewhofollowevolutionarybiology,thatthereisadeepanddisturbingdiscontinuityattheveryheartofbiology.

To a point, we can blame DNA for this sorry state of affairs. Ironically, the modern era ofmolecularbiology,andall theextraordinaryDNAtechnology that itentails,arguablybeganwithaphysicist, specifically with the publication of Erwin Schrödinger ’s bookWhat is Life? in 1944.Schrödingermadetwokeypoints:first,thatlifesomehowresiststheuniversaltendencytodecay,theincrease inentropy (disorder) that is stipulatedby the second lawof thermodynamics; and second,thatthetricktolife’slocalevasionofentropyliesinthegenes.Heproposedthatthegeneticmaterialisan‘aperiodic’crystal,whichdoesnothaveastrictlyrepeatingstructure,hencecouldactasa‘code-script’–reputedlythefirstuseoftheterminthebiologicalliterature.Schrödingerhimselfassumed,alongwithmostbiologistsatthetime,thatthequasicrystalinquestionmustbeaprotein;butwithinafrenzieddecade,CrickandWatsonhad inferred thecrystal structureofDNA itself. In their secondNaturepaperof1953,theywrote:‘Itthereforeseemslikelythattheprecisesequenceofthebasesisthe code which carries the genetical information.’ That sentence is the basis of modern biology.Todaybiologyisinformation,genomesequencesarelaidoutinsilico,andlifeisdefinedintermsofinformationtransfer.

Genomesare thegatewaytoanenchantedland.Thereamsofcode,3billionletters inourowncase,readlikeanexperimentalnovel,anoccasionallycoherentstoryinshortchaptersbrokenupbyblocksofrepetitivetext,verses,blankpages,streamsofconsciousness:andpeculiarpunctuation.A

tinyproportionofourowngenome,lessthan2%,codesforproteins;alargerportionisregulatory;andthefunctionoftherestisliabletocauseintemperaterowsamongotherwisepolitescientists.1 Itdoesn’tmatterhere.Whatisclearisthatgenomescanencodeuptotensofthousandsofgenesandagreatdealof regulatorycomplexity,capableofspecifyingeverything that isneeded to transformacaterpillarintoabutterflyorachildintoanadulthuman.Comparingthegenomesofanimals,plants,fungi,andsingle-celledamoebaeshowsthatthesameprocessesareatplay.Wecanfindvariantsofthesamegenes,thesameregulatoryelements,thesameselfishreplicators(suchasviruses)andthesamestretchesof repetitivenonsense ingenomesofvastlydifferentsizesand types.Onions,wheatandamoebaehavemoregenesandmoreDNAthanwedo.Amphibianssuchasfrogsandsalamandershavegenomesizesthatrangeovertwoordersofmagnitude,withsomesalamandergenomesbeing40timeslargerthanourown,andsomefrogsbeinglessthanathirdofoursize.Ifwehadtosumupthearchitecturalconstraintsongenomesinasinglephrase,itwouldhavetobe‘anythinggoes’.

That’s important. If genomes are information, and there are no fundamental constraints ongenome size and structure, then there are no constraints on information either. That doesn’tmeanthere are no constraints on genomes at all. Obviously there are. The forces acting on genomesinclude natural selection as well as more random factors – accidental duplications of genes,chromosomesorwholegenomes,inversions,deletions,andinvasionsofparasiticDNA.Howallofthis pans out depends on factors such as niche, competition between species, and population size.Fromourpointofview,allthesefactorsareunpredictable.Theyarepartoftheenvironment.Iftheenvironment isspecifiedprecisely,wemight justbeable topredict thegenomesizeofaparticularspecies.Butaninfinitenumberofspeciesliveinanendlessvarietyofmicroenvironments,rangingfromtheinsidesofothercells,tohumancities,topressurisedoceandepths.Notsomuch‘anythinggoes’as‘everythinggoes’.Weshouldexpecttofindasmuchvarietyingenomesastherearefactorsactingontheminthesediverseenvironments.Genomesdonotpredictthefuturebutrecallthepast:theyreflecttheexigenciesofhistory.

Considerotherworldsagain.Iflifeisaboutinformation,andinformationisunconstrained,thenwecan’tpredictwhatlifemightlooklikeonanotherplanet,onlythatitwillnotcontravenethelawsofphysics.Assoonassomeformofhereditarymaterialhasarisen–whetherDNAorsomethingelse–thenthetrajectoryofevolutionbecomesunconstrainedbyinformationandunpredictablefromfirstprinciples.Whatactuallyevolveswilldependontheexactenvironment,thecontingenciesofhistory,andtheingenuityofselection.Butlookbacktoearth.Thisstatementisreasonablefortheenormousvarietyoflifeasitexiststoday;butitissimplynottrueformostofthelonghistoryoftheearth.Forbillionsofyears,itseemsthatlifewasconstrainedinwaysthatcan’teasilybeinterpretedintermsofgenomes, historyor environment.Until recently, thepeculiar historyof life onourplanetwas farfromclear,andevennowthereismuchadoaboutthedetails.Letmesketchouttheemergingview,andcontrastitwitholderversionsthatnowlooktobewrong.

Abriefhistoryofthefirst2billionyearsoflifeOurplanetisabout4.5billionyearsold(that’stosay4,500millionyearsold).Itwaswrackedduringitsearlyhistory,forsome700millionyears,byaheavyasteroidbombardment,asthenascentsolarsystem settled itself down. A colossal early impact with aMars-sized object probably formed themoon.Unliketheearth,whoseactivegeologycontinuouslyturnsoverthecrust,thepristinesurfaceof themoonpreserves evidence for this early bombardment in its craters, dated by rocks broughthomebytheApolloastronauts.

Despitetheabsenceofearthlyrocksofacomparableage,therearestillafewcluestoconditionson the early earth. In particular, the composition of zircons (tiny crystals of zirconium silicate,

smallerthansandgrains,foundinmanyrocks)suggeststhattherewereoceansmuchearlierthanwehad thought.We can tell from uranium dating that some of these amazingly robust crystals wereformedbetween4and4.4billionyearsago,andlateraccumulatedasdetritalgrainsinsedimentaryrocks. Zircon crystals behave like tiny cages that trap chemical contaminants, reflecting theenvironment in which they were formed. The chemistry of early zircons suggests that they wereformedatrelativelylowtemperatures,andinthepresenceofwater.Farfromtheimageofavolcanichell,with oceans of boiling lava captured vividly in artists’ impressions of thewhat is technicallytermedthe‘Hadean’period,zirconcrystalspointtoamoretranquilwaterworldwithalimitedlandsurface.

Likewise,theoldideaofaprimordialatmosphererepletewithgasessuchasmethane,hydrogenandammonia,whichreacttogethertoformorganicmolecules,doesnotstandthescrutinyofzircons.Traceelementssuchasceriumare incorporated intozirconcrystalsmostly in theiroxidised form.The high content of cerium in the earliest zircons suggests that the atmospherewas dominated byoxidisedgasesemanating fromvolcanoes,notablycarbondioxide,watervapour,nitrogengasandsulphur dioxide. Thismixture is not dissimilar in composition to the air today, except that it wasmissingoxygen itself,whichwasnot abundantuntilmuch later, after the adventof photosynthesis.Reading themake-up of a long-vanishedworld from a few scattered zircon crystals puts a lot ofweight on what amounts to grains of sand, but it is better than no evidence at all. That evidenceconsistently conjures up a planet that was surprisingly similar to the one we know today. Theoccasionalasteroidimpactmighthavepartiallyvaporisedtheoceans,butisunlikelytohaveupsetanybacterialivinginthedeepoceans–iftheyhadalreadyevolved.

Theearliestevidenceforlifeisequallyflimsy,butmaydatebacktosomeoftheearliestknownrocksatIsuaandAkiliainsouth-westGreenland,whicharearound3.8billionyearsold(seeFigure2foratimeline).Thisevidenceisnotintheformoffossilsorcomplexmoleculesderivedfromlivingcells(‘biomarkers’)butissimplyanon-randomsortingofcarbonatomsingraphite.Carboncomesin two stable forms, or isotopes,which havemarginally differentmasses.2 Enzymes (proteins thatcatalyse reactions in living cells) have a slight preference for the lighter form, carbon-12, whichthereforetendstoaccumulateinorganicmatter.Youcouldthinkofcarbonatomsastinyping-pongballs – the slightly smaller balls bounce around slightly faster, so are more likely to bump intoenzymes, so are more likely to be converted into organic carbon. Conversely, the heavier form,carbon-13, which constitutes just 1.1% of the total carbon, is more likely to be left behind in theoceansandcan insteadaccumulatewhencarbonate isprecipitatedout insedimentary rockssuchaslimestone.Thesetinydifferencesareconsistenttothepointthattheyareoftenseenasdiagnosticoflife.Notonlycarbonbutotherelementssuchasiron,sulphurandnitrogenarealsofractionatedbylivingcellsinasimilarway.SuchisotopicfractionationisreportedinthegraphiteinclusionsatIsuaandAkilia.

Figure2AtimelineoflifeThetimelineshowsapproximatedatesforsomekeyeventsinearlyevolution.Manyofthesedatesareuncertainanddisputed,butmostevidencesuggeststhatthebacteriaandarchaeaarosearound1.5to2billionyearsbeforetheeukaryotes.

Everyaspectofthiswork,fromtheageoftherocksthemselvestotheveryexistenceofthesmallcarbongrainspurportedtosignify life,hasbeenchallenged.What’smore, ithasbecomeclear thatisotopicfractionationisnotuniquetolifeatall,butcanbemimicked,ifmoreweakly,bygeologicalprocesses inhydrothermalvents. If theGreenland rocks reallyareasancientas they seem,anddoindeedcontainfractionatedcarbon,thatisstillnoproofoflife.Thismightseemdiscouraging,butinanother sense is no less thanwe should expect. I shall argue that the distinction between a ‘livingplanet’–onethatisgeologicallyactive–andalivingcellisonlyamatterofdefinition.Thereisnohardandfastdividingline.Geochemistrygivesriseseamlesslytobiochemistry.Fromthispointofview,thefactthatwecan’tdistinguishbetweengeologyandbiologyintheseoldrocksisfitting.Here

isalivingplanetgivingrisetolife,andthetwocan’tbeseparatedwithoutsplittingacontinuum.Moveforwardafewhundredmillionyearsandtheevidenceforlifeismoretangible–assolid

and scrutable as the ancient rocks ofAustralia and SouthAfrica.Here, there aremicrofossils thatlookalotlikecells,althoughtryingtoplacetheminmoderngroupsisathanklesstask.Manyofthesetinyfossilsarelinedwithcarbon,againfeaturingtelltaleisotopicsignatures,butnowsomewhatmoreconsistent and pronounced, suggesting organised metabolism rather than haphazard hydrothermalprocesses.Andtherearestructuresresemblingstromatolites,thosedomedcathedralsofbacteriallife,in which cells grow layer upon layer, the buried layers mineralising, turning to stone, ultimatelybuildingupintostrikinglylaminatedrockstructures,ametreinheight.Beyondthesedirectfossils,by3.2billionyearsagotherearelarge-scalegeologicalfeatures,hundredsofsquaremilesinareaandtens of metres deep, notably banded iron formations and carbonrich shales. We tend to think ofbacteria and minerals as occupying different realms, living versus inanimate, but in fact manysedimentaryrocksaredeposited,onacolossalscale,bybacterialprocesses. In thecaseofbanded-ironformations–stunninglybeautifulintheirstripesofredandblack–bacteriastripelectronsfromirondissolvedintheoceans(such‘ferrous’ironisplentifulintheabsenceofoxygen)leavingbehindtheinsolublecarcass,rust,tosinkdowntothedepths.Whytheseiron-richrocksarestripedremainspuzzling,butisotopesignaturesagainbetraythehandofbiology.

These vast deposits indicate not just life but photosynthesis. Not the familiar form ofphotosynthesisthatweseearoundusinthegreenleavesofplantsandalgae,butasimplerprecursor.Inallformsofphotosynthesis,theenergyoflightisusedtostripelectronsfromanunwillingdonor.Theelectronsarethenforcedontocarbondioxidetoformorganicmolecules.Thevariousformsofphotosynthesisdifferintheirsourceofelectrons,whichcancomefromallkindsofdifferentplaces,most commonly dissolved (ferrous) iron, hydrogen sulphide, orwater. In each case, electrons aretransferred to carbon dioxide, leaving behind the waste: rusty iron deposits, elemental sulphur(brimstone)andoxygen,respectively.Thehardestnuttocrack,byfar,iswater.By3.2billionyearsago, life was extracting electrons from almost everything else. Life, as biochemist Albert Szent-Györgyiobserved,isnothingbutanelectronlookingforaplacetorest.Quitewhenthefinalsteptoextracting electrons from water took place is contentious. Some claim it was an early event inevolution,buttheweightofevidencenowsuggeststhat‘oxygenic’photosynthesisarosebetween2.9and2.4billionyearsago,notsolongbeforeacataclysmicperiodofglobalunrest,theearth’smidlifecrisis. Worldwide glaciations, known as a ‘snowball earth’, were followed by the widespreadoxidationof terrestrial rocks,around2.2billionyearsago, leaving rusty ‘redbeds’asadefinitivesignofoxygenintheair–the‘GreatOxidationEvent’.Eventheglobalglaciationsindicateariseofatmosphericoxygen.Byoxidisingmethane,oxygenremovedapotentgreenhousegasfromtheair,triggeringtheglobalfreeze.3

Withtheevolutionofoxygenicphotosynthesis,life’smetabolictoolkitwasessentiallycomplete.Ourwhistle-stoptourthroughnearly2billionyearsofearth’shistory–threetimeslongerthantheentire duration of animals – is unlikely to be accurate in all its details, but it is worth pausing amoment to consider what the bigger picture says about our world. First, life arose very early,probablybetween3.5and4billionyearsago, ifnot earlier,onawaterworldnotunlikeourown.Second, by 3.5 to 3.2 billion years ago, bacteria had already inventedmost forms ofmetabolism,including multiple forms of respiration and photosynthesis. For a billion years the world was acauldron of bacteria, displaying an inventiveness of biochemistry that we can only wonder at.4Isotopicfractionationsuggeststhatallthemajornutrientcycles–carbon,nitrogen,sulphur,iron,andsoon–wereinplacebefore2.5billionyearsago.Yetonlywiththeriseofoxygen,from2.4billionyearsago,didlifetransfigureourplanettothepointthatthisthrivingbacterialworldcouldhavebeendetectedasalivingplanetfromspace.Onlythendidtheatmospherebegintoaccumulateareactive

mixtureofgases, suchasoxygenandmethane,whichare replenishedcontinuouslyby livingcells,betrayingthehandofbiologyonaplanetaryscale.

TheproblemwithgenesandenvironmentTheGreatOxidationEventhaslongbeenrecognisedasapivotalmomentinthehistoryofourlivingplanet,butitssignificancehasshiftedradicallyinrecentyears,andthenewinterpretationiscriticaltomyargumentinthisbook.Theoldversionseesoxygenasthecriticalenvironmentaldeterminantoflife.Oxygendoesnotspecifywhatwillevolve,theargumentgoes,butitpermitstheevolutionoffargreater complexity – it releases the brakes.Animals, for example,make their living by physicallymovingaround,chasingpreyorbeingchasedthemselves.Obviouslythisrequiresalotofenergy,soit’seasytoimaginethatanimalscouldnotexistintheabsenceofoxygen,whichprovidesnearlyanorder of magnitude more energy than other forms of respiration.5 This statement is so blandlyuninteresting it is hardlyworth challenging. That is part of the problem: it does not invite furtherconsideration.Wecan take it forgranted thatanimalsneedoxygen (even though that isnotalwaystrue),andhaveit,sooxygenisacommondenominator.Therealproblemsinevolutionarybiologyarethenaboutthepropertiesandbehaviourofanimalsorplants.Orsoitwouldseem.

Thisview implicitlyunderpins the textbookhistoryof theearth.We tend to thinkofoxygenaswholesomeandgood,butinfactfromthepointofviewofprimordialbiochemistryitisanythingbut:itistoxicandreactive.Asoxygenlevelsrose,thetextbookstorygoes,thisdangerousgasputaheavyselectionpressureonthewholemicrobialworld.Therearestarktalesofthemassextinctiontoendthemall–whatLynnMargulistermedtheoxygen‘holocaust’.Thefactthatthereisnotraceofthiscataclysm in the fossil recordneednotworryus toomuch (weareassured): thesebugswereverysmall and it was all a terribly long time ago. Oxygen forced new relationships between cells –symbiosesandendosymbioses, inwhichcells tradedamongandwithin themselves for the toolsofsurvival.Over hundreds ofmillions of years, complexity gradually increased, as cells learnednotonly to deal with oxygen, but also to profit from its reactivity: they evolved aerobic respiration,givingthemfarmorepower.Theselarge,complex,aerobiccellspackagetheirDNAinaspecialisedcompartment called the nucleus, bequeathing their name ‘eukaryotes’ – literally, ‘true nucleus’. Ireiterate,thisisatextbookstory:Ishallarguethatitiswrong.

Today,allthecomplexlifeweseearoundus–allplants,animals,algae,fungiandprotists(largecells like amoebae) – are composed of these eukaryotic cells. The eukaryotes rose steadily todominance over a billion years, the story continues, in a period known, ironically, as the ‘boringbillion’,aslittlehappenedinthefossilrecord.Still,between1.6and1.2billionyearsagowedobeginto find fossils of single cells that look a lot like eukaryotes, some of which even fit snugly intomoderngroupssuchasredalgaeandfungi.

Thencameanotherperiodofglobalunrestandasuccessionofsnowballearthsaround750–600millionyearsago.Soonafterthat,oxygenlevelsroserapidlytonearlymodernlevels–andthefirstfossilsofanimalsappearabruptly in thefossil record.Theearliest largefossils–up toametre indiameter – are a mysterious group of symmetrical frond-like forms that most palaeontologistsinterpret as filter-feeding animals, though some insist aremerely lichens: theEdiacarans, ormoreaffectionately, vendobionts. Then, as abruptly as they appeared,most of these forms vanished in amassextinctionoftheirown,tobereplacedatthedawnoftheCambrianera,541millionyearsago–adateasiconicamongbiologistsas1066or1492–byanexplosionofmorerecognisableanimals.Large and motile, with complex eyes and alarming appendages, these fierce predators and theirfearsome armour-plated prey burst on to the evolutionary scene, red in tooth and claw,Darwin inmodernguise.

Howmuchofthisscenarioisinfactwrong?Atfacevalueitseemstobeplausible.Butinmyviewthesubtextiswrong;andaswelearnmore,soareagoodmanyofthedetails.Thesubtextrelatestothe interplay between genes and environment. The entire scenario revolves around oxygen,supposedlythekeyenvironmentalvariable,whichpermittedgeneticchange,releasingthebrakesoninnovation.Oxygen levels rose twice, in theGreatOxidationEvent2.4billionyearsagoandagaintowardstheendoftheeternalPrecambrianperiod,600millionyearsago(Figure2).Eachtime,thestorygoes, risingoxygen releasedconstraintson structureand function.After theGreatOxidationEvent, with its new threats and opportunities, cells traded among themselves in a series ofendosymbioses,graduallyaccruingthecomplexityoftrueeukaryoticcells.Whenoxygenlevelsroseasecondtime,beforetheCambrianexplosion,thephysicalconstraintsweresweptasidecompletely,as ifwith a flourish of amagician’s cloak, revealing the possibility of animals for the first time.Nobody claims that oxygen physically drove these changes; rather, it transformed the selectivelandscape. Across the magnificent vistas of this unconstrained new landscape, genomes expandedfreely,theirinformationcontentfinallyunfettered.Lifeflourished,fillingallconceivableniches,ineverywhichway.

Thisviewofevolutioncanbeseen in termsofdialecticalmaterialism, true to theprinciplesofsome leading evolutionary biologists during the neo-Darwinian synthesis of the early to midtwentieth century. The interpenetrating opposites are genes and environment, otherwise known asnatureandnurture.Biologyisallaboutgenes,andtheirbehaviourisallabouttheenvironment.Whatelseisthere,afterall?Well,biologyisnotonlyaboutgenesandenvironment,butalsocellsandtheconstraints of their physical structure, which we shall see have little to do with either genes orenvironment directly. The predictions that arise from these disparate world views are strikinglydifferent.

Takethefirstpossibility,interpretingevolutionintermsofgenesandtheenvironment.Thelackof oxygen on the early earth is a major environmental constraint. Add oxygen and evolutionflourishes.Alllifethatisexposedtooxygenisaffectedinonewayoranotherandmustadapt.Somecells justhappen tobebetter suited toaerobicconditions,andproliferate;othersdie.But therearemany different microenvironments. Rising oxygen doesn’t simply flood the whole world withoxygeninakindofmonomaniacalglobalecosystem,butitoxidisesmineralsonlandandsolutesinthe oceans, and that enriches anaerobic niches too. The availability of nitrate, nitrite, sulphate,sulphite,andsoon, rises.Thesecanallbeused insteadofoxygen incell respiration,soanaerobicrespiration flourishes inanaerobicworld.Allof thataddsup tomanydifferentwaysofmakingalivinginthenewworld.

Imaginea randommixtureofcells inanenvironment.Somecells suchasamoebaemake theirlivingbyphysically engulfingother cells, aprocess calledphagocytosis.Somearephotosynthetic.Others,suchasfungi,digesttheirfoodexternally–osmotrophy.Assumingthatcellstructuredoesn’timposeinsuperableconstraints,wewouldpredictthatthesedifferentcelltypeswoulddescendfromvariousdifferentbacterialancestors.Oneancestralcellhappenedtobeabitbetteratsomeprimitiveformofphagocytosis,anotheratasimpleformofosmotrophy,anotheratphotosynthesis.Overtimetheirdescendantsbecamemorespecialisedandbetteradaptedtothatparticularmodeoflife.

Toputthatmoreformally,ifrisingoxygenlevelspermittedflourishingnewlifestyles,wewouldexpecttoseeapolyphyleticradiation, inwhichunrelatedcellsororganisms(fromdifferentphyla)adaptswiftly,radiatingnewspeciesthatfillunoccupiedniches.Thiskindofpatternisexactlywhatwedo see – sometimes. Dozens of different animal phyla radiated in the Cambrian explosion, forexample, from sponges and echinoderms to arthropods andworms.These great animal radiationswereaccompaniedbymatchingradiationsamongalgaeandfungi,aswellasprotistssuchasciliates.Ecologybecameenormouslymorecomplex,andthisinitselfdrovefurtherchanges.Whetherornot

itwasspecifically therisingtideofoxygenthat triggeredtheCambrianexplosion, there isgeneralagreementthatenvironmentalchangesdidindeedtransformselection.Somethinghappened,andtheworldchangedforever.

Contrastthispatternwithwhatwewouldexpecttoseeifconstraintsofstructuredominated.Untiltheconstraintisovercome,weshouldseelimitedchangeinresponsetoanyenvironmentalshifts.Wewould expect long periods of stasis, impervious to environmental changes, with very occasionalmonophyleticradiations.That’stosaythatif,onarareoccasion,oneparticulargroupovercomesitsintrinsicstructuralconstraints,italonewillradiatetofillvacantniches(albeitpossiblydelayeduntilpermittedbyachangeintheenvironment).Ofcourse,weseethistoo.IntheCambrianexplosionweseetheradiationofdifferentanimalgroups–butnotmultipleoriginsofanimals.Allanimalgroupsshareacommonancestor,as indeeddoallplants.Complexmulticellulardevelopment, involvingadistinctgermlineandsoma(body)isdifficult.Theconstraintshererelateinparttotherequirementsforaprecisedevelopmentalprogram,whichexercisestightcontroloverthefateofindividualcells.At a looser level, though, somedegree ofmulticellular development is common,with asmany asthirtyseparateoriginsofmulticellularityamonggroupsincludingalgae(seaweeds),fungiandslimemoulds. But there is one place in which it seems that the constraints of physical structure – cellstructure – dominate to such a degree that they overwhelm everything else: the origin of theeukaryoticcell(largecomplexcells)frombacteria,intheaftermathoftheGreatOxidationEvent.

TheblackholeattheheartofbiologyIfcomplexeukaryoticcellsreallydidevolveinresponsetotheriseinatmosphericoxygen,wewouldpredict apolyphyletic radiation,with various different groups of bacteria begettingmore complexcell types independently.Wewould expect to see photosynthetic bacteria giving rise to larger andmorecomplexalgae,osmotrophicbacteriatofungi,motilepredatorycellstophagocytes,andsoon.Suchevolutionofgreatercomplexitycouldoccurthroughstandardgeneticmutations,geneswappingandnaturalselection,orbywayofthemergersandacquisitionsofendosymbiosis,asconceivedbyLynn Margulis in the well-known serial endosymbiosis theory. Either way, if there are nofundamental constraints on cell structure, then rising oxygen levels should have made greatercomplexity possible regardless of how exactly it evolved. We would predict that oxygen wouldrelease the constraints on all cells, enabling a polyphyletic radiation with all kinds of differentbacteriabecomingmorecomplexindependently.Butthat’snotwhatwesee.

Let me spell this out in more detail, as the reasoning is critical. If complex cells arose via‘standard’naturalselection,inwhichgeneticmutationsgiverisetovariationsacteduponbynaturalselection, thenwewouldexpect to seeamixedbagof internal structures, asvariedas theexternalappearanceofcells.Eukaryoticcellsarewonderfullyvariedintheirsizeandshape,fromgiantleaf-likealgalcellstospindlyneurons,tooutstretchedamoebae.Ifeukaryoteshadevolvedmostoftheircomplexityinthecourseofadaptingtodistinctwaysoflifeindivergentpopulations,thenthislonghistoryshouldbereflectedintheirdistinctiveinternalstructurestoo.Butlookinside(aswe’llsoondo)andyou’llseethatalleukaryotesaremadeofbasicallythesamecomponents.Mostofuscouldn’tdistinguishbetween aplant cell, a kidney cell and aprotist from the local ponddown the electronmicroscope:theyalllookremarkablysimilar.Justtryit(Figure3).Ifrisingoxygenlevelsremovedconstraints on complexity, the prediction from ‘standard’ natural selection is that adaptation todifferentwaysof life indifferentpopulations should lead toapolyphyletic radiation.But that isn’twhatwesee.

Figure3ThecomplexityofeukaryotesFourdifferenteukaryoticcellsshowingequivalentmorphologicalcomplexity.Ashowsananimalcell(aplasmacell),withalargecentralnucleus(N),extensiveinternalmembranes(endoplasmicreticulum,ER)studdedwithribosomes,andmitochondria(M).BistheunicellularalgaEuglena,foundinmanyponds,showingacentralnucleus(N),chloroplasts(C),andmitochondria(M).Cisaplantcellboundedbyacellwall,withavacuole(V),chloroplasts(C),anucleus(N),andmitochondria(M).Disachytridfunguszoospore,implicatedintheextinctionof150frogspecies;(N)isthenucleus,(M)mitochondria,(F)theflagellum,and(G)gammabodiesofunknownfunction.

Fromthelate1960s,LynnMargulisarguedthatthisviewisinanycasemisguided:thateukaryoticcellsdidnotariseviastandardnaturalselection,butthroughaseriesofendosymbioses,inwhichanumberofbacteriacooperatedtogethersocloselythatsomecellsphysicallygotinsideothers.Suchideas trace their roots back to the early twentieth century to Richard Altmann, KonstantinMereschkowski, George Portier, IvanWallin and others, who argued that all complex cells arosethrough symbioses between simpler cells. Their ideaswere not forgotten butwere laughed out ofhouseas‘toofantasticforpresentmentioninpolitebiologicalsociety’.Bythetimeofthemolecularbiologyrevolution in the1960s,Marguliswasonfirmer,albeit stillcontroversial,ground,andwenowknowthatatleasttwocomponentsofeukaryoticcellswerederivedfromendosymbioticbacteria– themitochondria (theenergy transducers incomplexcells),whichderive fromα-proteobacteria;and thechloroplasts (thephotosyntheticmachineryofplants),derivingfromcyanobacteria.Almostall the other specialised ‘organelles’ of eukaryotic cells have at one time or another also been

claimed tobeendosymbionts, including thenucleus itself, thecilia and flagella (sinuousprocesseswhoserhythmicbeatdrivesthemovementofcells)andperoxisomes(factoriesfortoxicmetabolism).Thus the serial endosymbiosis theory claims that eukaryotes are composed of an ensemble ofbacteria, forged in a communal enterprise over hundreds of millions of years after the GreatOxidationEvent.

It’sapoeticnotion,buttheserialendosymbiosistheorymakesanimplicitpredictionequivalenttothatofstandardselection.Ifitweretrue,wewouldexpecttoseepolyphyleticorigins–amixedbagofinternalstructures,asvariedastheexternalappearanceofcells.Inanyseriesofendosymbioses,where the symbiosis depends on some kind of metabolic trading in a particular environment, wewouldexpecttofinddisparatetypesofcellinteractingindifferentenvironments.Ifthesecellslaterbecamefashionedintotheorganellesofcomplexeukaryoticcells,thehypothesispredictsthatsomeeukaryotesshouldpossessonesetofcomponents,andothersadifferentset.Weshouldexpecttofindallkindsofintermediatesandunrelatedvariantslurkinginobscurehidingplaceslikestagnantmuds.Rightuptoherprematuredeathfromastrokein2011,Margulisdidindeedholdfirmtoherbeliefthateukaryotesarearichandvariedtapestryofendosymbioses.Forher,endosymbiosiswasawayoflife,anunderexplored‘feminine’avenueofevolution, inwhichcooperation–‘networking’,asshecalledit–trumpedtheunpleasantlymasculinecompetitionbetweenthehuntersandhunted.Butinhervenerationof‘real’livingcells,Margulisturnedherbackonthemorearidcomputationaldisciplineof phylogenetics, the study of gene sequences andwhole genomes,which has the power to tell usexactlyhowdifferenteukaryotesrelatetoeachother.Andthattellsaverydifferent–andultimatelyfarmorecompelling–story.

Thestoryhingesonalargegroupofspecies(athousandormoreinnumber)ofsimplesingle-celledeukaryotesthat lackmitochondria.Thisgroupwasoncetakentobeaprimitiveevolutionary‘missing link’ between bacteria and more complex eukaryotes – exactly the kind of intermediatepredictedbytheserialendosymbiosistheory.ThegroupincludesthenastyintestinalparasiteGiardia,which in Ed Yong’s words resembles a malevolent teardrop (Figure 4). It lives up to its looks,causingunpleasantdiarrhoea.Ithasnotjustonenucleusbuttwo,andsoisunquestionablyeukaryotic,butitlacksotherarchetypaltraits,notablymitochondria.Inthemid1980s,theiconoclasticbiologistTom Cavalier-Smith argued that Giardia and other relatively simple eukaryotes were probablysurvivors from theearliestperiodof eukaryotic evolution,before theacquisitionofmitochondria.WhileCavalier-Smithacceptedthatmitochondriadoindeedderivefrombacterialendosymbionts,hehad little time forMargulis’s serial endosymbiosis theory; instead, he pictured (and still does) theearliesteukaryotesasprimitivephagocytes,similartomodernamoeba,whichearnedtheirlivingbyengulfingothercells.Thecellsthatacquiredmitochondria,heargued,alreadyhadanucleus,andadynamicinternalskeletonthathelpedthemtochangeshapeandmovearound,andproteinmachineryforshiftingcargoabouttheirinsides,andspecialisedcompartmentsfordigestingfoodinternally,andso on. Acquiring mitochondria helped, certainly – they turbocharged those primitive cells. Butsouping-up a car does not alter the structure of the car: you still beginwith an automobile,whichalready has an engine, gearbox, brakes, everything that makes it a car. Turbocharging changesnothing but the power output. Likewise in the case of Cavalier-Smith’s primitive phagocytes –everythingwasalreadyinplaceexceptmitochondria,whichmerelygavecellsmorepower.Ifthereisatextbookviewofeukaryoticorigins–eventoday–thisisit.

Figure4TheArchezoa–thefabled(butfalse)missinglinkAAnoldandmisleadingtreeoflifebasedonribosomalRNA,showingthethreedomainsofbacteria,archaeaandeukaryotes.Thebarsmark(1)thesupposedearlyevolutionofthenucleus;and(2)thepresumedlateracquisitionofmitochondria.ThethreegroupsthatbranchbetweenthebarsconstitutetheArchezoa,supposedlyprimitiveeukaryotesthatnotyetacquiredmitochondria,suchasGiardia(B).WenowknowthattheArchezoaarenotprimitiveeukaryotesatall,butderivefrommorecomplexancestorsthatalreadyhadmitochondria–theyactuallybranchwithinthemainpartoftheeukaryotictree(N=nucleus;ER=endoplasmicreticulum;V=vacuoles;F=flagella).

Cavalier-Smithdubbedtheseearlyeukaryotesthe‘archezoa’(meaningancientanimals),toreflecttheirsupposedantiquity (Figure4).Several areparasites that causediseases, so their biochemistryandgenomeshaveattractedtheinterestofmedicalresearchandthefundingthatgoeswithit.Thatinturnmeanswenowknowagreatdealaboutthem.Overthepasttwodecades,wehavelearnedfromtheirgenomesequencesanddetailedbiochemistry thatnone of thearchezoa is a realmissing link,whichistosaythattheyarenottrueevolutionaryintermediates.Onthecontrary,allofthemderivefrom more complex eukaryotes, which once had a full quota of everything, including, and inparticular, mitochondria. They had lost their erstwhile complexity while specialising to live insimpler niches.All of them retain structures that are now known to derive frommitochondria byreductive evolution – either hydrogenosomes or mitosomes. These don’t look much likemitochondria,eventhoughtheyhaveanequivalentdouble-membranestructure,hencetheerroneousassumption that archezoa never possessed mitochondria. But the combination of molecular and

phylogeneticdatashowsthathydrogenosomesandmitosomesareindeedderivedfrommitochondria,not some other bacterial endosymbiont (as predicted by Margulis). Thus all eukaryotes havemitochondriainoneformoranother.Wecaninferthatthelasteukaryoticcommonancestoralreadyhadmitochondria,ashadbeenpredictedbyBillMartinin1998(seetheIntroduction).Thefactthatalleukaryotes have mitochondria may seem to be a trivial point, but when combined with theproliferationofgenomesequencesfromacrossthewidermicrobialworld,thisknowledgehasturnedourunderstandingofeukaryoticevolutiononitshead.

Wenowknowthateukaryotesallshareacommonancestor,whichbydefinitionarosejustonceinthe 4 billion years of life on earth.Letme reiterate this point, as it is crucial.All plants, animals,algae,fungiandprotistsshareacommonancestor–theeukaryotesaremonophyletic.Thismeansthatplants did not evolve from one type of bacteria, and animals or fungi from other types. On thecontrary,apopulationofmorphologicallycomplexeukaryoticcellsaroseonasingleoccasion–andallplants,animals,algaeandfungievolvedfromthisfounderpopulation.Anycommonancestorisbydefinitionasingularentity–notasinglecell,butasinglepopulationofessentiallyidenticalcells.Thatdoesnotinitselfmeanthattheoriginofcomplexcellswasarareevent.Inprinciple,complexcellscouldhavearisenonnumerousoccasions,butonlyonegrouppersisted–all therestdiedoutforsomereason.Ishallarguethatthiswasnotthecase,butfirstwemustconsiderthepropertiesofeukaryotesinalittlemoredetail.

The common ancestor of all eukaryotes quickly gave rise to five ‘supergroups’ with diversecellular morphologies, most of which are obscure even to classically trained biologists. Thesesupergroupshavenames likeunikonts (comprisinganimalsand fungi), excavates, chromalveolatesandplantae(includinglandplantsandalgae).Theirnamesdon’tmatter,buttwopointsareimportant.First there is farmoregeneticvariationwithineachof thesesupergroups than there isbetween theancestors of each group (Figure 5). That implies an explosive early radiation – specifically amonophyletic radiation that hints at a release from structural constraints. Second, the commonancestor was already a strikingly complex cell. By comparing traits common to each of thesupergroups,wecan reconstruct the likelypropertiesof thecommonancestor.Any traitpresent inessentiallyall thespeciesofallsupergroupswaspresumablyinheritedfromthatcommonancestor,whereas any traits that areonlypresent inoneor twogroupswerepresumablyacquired later, andonlyinthatgroup.Chloroplastsareagoodexampleofthelatter:theyarefoundonlyinplantaeandchromalveolates, as a result of well-known endosymbioses. They were not part of the eukaryoticcommonancestor.

So what does phylogenetics tell us was part of the common ancestor? Shockingly, nearlyeverythingelse.Letmerunthroughafewitems.Weknowthatthecommonancestorhadanucleus,where it stored itsDNA.Thenucleushasagreatdealofcomplexstructure that isagainconservedrightacrosseukaryotes.Itisenclosedbyadoublemembrane,orratheraseriesofflattenedsacsthatlooklikeadoublemembranebutareinfactcontinuouswithothercellularmembranes.Thenuclearmembrane is studded with elaborate protein pores and lined by an elastic matrix; and within thenucleus,otherstructuressuchasthenucleolusareagainconservedacrossalleukaryotes.It’sworthstressing thatdozensofcoreproteins in thesecomplexesareconservedacross thesupergroups,asare the histone proteins that wrap DNA. All eukaryotes have straight chromosomes, capped with‘telomeres’,whichpreventtheendsfromfrayinglikethetipsofshoelaces.Eukaryoteshave‘genesinpieces’, inwhich short sectionsofDNAencodingproteins are interspersedby longnon-codingregions,calledintrons.Theseintronsaresplicedoutbeforebeingincorporatedintoproteins,usingmachinerycommontoalleukaryotes.Eventhepositionoftheintronsisfrequentlyconserved,withinsertionsfoundatthesamepositionofthesamegeneacrosseukaryotes.

Figure5The‘supergroups’ofeukaryotesAtreeofeukaryotes,basedonthousandsofsharedgenes,showingthefive‘supergroups’asdepictedbyEugeneKooninin2010.ThenumbersrefertothenumberofgenessharedbyeachofthesesupergroupswithLECA(thelasteukaryoticcommonancestor).Eachgrouphasindependentlylostorgainedmanyothergenes.Mostvariationhereisbetweensingle-celledprotists;animalsfallwithintheMetazoans(nearthebottom).Noticethatthereisfarmorevariationwithineachsupergroupthanbetweentheancestorsofthesegroups,suggestinganexplosiveearlyradiation.Ilikethesymbolicblackholeatthecentre:LECAhadalreadyevolvedallthecommoneukaryotictraits,butphylogeneticsgiveslittleinsightintohowanyofthesearosefrombacteriaorarchaea–anevolutionaryblackhole.

Outside thenucleus, the story continues in the samevein.Barring the simpler archezoa (whichturn out to be scatteredwidely across the five supergroups, again demonstrating their independentloss of earlier complexity), all eukaryotes share essentially the same cellularmachinery.All havecomplexinternalmembranestructuressuchastheendoplasmicreticulumandGolgiapparatus,whichare specialised for packaging and exporting proteins. All have a dynamic internal cytoskeleton,capable of remodelling itself to all shapes and requirements. All havemotor proteins that shuttleobjects back and forth on cytoskeletal tracks across the cell. All have mitochondria, lysosomes,peroxisomes,themachineryofimportandexport,andcommonsignallingsystems.Thelistgoeson.

All eukaryotes divide bymitosis, inwhich chromosomes are separatedon amicrotubular spindle,using a common set of enzymes. All are sexual, with a life cycle involving meiosis (reductivedivision)toformgameteslikethespermandegg,followedbythefusionofthesegametes.Thefeweukaryotes that lose their sexuality tend to fallquicklyextinct (quickly in thiscasemeaningoverafewmillionyears).

Wehaveunderstoodmuchofthisforalongtimefromthemicroscopicstructureofcells,butthenew era of phylogenomics illuminates two aspects vividly. First, the structural similarities are notsuperficial resemblances, appearances that flatter to deceive, but are written out in the detailedsequencesofgenes,inmillionsandbillionsoflettersofDNA–andthatallowsustocomputetheirancestry as a branching tree with unprecedented precision. Second, the advent of high-throughputgenesequencingmeansthatsamplingofthenaturalworldnolongerreliesonpainstakingattemptstoculturecellsortopreparemicroscopicsections,butisasfastandreliableasashotgunsequencer.Wehave discovered several unexpected new groups, including eukaryotic extremophiles capable ofdealingwithhighconcentrationsoftoxicmetalsorhightemperatures,andtinybutperfectlyformedcells known as picoeukaryotes, as small as bacteria yet still featuring a scaled-down nucleus andmidgetmitochondria.Allofthismeanswehaveamuchclearerideaofthediversityofeukaryotes.Alltheseneweukaryotesfitcomfortablywithinthefiveestablishedsupergroups–theydonotopenupnewphylogeneticvistas.Thekillerfactthatemergesfromthisenormousdiversityishowdamnedsimilar eukaryotic cells are.We do not find all kinds of intermediates and unrelated variants. Thepredictionoftheserialendosymbiosistheory,thatweshould,iswrong.

That poses a different problem. The stunning success of phylogenetics and the informationalapproach to biology can easily blind us to its limitations. The problemhere iswhat amounts to aphylogenetic ‘event horizon’ at the origin of eukaryotes. All these genomes lead back to the lastcommonancestor of eukaryotes,whichhadmoreor less everything.Butwheredid all thesepartscomefrom?Theeukaryoticcommonancestormightaswellhavejumped,fullyformed,likeAthenafrom the head of Zeus.We gain little insight into traits that arose before the common ancestor –essentiallyallofthem.Howandwhydidthenucleusevolve?Whataboutsex?Whydovirtuallyalleukaryoteshavetwosexes?Wheredidtheextravagantinternalmembranescomefrom?Howdidthecytoskeleton become so dynamic and flexible? Why does sexual cell division (‘meiosis’) halvechromosomenumbersby first doubling themup?Whydoweage, get cancer, anddie?For all itsingenuity,phylogeneticscantelluslittleaboutthesecentralquestionsinbiology.Almostallthegenesinvolved (encoding so-called eukaryotic ‘signature proteins’) are not found in prokaryotes. Andconversely,bacteriashowpracticallyno tendency toevolveanyof thesecomplexeukaryotic traits.There are no known evolutionary intermediates between the morphologically simple state of allprokaryotes and the disturbingly complex common ancestor of eukaryotes (Figure 6). All theseattributesofcomplexlifearoseinaphylogeneticvoid,ablackholeattheheartofbiology.

Figure6TheblackholeattheheartofbiologyThecellatthebottomisNaegleria,takentobesimilarinsizeandcomplexitytothecommonancestorofalleukaryotes.Ithasanucleus(N),endoplasmicreticulum(ER),Golgicomplex(Gl),mitochondria(Mi),foodvacuole(Fv),phagosomes(Ps)andperoxisomes(P).Atthetopisarelativelycomplexbacterium,Planctomycetes,shownroughlytoscale.IamnotsuggestingthattheeukaryotesderivedfromPlanctomycetes(theycertainlydidnot),merelyshowingthescaleofthegulfbetweenarelativelycomplexbacteriumandarepresentativesingle-celledeukaryote.Therearenosurvivingevolutionaryintermediatestotellthetale(indicatedbytheskullsandcrossbones).

ThemissingstepstocomplexityEvolutionarytheorymakesasimpleprediction.Complextraitsariseviaaseriesofsmallsteps,eachnewstepofferingasmalladvantageoverthelast.Selectionofthebest-adaptedtraitsmeanslossofthelesswell-adaptedtraits,soselectioncontinuouslyeliminatesintermediates.Overtime,traitswilltendtoscalethepeaksofanadaptivelandscape,soweseetheapparentperfectionofeyes,butnottheless

perfectintermediatestepsenroutetotheirevolution.InTheOriginofSpeciesDarwinmadethepointthat natural selection actually predicts that intermediates should be lost. In that context, it is notterriblysurprisingthattherearenosurvivingintermediatesbetweenbacteriaandeukaryotes.Whatismore surprising, though, is that the same traits donot keepon arising, time and time again– likeeyes.

Wedonotseethehistoricalstepsintheevolutionofeyes,butwedoseeanecologicalspectrum.From a rudimentary light-sensitive spot on some early worm-like creature, eyes have arisenindependentlyonscoresofoccasions.Thatisexactlywhatnaturalselectionpredicts.Eachsmallstepoffersasmalladvantageinoneparticularenvironment,withthepreciseadvantagedependingontheprecise environment. Morphologically distinct types of eye evolve in different environments, asdivergentasthecompoundeyesoffliesandmirroreyesofscallops,orasconvergentasthecameraeyesthataresosimilarinhumansandoctopuses.Everyconceivableintermediate,frompinholestoaccommodatinglenses,isfoundinonespeciesoranother.Weevenseeminiatureeyes,repletewitha‘lens’anda ‘retina’, insomesingle-celledprotists. Inshort,evolutionary theorypredicts that thereshouldbemultiple–polyphyletic–originsoftraitsinwhicheachsmallstepoffersasmalladvantageover the last step.Theoretically thatapplies toall traits, and it is indeedwhatwegenerally see.Sopoweredflightaroseonatleastsixdifferentoccasionsinbats,birds,pterosaursandvariousinsects;multicellularity about 30 times, as noted earlier; different forms of endothermy (warm blood) inseveral groups including mammals and birds, but also some fish, insects and plants;6 and evenconsciousawarenessappears tohavearisenmoreor less independently inbirdsandmammals.Aswith eyes,we see amyriad of different forms reflecting the different environments inwhich theyarose.Certainly therearephysical constraints,but theyarenot strongenough toprecludemultipleorigins.

Sowhataboutsex,orthenucleus,orphagocytosis?Thesamereasoningoughttoapply.Ifeachofthese traits arose by natural selection –which they undoubtedly did – and all of the adaptive stepsofferedsomesmalladvantage–whichtheyundoubtedlydid–thenweshouldseemultipleoriginsofeukaryotictraitsinbacteria.Butwedon’t.Thisislittleshortofanevolutionary‘scandal’.Weseenomorethanthebeginningsofeukaryotictraitsinbacteria.Takesex,forexample.Somemayarguethatbacteriapractiseaformofconjugationequivalenttosex,transferringDNAfromonetoanotherby‘lateral’genetransfer.BacteriahaveallthemachineryneededtorecombineDNA,enablingthemtoforge new and varied chromosomes, which is usually taken to be the advantage of sex. But thedifferencesareenormous.Sexinvolvesthefusionoftwogametes,eachwithhalfthenormalquotaofgenes, followed by reciprocal recombination across the entire genome. Lateral gene transfer isneitherreciprocalnorsystematicinthisway,butpiecemeal.Ineffect,eukaryotespractise‘totalsex’,bacteriaapallidhalf-heartedform.Plainlytheremustbesomeadvantagetoeukaryotesindulgingintotalsex;butifso,wewouldexpectthatatleastsometypesofbacteriawoulddosomethingsimilar,even if the detailedmechanismswere different.To the best of our knowledge, none ever did.Thesamegoesforthenucleusandphagocytosis–andmoreorlessalleukaryotictraits.Thefirststepsarenottheproblem.Weseesomebacteriawithfoldedinternalmembranes,otherswithnocellwallandamodestlydynamiccytoskeleton,yetotherswithstraightchromosomes,ormultiplecopiesoftheirgenome,orgiantcellsize:allthebeginningsofeukaryoticcomplexity.Butbacteriaalwaysstopwell short of the baroque complexity of eukaryotes, and rarely if ever combinemultiple complextraitsinthesamecell.

Theeasiestexplanationforthedeepdifferencesbetweenbacteriaandeukaryotesiscompetition.Once the first true eukaryotes had evolved, the argument goes, theywere so competitive that theydominated the niche ofmorphological complexity. Nothing else could compete. Any bacteria that‘tried’ to invade thiseukaryoticnicheweregivenshortshriftby thesophisticatedcells thatalready

lived there.To use the parlance, theywere outcompeted to extinction.We are all familiarwith themassextinctionsofdinosaursandotherlargeplantsandanimals,sothisexplanationseemsperfectlyreasonable.Thesmall,furryancestorsofmodernmammalswereheldincheckbythedinosaursformillionsofyears,onlyradiatingintomoderngroupsafterthedinosaurs’demise.Yettherearesomegood reasons toquestion this comfortablebutdeceptive idea.Microbesarenot equivalent to largeanimals: their population sizes are enormously larger, and they pass around useful genes (such asthose for antibiotic resistance) by lateral gene transfer,making themverymuch less vulnerable toextinction.Thereisnohintofanymicrobialextinction,evenintheaftermathoftheGreatOxidationEvent.The‘oxygenholocaust’,whichsupposedlywipedoutmostanaerobiccells,can’tbetracedatall:thereisnoevidencefromeitherphylogeneticsorgeochemistrythatsuchanextinctionevertookplace.Onthecontrary,anaerobesprospered.

More significantly, there is very strong evidence that the intermediates were not, in fact,outcompetedtoextinctionbymoresophisticatedeukaryotes.Theystillexist.Wemetthemalready–the‘archezoa’,thatlargegroupofprimitiveeukaryotesthatwereoncemistakenforamissinglink.Theyarenottrueevolutionaryintermediates,butthey’rerealecologicalintermediates.Theyoccupythesameniche.Anevolutionaryintermediateisamissinglink–afishwithlegs,suchasTiktaalik,oradinosaurwithfeathersandwings,suchasArchaeopteryx.Anecological intermediate isnota truemissinglinkbutitprovesthatacertainniche,awayoflife,isviable.Aflyingsquirrelisnotcloselyrelatedtootherflyingvertebratessuchasbatsorbirds,butitdemonstratesthatglidingflightbetweentrees ispossiblewithout fully fledgedwings.Thatmeans it’snotpuremake-believe tosuggest thatpoweredflightcouldhavestartedthatway.Andthatistherealsignificanceofthearchezoa–theyareecologicalintermediates,whichprovethatacertainwayoflifeisviable.

Imentionedearlier that therearea thousandormoredifferentspeciesofarchezoa.Thesecellsarebonafideeukaryotes,whichadaptedtothis‘intermediate’nichebybecomingsimpler,notbacteriathatbecameslightlymorecomplex.Letmestressthepoint.Thenicheisviable.Ithasbeeninvadedonnumerousoccasionsbymorphologicallysimplecells,whichthrivethere.Thesesimplecellswerenotoutcompetedtoextinctionbymoresophisticatedeukaryotes thatalreadyexistedandfilledthesameniche.Quite thereverse: theyflourishedpreciselybecause theybecamesimpler. Instatistical terms,all else being equal, the probability of only simple eukaryotes (rather than complex bacteria)invading thisnicheon1,000separateoccasions isaboutone in10300against–anumber thatcouldhavebeenconjuredupbyZaphodBeeblebrox’sInfiniteImprobabilityDrive.Evenifarchezoaaroseindependently on a farmore conservative 20 separate occasions (each time radiating to produce alargenumberofdaughterspecies),theprobabilityisstilloneinamillionagainst.Eitherthiswasaflukeoffreakishproportions,orallelsewasnotequal.Themostplausibleexplanationisthattherewas something about the structure of eukaryotes that facilitated their invasion of this intermediateniche, and conversely, something about the structure of bacteria that precluded their evolution ofgreatermorphologicalcomplexity.

That doesn’t seem particularly radical. In fact it chimes with everything else we know. I havetalked throughout thischapteraboutbacteria,butaswenoted in theIntroduction, thereareactuallytwo large groups, or domains, of cells that lack a nucleus, hence are designated ‘prokaryotes’(literally ‘before thenucleus’).Theseare thebacteriaand the ‘archaea’,not tobemistaken for thearchezoa,thesimpleeukaryoticcellsthatwe’vebeendiscussing.WhileIcanonlyapologisefortheconfusionofscientific terminology,whichsometimesseemstobecraftedbyalchemistswhocravenot tobeunderstood,please remember that thearchaeaand thebacteriaareprokaryotes, lackinganucleus,whereasthearchezoaareprimitiveeukaryotes,whichhaveanucleus.Infact,thearchaeaarestill sometimes called archaebacteria, or ‘ancient bacteria’, as opposed to eubacteria, or ‘truebacteria’, sobothgroupscan legitimatelybecalledbacteria.Forsimplicity, I’llcontinue touse the

word bacteria loosely to refer to both groups, except when I need to specify critical differencesbetweenthetwodomains.7

Thecrucialpointisthatthesetwodomains,thebacteriaandthearchaea,areextremelydifferentintheirgeneticsandintheirbiochemistry,butalmostindistinguishableintheirmorphology.Bothtypesaresmallsimplecellsthatlackanucleusandalltheothereukaryotictraitsthatdefinecomplexlife.Thefactthatbothgroupsfailedtoevolvecomplexmorphology,despitetheirextraordinarygeneticdiversityandbiochemicalingenuity,makesitlookasifanintrinsicphysicalconstraintprecludestheevolutionofcomplexityinprokaryotes,aconstraint thatwassomehowreleasedintheevolutionofeukaryotes. InChapter5, I’ll argue that this constraintwas released by a rare event – the singularendosymbiosisbetweentwoprokaryotesthatwediscussedintheIntroduction.Fornow,though,let’sjust note that some sort of structural constraint must have acted equally on both of the two greatdomains of prokaryotes, the bacteria and archaea, forcing both groups to remain simple in theirmorphologythroughoutanincomprehensible4billionyears.Onlyeukaryotesexploredtherealmofcomplexity, and they did so via an explosive monophyletic radiation that implies a release fromwhateverthesestructuralconstraintsmighthavebeen.Thatappearstohavehappenedjustonce–alleukaryotesarerelated.

ThewrongquestionThis,then,isourshorthistoryoflifethroughneweyes.Hereisaswiftsummary.Theearlyearthwasnot drastically different from our own world: it was a water world, with a moderate climate,dominated by volcanic gases such as carbon dioxide and nitrogen.While our early planet lackedoxygen,itwasnotrichingasesconducivetoorganicchemistry–hydrogen,methaneandammonia.That rules out tired old ideas of primordial soup; yet life started as early as could be, perhaps 4billionyearsago.Atfacevalue,somethingelsewasdrivingtheemergenceoflife;wewillcometothat.Bacteria soon tookover,colonisingevery inch,everymetabolicniche, remodelling theglobeover 2 billion years, depositing rocks and minerals on a colossal scale, transforming oceans,atmosphere and continents. They crashed the climate in global snowball earths; they oxidised theworld, filling theoceansandairwithreactiveoxygen.Yet inall this immenseduration,neither thebacterianorthearchaeabecameanythingelse:theyremainedstubbornlysimpleintheirstructureandwayoflife.Foraneternal4billionyears,throughextremesofenvironmentalandecologicalchange,bacteriachangedtheirgenesandbiochemistry,butneverchangedtheirform.Theynevergaverisetomorecomplexlifeforms,ofthekindwemighthopetodetectonanotherplanet,intelligentaliens–exceptjustonce.

Ononesingleoccasion,hereonearth,bacteriagaverise toeukaryotes.There isnothingin thefossilrecord,orinphylogenetics,tosuggestthatcomplexlifeactuallyaroserepeatedly,butthatonlyonegroup,thefamiliarmoderneukaryotes,survived.Onthecontrary,themonophyleticradiationofeukaryotessuggeststheiruniqueoriginwasdictatedbyintrinsicphysicalconstraintswhichhadlittleif anything to dowith environmental upheavals such as theGreatOxidationEvent.We’ll seewhatthese constraintsmight havebeen inPart III. For now, let’s just note that any proper accountmustexplainwhytheevolutionofcomplexlifehappenedonlyonce:ourexplanationmustbepersuasiveenoughtobebelievable,butnotsopersuasive thatweare leftwonderingwhyitdidnothappenonmanyoccasions.Anyattempttoexplainasingulareventwillalwayshavetheappearanceofaflukeabout it.Howcanweprove itonewayoranother?Theremightnotbemuchtogoon in theeventitself,buttheremaybecluesconcealedintheaftermath,asmokinggunthatgivessomeindicationofwhat happened. Once they cast off their bacterial shackles, the eukaryotes became enormouslycomplexanddiverse in theirmorphology.Yet theydidnot accrue this complexity in anobviously

predictableway:theycameupwithawholeseriesoftraits,fromsexandageingtospeciation,noneofwhichhaveeverbeen seen inbacteriaor archaea.Theearliest eukaryotesaccumulatedall thesesingular traits inacommonancestorwithoutpeer.Therearenoknownevolutionary intermediatesbetweenthemorphologicalsimplicityofbacteriaandthatenormouslycomplexeukaryoticcommonancestortotellthetale.Allofthisaddsuptoathrillingprospect–thebiggestquestionsinbiologyremain tobesolved! Is there somepattern to these traits thatmightgivean indicationofhow theyevolved?Ithinkso.

Thispuzzlerelatesbacktothequestionthatweaskedatthebeginningofthischapter.Howmuchof life’s history and properties can be predicted from first principles? I suggested that life isconstrainedinwaysthatcan’teasilybeinterpretedintermsofgenomes,historyorenvironment.Ifweconsiderlifeintermsofinformationalone,mycontentionwasthatwecouldpredictnoneofthisinscrutablehistory.Whydid life start soearly?Whydid it stagnate inmorphological structure forbillions of years? Why were bacteria and archaea unaffected by environmental and ecologicalupheavals on a global scale?Why is all complex lifemonophyletic, arising just once in 4 billionyears?Whydoprokaryotesnotcontinuously,orevenoccasionally,giverisetocellsandorganismswith greater complexity? Why do individual eukaryotic traits such as sex, the nucleus andphagocytosisnotariseinbacteriaorarchaea?Whydideukaryotesaccumulateallthesetraits?

If life is all about information, these are deep mysteries. I don’t believe this story could beforetold,predictedasscience,onthebasisofinformationalone.Thequirkypropertiesoflifewouldhavetobeascribedtothecontingenciesofhistory,theslingsandarrowsofoutrageousfortune.Wewouldhavenopossibilityofpredictingthepropertiesoflifeonotherplanets.YetDNA,thebeguilingcode-scriptwhich seems topromise every answer, hasmadeus forgetSchrödinger ’sother centraltenet–thatliferesistsentropy,thetendencytodecay.InafootnotetoWhatisLife?Schrödingernotedthatifhehadbeenwritingforanaudienceofphysicists,hewouldhaveframedhisargumentnotintermsofentropy,butoffreeenergy.Thatword‘free’hasaspecificmeaning,whichwewillconsiderinthenextchapter;sufficefornowtosaythatenergyispreciselywhatwasmissingfromthischapter,and indeed fromSchrödinger ’s book.His iconic title asked thewrong question altogether.Add inenergy,andthequestionismuchmoretelling:WhatisLiving?ButSchrödingermustbeforgiven.Hecouldnothaveknown.Whenhewaswriting,nobodyknewmuchabout thebiological currencyofenergy.Nowwe know how it all works in exquisite detail, right down to the level of atoms. Thedetailedmechanismsofenergyharvestingturnouttobeconservedasuniversallyacrosslifeasthegenetic code itself, and thesemechanisms exert fundamental structural constraints on cells.Butwehavenoideahowtheyevolved,norhowbiologicalenergyconstrainedthestoryoflife.Thatisthequestionofthisbook.

Footnotes1Thereisanoisydisputeaboutwhetherallthisnon-codingDNAservesanyusefulpurpose.Someclaimthatitdoes,andthattheterm‘junkDNA’shouldbedismissed.Othersposethe‘oniontest’:ifmostnon-codingDNAdoesserveausefulpurpose,whydoesanonionneedfivetimesmoreofitthanahumanbeing?Inmyviewit’sprematuretoabandontheterm.Junkisnotthesamethingasgarbage.Garbageisthrownawayimmediately;junkisretainedinthegarage,inthehopethatitmightturnouttobeusefuloneday.2There’salsoathird,unstableisotope,carbon-14,whichisradioactive,breakingdownwithahalf-lifeof5,570years.Thisisoftenusedfordatinghumanartefacts,butisnouseovergeologicalperiods,andsonotrelevanttoourstoryhere.3Thismethanewasproducedbymethanogenicbacteria,ormorespecificallyarchaea,whichifcarbonisotopesignaturesaretobebelieved(methanogensproduceaparticularlystrongsignal),werethrivingbefore3.4billionyearsago.Asnotedearlier,methanewasnotasignificantconstituentoftheearth’sprimordialatmosphere.4FormostofthischapterIwillreferonlytobacteriaforsimplicity,althoughImeanprokaryotes,includingbothbacteriaandarchaea,asdiscussedintheIntroduction.We’llreturntothesignificanceofarchaeatowardstheendofthechapter.5Thisisnotstrictlytrue.Aerobicrespirationdoesproducenearlyanorderofmagnitudemoreusableenergythanfermentation,butfermentationisnottechnicallyaformofrespirationatall.Trueanaerobicrespirationusessubstancesotherthanoxygen,suchasnitrate,as

anelectronacceptor,andtheseprovidenearlyasmuchenergyasoxygenitself.Buttheseoxidantscanonlyaccumulateatlevelssuitableforrespirationinanaerobicworld,astheirformationdependsonoxygen.Soevenifaquaticanimalscouldrespireusingnitrateinsteadofoxygen,theycouldstillonlydosoinanoxygenatedworld.6Theideaofendothermyinplantsmightseemsurprising,butitisknowninmanydifferentflowers,probablyhelpingtoattractpollinatorsbyaidingthereleaseofattractantchemicals;itmayalsoprovidea‘heatreward’forpollinatinginsects,promoteflowerdevelopmentandprotectagainstlowtemperatures.Someplantslikethesacredlotus(Nelumbonucifera)areevencapableofthermoregulation,sensingchangesintemperatureandregulatingcellularheatproductiontomaintaintissuetemperaturewithinanarrowrange.7Allofthesewordsareheavilyloadedwithintellectualandemotionalbaggage,accumulatedoverdecades.Thetermsarchaebacteriaandarchaeaaretechnicallyincorrectanywayasthedomainisnoolderthanthebacteria.Iprefertousethetermsarchaeaandbacteria,inpartbecausetheyemphasisethesurprisinglyfundamentaldifferencesbetweenthetwodomains,andinpartbecausetheyarejustsimpler.

I

2

WHATISLIVING?

tisacoldkiller,withacalculatedcunninghonedovermillionsofgenerations.Itcaninterferewiththesophisticated immunesurveillancemachineryofanorganism,meltingunobtrusively into the

backgroundlikeadoubleagent.Itcanrecogniseproteinsonthecellsurface,andlockontothemasifitwerean insider,gainingentrance to the innersanctum.Itcanhomeinunerringlyon thenucleus,andincorporateitselfintoahostcell’sDNA.Sometimesitremainsthereinhidingforyears,invisibletoallaround.Onotheroccasionsittakesoverwithoutdelay,sabotagingthehostcell’sbiochemicalmachinery, making thousands upon thousands of copies of itself. It dresses up these copies in acamouflagedtunicof lipidsandproteins,shipsthemtothesurface,andburstsout tobeginanotherroundofguileanddestruction.Itcankillahumanbeingcellbycell,personbyperson,indevastatingepidemic,ordissolveentireoceanicbloomsextendingoverhundredsofmiles,overnight.Yetmostbiologistswouldnotevenclassifyitasalive.Thevirusitselfdoesn’tgiveadamn.

Whywouldavirusnotbealive?Because itdoesnothaveanyactivemetabolismof itsown; itreliesentirelyon thepowerof itshost.That raises thequestion– ismetabolicactivityanecessaryattribute of life? The pat answer is yes, of course; but why, exactly? Viruses use their immediateenvironmenttomakecopiesofthemselves.Butthensodowe:weeatotheranimalsorplants,andwebreatheinoxygen.Cutusofffromourenvironment,saywithaplasticbagoverthehead,andwedieinafewminutes.Onecouldsaythatweparasitiseourenvironment–likeviruses.Sodoplants.Plantsneedusnearlyasmuchasweneedthem.Tophotosynthesisetheirownorganicmatter,togrow,plantsneedsunlight,waterandcarbondioxide(CO2).Ariddesertsordarkcavesprecludegrowth,butsotoowoulda shortageofCO2.Plants arenot shortof thegaspreciselybecauseanimals (and fungi andvariousbacteria)continuouslybreakdownorganicmatter,digestingit,burningit,finallyreleasingitback into the atmosphere as CO2. Our extra efforts to burn all the fossil fuelsmay have horribleconsequencesfor theplanet,butplantshavegoodcause tobegrateful.For them,moreCO2meansmoregrowth.So,likeus,plantsareparasitesoftheirenvironment.

Fromthispointofview,thedifferencebetweenplantsandanimalsandvirusesislittlemorethanthe largesse of that environment.Within our cells, viruses are cosseted in the richest imaginablewomb,aworldthatprovidestheireverylastwant.Theycanaffordtobesopareddown–whatPeterMedawaroncecalled‘apieceofbadnewswrappedinaproteincoat’–onlybecausetheirimmediateenvironment is so rich. At the other extreme, plants place very low demands on their immediateenvironment.Theywillgrowalmostanywherewithlight,waterandair.Toekeoutanexistencewithso few external requirements forces them to be internally sophisticated. In terms of theirbiochemistry, plants canproduce everything theyneed to grow, literally synthesising it out of thin

air.1Weourselvesaresomewhereinthemiddle.Beyondageneralrequirementforeating,weneedspecificvitamins inourdiet,withoutwhichwesuccumbtonastydiseases likescurvy.Vitaminsarecompounds that we can’t make for ourselves from simple precursors, because we have lost ourancestors’ biochemical machinery for synthesising them from scratch.Without the external propsprovidedbyvitamins,weareasdoomedasaviruswithoutahost.

Soweallneedpropsfromtheenvironment,theonlyquestionishowmany?Virusesareactuallyextremely sophisticated in relation to some parasites of DNA, such as retrotransposons (jumpinggenes) and the like. These never leave the safety of their host, yet copy themselves across wholegenomes.Plasmids – typically small independent rings ofDNAcarrying a handful of genes – canpass directly from one bacterium to another (via a slender connecting tube) without any need tofortifythemselvestotheouterworld.Areretrotransposons,plasmidsandvirusesalive?Allshareakindof‘purposeful’cunning,anabilitytotakeadvantageoftheirimmediatebiologicalenvironment,tomakecopiesof themselves.Plainly there isacontinuumbetweennon-livingand living,and it ispointlesstotrytodrawalineacrossit.Mostdefinitionsoflifefocusonthelivingorganismitself,andtendtoignorelife’sparasitisingofitsenvironment.TaketheNASA‘workingdefinition’oflife,for example: life is ‘a self-sustaining chemical system capable ofDarwinian evolution’.Does thatinclude viruses? Probably not, but it depends on what we read into that slippery phrase ‘self-sustaining’. Either way, life’s dependence on its environment is not exactly emphasised. Theenvironment,by itsverynature, seemsextraneous to life;weshall see that it isnotat all.The twoalwaysgohandinhand.

What happenswhen life is cut off from its preferred environment?We die, of course:we areeither living or dead. But that’s not always true.When cut off from the resources of a host cell,virusesdonotinstantlydecayand‘die’:theyarefairlyimpervioustothedepredationsoftheworld.Ineverymillilitreofseawater, therearetentimesasmanyviruses,waitingfortheirmoment,astherearebacteria.Avirus’sresistancetodecayisreminiscentofabacterialspore,whichisheldinastateofsuspendedanimationandcanremainthatwayformanyyears.Sporessurvivethousandsofyearsinpermafrost,oreveninouterspace,withoutmetabolisingatall.They’renotalone:seedsandevenanimalsliketardigradescanwithstandextremeconditionssuchascompletedehydration,radiationathousandtimesthedosethatwouldkillahuman,intensepressuresatthebottomoftheocean,orthevacuumofspace–allwithoutfoodorwater.

Why do viruses, spores and tardigrades not fall to pieces, conforming to the universal decaydictatedbythesecondlawofthermodynamics?Theymightdointheend–iffrazzledbythedirecthitofacosmicrayorabus–butotherwisetheyarealmostcompletelystableintheirnon-livingstate.That tells us something important about the difference between life and living. Spores are nottechnicallyliving,eventhoughmostbiologistswouldclassifythemasalive,becausetheyretainthepotentialtorevive.Theycangobacktoliving,sothey’renotdead.Idon’tseewhyweshouldviewvirusesinanydifferentlight: theytoorevert tocopyingthemselvesassoonastheyareintherightenvironment. Likewise tardigrades. Life is about its structure (dictated in part by genes andevolution), but living – growing, proliferating – is as much about the environment, about howstructure and environment interrelate.Weknowa tremendous amount about howgenes encode thephysical components of cells, but far less about how physical constraints dictate the structure andevolutionofcells.

Energy,entropyandstructureThesecondlawofthermodynamicsstatesthatentropy–disorder–mustincrease,soitseemsoddatfirstglancethatasporeoravirusshouldbesostable.Entropy,unlikelife,hasaspecificdefinition

and canbemeasured (it hasunits of joulesper kelvinpermole, sinceyou ask).Take a spore andsmash it to smithereens:grind itup intoallof itsmolecularcomponents, andmeasure theentropychange.Surelyentropymusthaveincreased!Whatwasonceabeautifullyorderedsystem,capableofresuminggrowthassoonasitfoundsuitableconditions,isnowarandomnon-functionalassortmentof bits – high entropy by definition. But no! According to the careful measurements of thebioenergeticistTedBattley,entropybarelychanged.That’sbecausethereismoretoentropythanjustthespore;wemustalsoconsideritssurroundings,andtheyhavesomelevelofdisordertoo.

Asporeiscomposedofinteractingpartsthatfitsnuglytogether.Oily(lipid)membranespartitionthemselvesfromwaternaturallybecauseofphysicalforcesactingbetweenmolecules.Amixtureofoilylipidsshakenupinwaterwillspontaneouslysortitselfintoathinbilayer,abiologicalmembraneenclosingawateryvesicle,becausethatisthemoststablestate(Figure7).Forrelatedreasons,anoilslickwill spread into a thin layer across the surface of the ocean, causingdevastation to life overhundredsofsquaremiles.Oilandwateraresaidtobeimmiscible–thephysicalforcesofattractionandrepulsionmeantheyprefertointeractwiththemselvesratherthaneachother.Proteinsbehaveinmuch the sameway: thosewith lots of electrical charges dissolve inwater; thosewithout chargesinteractmuchbetterwithoils–theyarehydrophobic,literally‘water-hating’.Whenoilymoleculesnestledowntogether,andelectricallychargedproteinsdissolveinwater,energyisreleased:thatisaphysically stable, low-energy, ‘comfortable’ state ofmatter.Energy is released as heat.Heat is themotionofmolecules,theirjostlingandmoleculardisorder.Entropy.Sothereleaseofheatwhenoilandwaterseparateactuallyincreasesentropy.Intermsofoverallentropy, then,and takingall thesephysicalinteractionsintoconsideration,anorderedoilymembranearoundacellisahigherentropystatethanarandommixtureofimmisciblemolecules,eventhoughitlooksmoreordered.2

Grind up a spore and the overall entropy hardly changes, because although the crushed sporeitselfismoredisordered,thecomponentpartsnowhaveahigherenergythantheydidbefore–oilsare mixed with water, immiscible proteins are rammed hard together. This physically‘uncomfortable’ state costs energy. If a physically comfortable state releases energy into thesurroundingsasheat,aphysicallyuncomfortablestatedoestheopposite.Energyhastobeabsorbedfromthesurroundings,loweringtheirentropy,coolingthemdown.Writersofhorrorstoriesgraspthecentralpoint in theirchillingnarratives–almost literally.Spectres,poltergeistsandDementorschill, or even freeze, their immediate surroundings, sucking out energy to pay for their unnaturalexistence.

Figure7StructureofalipidmembraneTheoriginalfluid-mosaicmodelofthelipidbilayer,asdepictedbySingerandNicholsonin1972.Proteinsfloat,submergedinaseaoflipids,somepartiallyembedded,andothersextendingacrosstheentiremembrane.Thelipidsthemselvesarecomposedofhydrophilic(water-loving)head-groups,typicallyglycerolphosphate,andhydrophobic(water-hating)tails,generallyfattyacidsinbacteriaandeukaryotes.Themembraneisorganisedasabilayer,withhydrophilicheadsinteractingwiththewaterycontentsofthecytoplasmandthesurroundings,andthehydrophobictailspointinginwardsandinteractingwitheachother.Thisisalow-energy,physically‘comfortable’state:despiteitsorderedappearance,theformationoflipidbilayersactuallyincreasesoverallentropybyreleasingenergyasheatintothesurroundings.

When all this is taken into consideration in the case of the spore, the overall entropy barelychanges. At the molecular level, the structure of polymers minimises energy locally, with excessenergybeing releasedasheat to the surroundings, increasing their entropy.Proteinsnaturally foldintoshapeswiththelowestpossibleenergy.Theirhydrophobicpartsareburiedfarfromwateratthesurface. Electrical charges attract or repel each other: positive charges are fixed in their place bycounterbalancing negative charges, stabilising the three-dimensional structure of the protein. Soproteinsfoldspontaneouslyintoparticularshapes,albeitnotalwaysinahelpfulmanner.Prionsareperfectly normal proteins that spontaneously refold into semicrystalline structures that act as atemplateformorerefoldedprions.Theoverallentropybarelychanges.Theremaybeseveralstablestatesforaprotein,onlyoneofthemusefulforacell;butintermsofentropythereislittledifferencebetween them. Perhaps most surprisingly, there is little difference in overall entropy between adisorderedsoupofindividualaminoacids(thebuildingblocksofproteins)andabeautifullyfoldedprotein.Tounfoldtheproteinreturnsittoastatemoresimilartoasoupofaminoacids,increasingits entropy. But doing so also exposes the hydrophobic amino acids to water, and this physicallyuncomfortable state sucks in energy from outside, decreasing the entropy of the surroundings,coolingthemdown–whatwemightcall the‘poltergeisteffect’.Theideathat lifeisa low-entropystate–thatitismoreorganisedthanasoup–isnotstrictlytrue.Theorderandorganisationoflifeismorethanmatchedbytheincreaseddisorderofthesurroundings.

SowhatwasErwinSchrödingertalkingaboutwhenhesaidthatlife‘sucks’negativeentropyfromits environment, bywhich hemeant that life somehow extracts order from its surroundings.Well,eventhoughabrothofaminoacidsmighthavethesameentropyasaperfectlyfoldedprotein,there

aretwosensesinwhichtheproteinislessprobable,andthereforecostsenergy.First,thebrothofaminoacidswillnotspontaneouslyjointogethertoformachain.Proteinsare

chainsof linkedaminoacids,but theaminoacidsarenot intrinsicallyreactive.Toget themtojointogether,livingcellsneedfirsttoactivatethem.Onlythenwilltheyreactandformintoachain.Thisreleasesroughlythesameamountofenergythatwasusedtoactivatetheminthefirstplace,sotheoverallentropyinfactremainsaboutthesame.Theenergyreleasedastheproteinfoldsitselfislostasheat, increasing theentropyof the surroundings.Andso there is anenergybarrier between twoequivalentlystablestates.Justastheenergybarriermeansitistrickytogetproteinstoform,sotoothereisabarriertotheirdegradation.Ittakessomeeffort(anddigestiveenzymes)tobreakproteinsbackdownintotheircomponentparts.Wemustappreciatethatthetendencyoforganicmoleculestointeractwitheachother,toformlargerstructures,whetherproteins,DNAormembranes,isnomoremysterious than the tendency of large crystals to form in cooling lava. Given enough reactivebuildingblocks,theselargerstructuresarethemoststablestate.Therealquestionis:wheredoallthereactivebuildingblockscomefrom?

That brings us to the second problem.A broth of amino acids, let alone activated ones, is notexactlyprobable in today’senvironmenteither. If left standingaround, itwill eventually reactwithoxygenandreverttoasimplermixtureofgases–carbondioxide,nitrogenandsulphuroxides,andwatervapour. Inotherwords, it takes energy to form these aminoacids in the first place, and thatenergy is releasedwhen they are broken down again. That’swhywe can survive starvation for awhile,bybreakingdowntheproteininourmusclesandusingitasafuel.Thisenergydoesnotcomefrom the protein itself, but from burning up its constituent amino acids. Thus, seeds, spores andvirusesarenotperfectlystableintoday’soxygen-richenvironment.Theircomponentswillreactwithoxygen – oxidise – slowly over time, and that ultimately erodes their structure and function,preventing them from springing back to life in the right conditions. Seeds die. But change theatmosphere, keep oxygen at bay, and they are stable indefinitely.3 Because organisms are ‘out ofequilibrium’withtheoxygenatedglobalenvironment,theywilltendtooxidise,unlesstheprocessisactivelyprevented.(We’llseeinthenextchapterthatthiswasnotalwaysthecase.)

Soundernormalcircumstances(inthepresenceofoxygen)itcostsenergytomakeaminoacidsandotherbiologicalbuildingblocks,suchasnucleotides,fromsimplemoleculeslikecarbondioxideandhydrogen.Anditcostsenergyto join themupinto longchains,polymerssuchasproteinsandDNA, even though there is little change in entropy. That’s what living is all about –making newcomponents, joining them all together, growing, reproducing. Growth also means activelytransportingmaterialsinandoutofthecell.Allofthisrequiresacontinuousfluxofenergy–whatSchrödingerreferredtoas‘freeenergy’.Theequationhehadinmindisaniconicone,whichrelatesentropyandheattofreeenergy.Itissimpleenough:

G= H–T S.

Whatdoesthismean?TheGreeksymbol (delta)signifiesachange. GisthechangeinGibbsfreeenergy,namedafterthegreatreclusivenineteenth-centuryAmericanphysicistJ.WillardGibbs.Itis the energy that is ‘free’ to drive mechanical work such as muscle contraction or anythinghappening in the cell. H is the change in heat,which is released into the surroundings,warmingthemup,andsoincreasingtheirentropy.Areactionthatreleasesheatintothesurroundingsmustcoolthesystemitself,becausethereisnowlessenergyinitthantherewasbeforethereaction.Soifheatisreleasedfromthesystemintothesurroundings, H,whichreferstothesystem,takesanegativesign.T is the temperature. It matters just for context. Releasing a fixed amount of heat into a coolenvironmenthasagreatereffectonthatenvironmentthantheexactsameamountofheatreleasedinto

awarmenvironment–therelativeinputisgreater.Finally, Sisthechangeinentropyofthesystem.This takes a negative sign if the entropy of the systemdecreases, becomingmore ordered, and ispositiveifentropyincreases,withthesystembecomingmorechaotic.

Overall,foranyreactiontotakeplacespontaneouslythefreeenergy, G,mustbenegative.Thisisequallytrueforthetotalsumofallthereactionsthatconstituteliving.That’stosay,areactionwilltakeplaceonitsownaccordonlyif Gisnegative.Forthattobethecase,eithertheentropyofthesystemmustrise(thesystembecomesmoredisordered)orenergymustbelostfromthesystemasheat,orboth.Thismeansthatlocalentropycandecrease–thesystemcanbecomemoreordered–solongas H is evenmorenegative,meaning that a lot of heat is released to the surroundings.Thebottom line is that, to drive growth and reproduction – living! – some reactionmust continuouslyreleaseheatintothesurroundings,makingthemmoredisordered.Justthinkofthestars.Theypayfortheirorderedexistencebyreleasingvastamountsofenergy into theuniverse. Inourowncase,wepayforourcontinuedexistencebyreleasingheatfromtheunceasingreactionthatisrespiration.Wearecontinuouslyburningfoodinoxygen,releasingheat intotheenvironment.Thatheat loss isnotwaste – it is strictly necessary for life to exist. The greater the heat loss, the greater the possiblecomplexity.4

Everything that happens in a living cell is spontaneous, andwill take place on its own accord,giventherightstartingpoint. Gisalwaysnegative.Energetically,it’sdownhillalltheway.Butthismeans that the starting point has to be very high up. To make a protein, the starting point is theimprobableassemblyofenoughactivatedaminoacidsinasmallspace.Theywillthenreleaseenergywhen they join and fold to form proteins, increasing the entropy of the surroundings. Even theactivatedaminoacidswillformspontaneously,givenenoughsuitablyreactiveprecursors.Andthosesuitablyreactiveprecursorswillalsoformspontaneously,givenahighlyreactiveenvironment.Thus,ultimately, the power for growth comes from the reactivity of the environment, which fluxescontinuouslythroughlivingcells(intheformoffoodandoxygeninourcase,photonsoflightinthecaseofplants).Livingcellscouplethiscontinuousenergyfluxtogrowth,overcomingtheirtendencyto break down again. They do so through ingenious structures, in part specified by genes. Butwhateverthosestructuresmaybe(we’llcometothat),theyarethemselvestheoutcomeofgrowthandreplication,naturalselectionandevolution,noneofwhichispossibleintheabsenceofacontinuousenergyfluxfromsomewhereintheenvironment.

ThecuriouslynarrowrangeofbiologicalenergyOrganisms require an extraordinary amount of energy to live. The energy ‘currency’ used by alllivingcellsisamoleculecalledATP,whichstandsforadenosinetriphosphate(butdon’tworryaboutthat).ATPworkslikeacoininaslotmachine.Itpowersoneturnonamachinethatpromptlyshutsdownagainafterwards.InthecaseofATP,the‘machine’istypicallyaprotein.ATPpowersachangefromonestablestatetoanother,likeflippingaswitchfromuptodown.Inthecaseoftheprotein,theswitchisfromonestableconformationtoanother.ToflipitbackagainrequiresanotherATP,justasyouhave to insertanothercoin in theslotmachine tohavea secondgo.Picture thecellasagiantamusementarcade,filledwithproteinmachinery,allpoweredbyATPcoinsinthisway.Asinglecellconsumesaround10millionmoleculesofATPeverysecond!Thenumberisbreathtaking.Thereareabout40trillioncellsinthehumanbody,givingatotalturnoverofATPofaround60–100kilogramsperday–roughlyourownbodyweight.Infact,wecontainonlyabout60gramsofATP,soweknowthateverymoleculeofATPisrechargedonceortwiceaminute.

Recharged?WhenATPis‘split’,itreleasesfreeenergythatpowerstheconformationalchange,aswellasreleasingenoughheattokeep Gnegative.ATPisusuallysplitintotwounequalpieces,ADP

(adenosinediphosphate)andinorganicphosphate(PO43–).Thisisthesamestuffweuseinfertilisers

andisusuallydepictedasPi.ItthencostsenergytoreformATPagainfromADPandPi.Theenergyof respiration– theenergyreleasedfromthereactionof foodwithoxygen– isused tomakeATPfromADPandPi.That’sit.Theendlesscycleisassimpleasthis:

ADP+Pi+energy ATP

Wearenothingspecial.BacteriasuchasE.colicandivideevery20minutes.TofuelitsgrowthE.coliconsumesaround50billionATPspercelldivision,some50–100timeseachcell’smass.That’saboutfourtimesourownrateofATPsynthesis.Convertthesenumbersintopowermeasuredinwattsandtheyarejustasincredible.Weuseabout2milliwattsofenergypergram–orsome130wattsforanaveragepersonweighing65kg,abitmorethanastandard100wattlightbulb.Thatmaynotsoundlikealot,butpergramitisafactorof10,000morethanthesun(onlyatinyfractionofwhich,atanyonemoment,isundergoingnuclearfusion).Lifeisnotmuchlikeacandle;moreofarocketlauncher.

Fromatheoreticalpointofview,then,lifeisnomystery.Itdoesn’tcontraveneanylawsofnature.Theamountofenergythat livingcellsget through,secondbysecond,isastronomical,butthentheamountofenergypouringinupontheearthassunlightismanyordersofmagnitudemore(becausethesunisenormouslylarger,eventhoughithaslesspowerpergram).Solongassomeportionofthisenergyisavailabletodrivebiochemistry,onemightthinkthat lifecouldoperateinalmostanywhich way. As we saw with genetic information in the last chapter, there doesn’t seem to be anyfundamentalconstraintonhowenergyisused,justthattherebeplentyofit.Thatmakesitallthemoresurprising,then,thatlifeonearthturnsouttobeextremelyconstrainedinitsenergetics.

Therearetwoaspectstotheenergyoflifethatareunexpected.First,allcellsderivetheirenergyfromjustoneparticulartypeofchemicalreactionknownasaredoxreaction,inwhichelectronsaretransferredfromonemoleculetoanother.Redoxstandsfor‘reductionandoxidation’.Itissimplythetransferofoneormoreelectronsfromadonortoareceptor.Asthedonorpassesonelectrons,itissaidtobeoxidised.Thisiswhathappenswhensubstancessuchasironreactwithoxygen–theypasselectrons on to oxygen, themselves becoming oxidised to rust. The substance that receives theelectrons,inthiscaseoxygen,issaidtobereduced.Inrespirationorafire,oxygen(O2)isreducedtowater(H2O)becauseeachoxygenatompicksuptwoelectrons(togiveO2–)plustwoprotons,whichbalancethecharges.Thereactionproceedsbecauseitreleasesenergyasheat,increasingentropy.Allchemistryultimatelyincreasestheheatofthesurroundingsandlowerstheenergyofthesystemitself;the reaction of iron or foodwith oxygen does that particularlywell, releasing a large amount ofenergy (as in a fire).Respiration conserves some of the energy released from that reaction in theformofATP,atleastfortheshortperioduntilATPissplitagain.ThatreleasestheremainingenergycontainedintheADP–PibondofATPasheat.Intheend,respirationandburningareequivalent;theslightdelayinthemiddleiswhatweknowaslife.

Becauseelectronsandprotonsareoften(butnotalways)coupledtogetherinthisway,reductionsaresometimesdefinedasthetransferofahydrogenatom.Butreductionsaremucheasiertograspifyou thinkprimarily in termsofelectrons.Asequenceofoxidationandreduction (redox) reactionsamountstothetransferofanelectrondownalinkedchainofcarriers,whichisnotunliketheflowofelectricalcurrentdownawire.Thisiswhathappensinrespiration.Electronsstrippedfromfoodarenotpasseddirectlytooxygen(whichwouldreleasealltheenergyinonego)buttoa‘steppingstone’–typicallyoneofseveralchargedironatoms(Fe3+)embeddedinarespiratoryprotein,oftenaspartof a small inorganic crystal known as an ‘iron–sulphur cluster ’ (see Figure 8). From there theelectron hops to a very similar cluster, but with a slightly higher ‘need’ for the electron. As the

electronisdrawnfromoneclustertothenext,eachoneisfirstreduced(acceptinganelectronsoanFe3+becomesFe2+)and thenoxidised(losing theelectronandreverting toFe3+) in turn.Ultimately,afteraround15ormoresuchhops,theelectronreachesoxygen.Formsofgrowththatatfirstglanceseemtohavelittleincommon,suchasphotosynthesisinplants,andrespirationinanimals,turnouttobe basically the same in that they both involve the transfer of electrons down such ‘respiratorychains’. Why should this be? Life could have been driven by thermal or mechanical energy, orradioactivity,orelectricaldischarges,orUVradiation,theimaginationisthelimit;butno,alllifeisdrivenbyredoxchemistry,viaremarkablysimilarrespiratorychains.

ThesecondunexpectedaspecttotheenergyoflifeisthedetailedmechanismbywhichenergyisconservedinthebondsofATP.Lifedoesn’tuseplainchemistry,butdrivestheformationofATPbytheintermediaryofprotongradientsacrossthinmembranes.We’llcometowhatthatmeans,andhowit is done, in a moment. For now, let’s just recall that this peculiar mechanism was utterlyunanticipated–‘themostcounterintuitiveideainbiologysinceDarwin’,accordingtothemolecularbiologist Leslie Orgel. Today, we know the molecular mechanisms of how proton gradients aregeneratedandtappedinastonishingdetail.Wealsoknowthattheuseofprotongradientsisuniversalacross lifeonearth–protonpower is asmuchpart andparcelof lifeasDNA itself, theuniversalgeneticcode.Yetweknownexttonothingabouthowthiscounterintuitivemechanismofbiologicalenergygenerationevolved.Forwhateverreason,itseemsthatlifeonearthusesastartlinglylimitedandstrangesubsetofpossibleenergeticmechanisms.Does this reflect thequirksofhistory,orarethesemethods somuch better than anything else that they eventually came to dominate?Ormoreintriguingly–couldthisbetheonlyway?

Figure8ComplexIoftherespiratorychainAIron-sulphurclustersarespacedatregulardistancesof14ångströmsorless;electronshopfromoneclustertothenextby‘quantumtunnelling’,withmostfollowingthemainpathofthearrows.Thenumbersgivethedistanceinångströmsfromcentretocentreofeachcluster;thenumbersinbracketsgivethedistancefromedgetoedge.BThewholeofcomplexIinbacteriainLeoSazanov’sbeautifulX-raycrystallographystructure.TheverticalmatrixarmtransferselectronsfromFMN,wheretheyentertherespiratorychain,tocoenzymeQ(alsocalledubiquinone),whichpassesthemontothenextgiantproteincomplex.Youcanjustmakeoutthepathwayofiron-sulphurclustersshowninAburiedwithintheprotein.CMammaliancomplexI,showingthesamecoresubunitsfoundinbacteria,butpartiallyconcealedbeneathanadditional30smallersubunits,depictedindarkshadesinJudyHirst’srevealingelectroncryo-microscopystructure.

Here’swhatishappeninginyourightnow.Takeadizzyingridedownintooneofyourcells,let’ssayaheartmusclecell.ItsrhythmiccontractionsarepoweredbyATP,whichisfloodingoutfromthemanylargemitochondria, thepowerhousesof thecell.Shrinkyourselfdownto thesizeofanATPmolecule,andzoominthroughalargeproteinporeintheexternalmembraneofamitochondrion.We find ourselves in a confined space, like the engine room of a boat, packed with overheatingproteinmachinery,stretchingasfaras theeyecansee.Thegroundisbubblingwithwhat looklikelittleballs,whichshootoutfromthemachines,appearinganddisappearinginmilliseconds.Protons!Thiswholespaceisdancingwiththefleetingapparitionsofprotons,thepositivelychargednucleiofhydrogenatoms.Nowonderyoucanbarelyseethem!Sneakthroughoneofthosemonstrousproteinmachines into the inner bastion, the matrix, and an extraordinary sight greets you. You are in acavernous space, a dizzyingvortexwhere fluidwalls sweeppast you in all directions, all jammedwithgigantic clankingand spinningmachines.Watchyourhead!Thesevastproteincomplexesaresunkdeeply into thewalls, andmovearound sluggishlyas if submerged in the sea.But theirpartsmoveatamazingspeed.Somesweepbackandforth,toofastfortheeyetosee,likethepistonsofasteamengine.Othersspinon theiraxis, threatening todetachandflyoffatanymoment,drivenbypirouettingcrankshafts.Tensofthousandsofthesecrazyperpetualmotionmachinesstretchoffinalldirections,whirringaway,allsoundandfury,signifying…what?

Youareatthethermodynamicepicentreofthecell,thesiteofcellularrespiration,deepwithinthe

mitochondria.Hydrogenisbeingstrippedfromthemolecularremainsofyourfood,andpassedintothefirstandlargestofthesegiantrespiratorycomplexes,complexI.Thisgreatcomplexiscomposedofasmanyas45separateproteins,eachoneachainofseveralhundredaminoacids.Ifyou,anATP,wereasbigasaperson,complexIisaskyscraper.Butnoordinaryskyscraper–adynamicmachineoperatinglikeasteamengine,aterrifyingcontraptionwithalifeofitsown.Electronsareseparatedfromprotonsandfedintothisvastcomplex,suckedinatoneendandspatoutoftheother,allthewayoverthere,deepinthemembraneitself.Fromtheretheelectronspassthroughtwomoregiantproteincomplexes,whichtogethercomprisetherespiratorychain.Eachindividualcomplexcontainsmultiple‘redox centres’ – about nine of them in complex I – that transiently hold an electron (Figure 8).Electronshop fromcentre tocentre. In fact, the regular spacingof thesecentres suggests that they‘tunnel’ by some formof quantummagic, appearing and disappearing fleetingly, according to therulesofquantumprobability.Allthattheelectronscanseeisthenextredoxcentre,solongasitisnotfaraway.Distancehereismeasuredinångströms(Å),roughlythesizeofanatom.5Solongaseachredoxcentreisspacedwithinabout14Åofthenext,andeachonehasaslightlystrongeraffinityforanelectronthanthelast,electronswillhopondownthispathwayofredoxcentres,asifcrossingariveronniceregularlyspacedsteppingstones.Theypassstraightthroughthethreegiantrespiratorycomplexes,butdon’tnoticethemanymorethanyouneedtonoticetheriver.Theyaredrawnonwardsbythepowerfultugofoxygen,itsvoraciouschemicalappetiteforelectrons.Thisisnotactionatadistance–itisallabouttheprobabilityofanelectronbeingonoxygenratherthansomewhereelse.Itamountstoawire,insulatedbyproteinsandlipids,channellingthecurrentofelectronsfrom‘food’tooxygen.Welcometotherespiratorychain!

The electrical current animates everything here. The electrons hop along their path, interestedonlyintheirroutetooxygen,andoblivioustotheclankingmachinesclingingtothelandscapelikepumpjackoilwells.Butthegiantproteincomplexesarefulloftripswitches.Ifanelectronsitsinaredox centre, the adjoining protein has a particular structure. When that electron moves on, thestructure shifts a fraction, a negative charge readjusts itself, a positive charge follows suit,wholenetworksofweakbondsrecalibratethemselves,andthegreatedificeswingsintoanewconformationinatinyfractionofasecond.Smallchangesinoneplaceopencavernouschannelselsewhereintheprotein.Thenanotherelectronarrives, and theentiremachine swingsback to its former state.Theprocess is repeated tensof times a second.Agreatdeal is knownnowabout the structureof theserespiratorycomplexes,downtoaresolutionofjustafewångströms,nearlythelevelofatoms.Weknow how protons bind to immobilised water molecules, themselves pinioned in their place bycharges on the protein.We know how these water molecules shift when the channels reconfigurethemselves.Weknowhowprotonsarepassedfromonewatermoleculetoanotherthroughdynamicclefts,openingandclosinginswiftsuccession,aperilousroutethroughtheproteinthatslamsclosedinstantlyafterthepassageoftheproton,preventingitsretreatasifinanIndianaJonesadventure,theProteinsofDoom.Thisvast,elaborate,mobilemachineryachievesjustonething:ittransfersprotonsfromonesideofthemembranetotheother.

For each pair of electrons that passes through the first complex of the respiratory chain, fourprotons cross the membrane. The electron pair then passes directly into the second complex(technicallycomplexIII;complexIIisanalternativeentrypoint),whichconveysfourmoreprotonsacross the barrier. Finally, in the last great respiratory complex, the electrons find their Nirvana(oxygen),butnotbeforeanothertwoprotonshavebeenshuttledacrossthemembrane.Foreachpairofelectronsstrippedfromfood,tenprotonsareferriedacrossthemembrane.Andthat’sit(Figure9).A little less than half the energy released by flow of electrons to oxygen is saved in the protongradient.All that power, all that ingenuity, all thevast protein structures, all of that is dedicated topumping protons across the inner mitochondrial membrane. One mitochondrion contains tens of

thousands of copies of each respiratory complex.A single cell contains hundreds or thousands ofmitochondria. Your 40 trillion cells contain at least a quadrillion mitochondria, with a combinedconvoluted surface area of about 14,000 squaremetres; about four football fields. Their job is topumpprotons,andtogethertheypumpmorethan1021ofthem–nearlyasmanyastherearestarsintheknownuniverse–everysecond.

Well, that’s half their job. The other half is to bleed off that power to make ATP.6 Themitochondrial membrane is very nearly impermeable to protons – that is the point of all thesedynamicchannelsthatslamshutassoonastheprotonhaspassedthrough.Protonsaretiny–justthenucleusofthesmallestatom,thehydrogenatom–soitisnomeanfeattokeepthemout.Protonspassthroughwatermoreorlessinstantaneously,sothemembranemustbecompletelysealedofftowaterinallplacesaswell.Protonsarealsocharged;theycarryasinglepositivecharge.Pumpingprotonsacross a sealed membrane achieves two things: first, it generates a difference in the protonconcentration between the two sides; and second, it produces a difference in electrical charge, theoutside being positive relative to the inside. That means there is an electrochemical potentialdifferenceacrossthemembrane,intheorderof150to200millivolts.Becausethemembraneisverythin(around6nmthick)thischargeisextremelyintenseacrossashortdistance.Shrinkyourselfbackdown to the size of an ATP molecule again, and the intensity of the electric field you wouldexperienceinthevicinityofthemembrane–thefieldstrength–is30millionvoltspermetre,equaltoaboltoflightning,orathousandtimesthecapacityofnormalhouseholdwiring.

Figure9HowmitochondriaworkAElectronmicrographofmitochondria,showingtheconvolutedinnermembranes(cristae)whererespirationtakesplace.BAcartoonoftherespiratorychain,depictingthethreemajorproteincomplexesembeddedintheinnermembrane.Electrons(e–)enterfromtheleftandpassthroughthreelargeproteincomplexestooxygen.ThefirstiscomplexI(seeFigure8foramorerealisticdepiction);electronsthenpassthroughcomplexIIIandIV.ComplexII(notshown)isaseparateentrypointintotherespiratorychain,andpasseselectronsstraighttocomplexIII.Thesmallcirclewithinthemembraneisubiquinone,whichshuttleselectronsfromcomplexesIandIItoIII;theproteinlooselyboundtothemembranesurfaceiscytochromec,whichshuttleselectronsfromcomplexIIItoIV.Thecurrentofelectronstooxygenisdepictedbythearrow.Thiscurrentpowerstheextrusionofprotons(H+)throughthethreerespiratorycomplexes(complexIIpassesonelectronsbutdoesnotpumpprotons).Foreachpairofelectronsthatpassesdownthechain,fourprotonsarepumpedatcomplexI,fouratcomplexIIIandtwoatcomplexIV.ThefluxofprotonsbackthroughtheATPsynthase(shownattheright)drivesthesynthesisofATPfromADPandPi.

This huge electrical potential, known as the proton-motive force, drives the most impressiveproteinnanomachineofthemall,theATPsynthase(Figure10).MotiveimpliesmotionandtheATPsynthase is indeed a rotarymotor, inwhich the flowof protons turns a crank shaft,which in turnrotatesacatalytichead.ThesemechanicalforcesdrivethesynthesisofATP.Theproteinworkslikeahydroelectric turbine,wherebyprotons,pentup ina reservoirbehind thebarrierof themembrane,flood through the turbine likewater cascadingdownhill, turning the rotatingmotor.This isbarelypoetic licencebut a precise description, yet it is hard to convey the astonishing complexity of thisproteinmotor.Westilldon’tknowexactlyhowitworks–howeachprotonbindson to theC-ringwithin the membrane, how electrostatic interactions spin this ring in one direction only, how thespinning ring twists the crank shaft, forcing conformational changes in the catalytichead, how thecleftsthatopenandcloseinthisheadclaspADPandPiandforcethemtogetherinmechanicalunion,topressanewATP.Thisisprecisionnanoengineeringofthehighestorder,amagicaldevice,andthemorewe learn about it themoremarvellous it becomes.Some see in it proof for the existenceofGod.Idon’t.Iseethewonderofnaturalselection.Butitisundoubtedlyawondrousmachine.

ForeverytenprotonsthatpassthroughtheATPsynthase,therotatingheadmakesonecompleteturn,andthreenewlymintedATPmoleculesarereleasedintothematrix.Theheadcanspinatoverahundredrevolutionspersecond.ImentionedthatATPiscalledtheuniversalenergy‘currency’oflife.TheATPsynthaseandtheproton-motiveforcearealsoconserveduniversallyacrosslife.AndImeanuniversally.TheATPsynthase is foundinbasicallyallbacteria,allarchaea,andalleukaryotes(thethreedomainsof lifewediscussed in thepreviouschapter),barringahandfulofbugs that relyonfermentationinstead.Itisasuniversalasthegeneticcodeitself.Inmybook,theATPsynthaseshouldbeassymbolicoflifeasthedoublehelixofDNA.Nowthatyoumentionit,thisismybook,anditis.

Figure10StructureoftheATPsynthaseTheATPsynthaseisaremarkablerotatingmotorembeddedinthemembrane(bottom).ThisbeautifulartisticrenditionbyDavidGoodsellistoscale,andshowsthesizeofanATPandevenprotonsrelativetothemembraneandtheproteinitself.Thefluxofprotonsthroughamembranesubunit(openarrow)drivestherotationofthestripedFOmotorinthemembrane,aswellasthedriveshaft(stalk)attachedabove(turningblackarrow).Therotationofthedriveshaftforcesconformationalchangesinthecatalytichead(F1subunit),drivingthesynthesisofATPfromADPandphosphate.Theheaditselfispreventedfromrotatingbythe‘stator’–therigidsticktotheleft–whichfixesthecatalyticheadinposition.Protonsareshownbelowthemembraneboundtowaterashydroniumions(H3O

+).

AcentralpuzzleinbiologyTheconceptoftheproton-motiveforcecamefromoneofthemostquietlyrevolutionaryscientistsofthetwentiethcentury,PeterMitchell.Quietonlybecausehisdiscipline,bioenergetics,was(andstillis)somethingofabackwaterinaresearchworldentrancedbyDNA.Thatfascinationbeganintheearly1950swithCrickandWatsoninCambridge,whereMitchellwasanexactcontemporary.Mitchell,too,wentontowintheNobelPrize,in1978,buthisideaswerefarmoretraumaticinthemaking.Unlike

thedoublehelix,whichWatsonimmediatelydeclaredtobe‘soprettyithastobetrue’–andhewasright – Mitchell’s ideas were extremely counterintuitive. Mitchell himself was irascible,argumentativeandbrilliantby turns.Hewasobliged to retirewithstomachulcers fromEdinburghUniversity in theearly1960s,soonafter introducinghis‘chemiosmotichypothesis’ in1961(whichwaspublished inNature, likeCrickandWatson’smore famousearlier treatise). ‘Chemiosmotic’ isthe termMitchellused to refer to the transferofprotonsacrossamembrane.Characteristically,heused theword ‘osmotic’ in its originalGreek sense,meaning ‘to push’ (not in themore familiarusage of osmosis, the passage of water across a semipermeable membrane). Respiration pushesprotonsacrossathinmembrane,againstaconcentrationgradient,andhenceischemiosmotic.

Withprivatemeansandapracticalbent,MitchellspenttwoyearsrefurbishingamanorhousenearBodmininCornwallasalabandhome,andopenedtheGlynnInstitutetherein1965.Forthenexttwodecades, he and a small number of other leading figures in bioenergetics set about testing thechemiosmotic hypothesis to destruction. The relationships between them took a similar battering.Thisperiodhasgonedown in the annalsofbiochemistryas the ‘oxphoswars’– ‘oxphos’beingshort for ‘oxidative phosphorylation’, themechanismbywhich the flowof electrons to oxygen iscoupledtothesynthesisofATP.It’shardtoappreciatethatnoneofthedetailsIhavegiveninthelastfewpageswereknownasrecentlyasthe1970s.Manyofthemarestillthefocusofactiveresearch.7

WhywereMitchell’sideassohardtoaccept?Inpartbecausetheyweresogenuinelyunexpected.ThestructureofDNAmakesperfectsense–thetwostrandseachactasatemplatefortheother,andthe sequence of letters encodes the sequence of amino acids in a protein. The chemiosmotichypothesis, incomparison, seemedquirky in theextreme, andMitchellhimselfmight aswellhavebeentalkingMartian.Lifeisaboutchemistry,weallknowthat.ATPisformedfromthereactionofADP and phosphate, so all thatwas neededwas the transfer of one phosphate from some reactiveintermediateontoADP.Cellsarefilledwithreactiveintermediates,soitwasjustacaseoffindingtherightone.Orsoitseemedforseveraldecades.ThenalongcameMitchellwithamadglintinhiseye,plainly an obsessive, writing out equations that nobody could understand, and declaring thatrespirationwas not about chemistry at all, that the reactive intermediatewhich everyone had beensearchingfordidnotevenexist,andthatthemechanismcouplingelectronflowtoATPsynthesiswasactually a gradient of protons across an impermeable membrane, the proton-motive force. Nowonderhemadepeoplecross!

Thisisthestuffoflegend:aniceexampleofhowscienceworksinunexpectedways,toutedasa‘paradigmshift’inbiologysupportingThomasKuhn’sviewofscientificrevolutions,butnowsafelyconfinedtothehistorybooks.Thedetailshavebeenworkedoutatatomicresolution,culminatinginJohnWalker ’sNobelPrizein1997forthestructureoftheATPsynthase.ResolvingthestructureofcomplexIisaneventallerorder,butoutsidersmightbeforgivenforthinkingthatthesearedetails,andthatbioenergeticsisnolongerhidinganyrevolutionarydiscoveriestocomparewithMitchell’sown.That’sironicbecauseMitchellarrivedathisradicalviewofbioenergeticsnotbythinkingaboutthedetailedmechanismofrespirationitself,butamuchsimplerandmoreprofoundquestion–howdo cells (he had bacteria in mind) keep their insides different from the outside? From the verybeginning, he saw organisms and their environment as intimately and inextricably linked throughmembranes, a view which is central to this whole book. He appreciated the importance of theseprocessestotheoriginandexistenceoflifeinawaythatveryfewothershavedonesince.Considerthispassagefromalecturethathegaveontheoriginoflifein1957,atameetinginMoscow,fouryearsbeforepublishinghischemiosmotichypothesis:

Icannotconsidertheorganismwithoutitsenvironment…Fromaformalpointofviewthetwomayberegardedasequivalentphasesbetweenwhichdynamiccontactismaintainedbythemembranesthatseparateandlinkthem.

ThislineofMitchell’sthinkingismorephilosophicalthanthenutsandboltsofthechemiosmotichypothesis,whichgrewfromit,but I think it isequallyprescient.Ourmodern focusonmolecularbiologymeanswe have all but forgottenMitchell’s preoccupationwithmembranes as a necessarylinkbetween inside andoutside,withwhatMitchell called ‘vectorial chemistry’– chemistrywith adirectioninspace,wherepositionandstructurematter.Nottest-tubechemistry,whereeverythingismixedinsolution.Essentiallyalllifeusesredoxchemistrytogenerateagradientofprotonsacrossamembrane.Whyonearthdowedothat?Iftheseideasseemlessoutrageousnowthantheydidinthe1960s, that is only because we have lived with them for 50 years, and familiarity breeds, if notcontempt,atleastdwindlinginterest.Theyhavecollecteddustandsettledintotextbooks,ne’ertobequestionedagain.Wenowknowthattheseideasaretrue;butareweanyclosertoknowingwhytheyare true? The question boils down to two parts: why do all living cells use redox chemistry as asourceof free energy?Andwhydoall cells conserve this energy in the formofprotongradientsovermembranes?Atamorefundamentallevel,thesequestionsare:whyelectrons,andwhyprotons?

LifeisallaboutelectronsSowhydoeslifeonearthuseredoxchemistry?Perhapsthisistheeasiestparttoanswer.Lifeasweknowitisbasedoncarbon,andspecificallyonpartiallyreducedformsofcarbon.Toanabsurdfirstapproximation(puttingasidetherequirementforrelativelysmallamountsofnitrogen,phosphorus,andotherelements),a‘formula’forlifeisCH2O.Giventhestartingpointofcarbondioxide(moreon this in the next chapter), then life must involve the transfer of electrons and protons fromsomething likehydrogen(H2)on toCO2. Itdoesn’tmatter inprinciplewhere thoseelectronscomefrom– theycouldbesnatched fromwater (H2O)orhydrogensulphide(H2S)oreven ferrous iron(Fe2+). The point is they are transferred on to CO2, and all such transfers are redox chemistry.‘Partiallyreduced’,incidentally,meansthatCO2isnotreducedcompletelytomethane(CH4).

Could lifehaveusedsomethingother thancarbon?Nodoubt it isconceivable.Weare familiarwith robotsmadefrommetalorsilicon,sowhat is specialaboutcarbon?Quitea lot, in fact.Eachcarbon atom can form four strong bonds, much stronger than the bonds formed by its chemicalneighbour silicon. These bonds allow an extraordinary variety of long-chain molecules, notablyproteins,lipids,sugarsandDNA.Siliconcan’tmanageanythinglikethiswealthofchemistry.What’smore,therearenogaseoussiliconoxidestocomparewithcarbondioxide.IimagineCO2asakindofaLegobrick.Itcanbepluckedfromtheairandaddedonecarbonatatimeontoothermolecules.Silicon oxides in contrast…well, you try buildingwith sand. Silicon or other elementsmight beamenabletousebyahigherintelligencesuchasourselves,butitishardtoseehowlifecouldhavebootstrappeditselffromthebottomupusingsilicon.That’snottosaythatsilicon-basedlifecouldn’tpossibly evolve in an infinite universe, who could say; but as a matter of probability andpredictability,which iswhat thisbook is about, that seemsoverwhelmingly less likely.Apart frombeingmuchbetter,carbonisalsomuchmoreabundantacrosstheuniverse.Toafirstapproximation,then,lifeshouldbecarbonbased.

But the requirement for partially reduced carbon is only a small part of the answer. In mostmodern organisms, carbon metabolism is quite separate from energy metabolism. The two areconnected byATP and a handful of other reactive intermediates such as thioesters (notably acetylCoA) but there is no fundamental requirement for these reactive intermediates to be produced byredox chemistry. A few organisms survive by fermentation, though this is neither ancient norimpressive in yield. But there is no shortage of ingenious suggestions about possible chemicalstartingpointsforlife,oneofthemostpopular(andperverse)beingcyanide,whichcouldbeformed

bytheactionofUVradiationongasessuchasnitrogenandmethane.Isthatfeasible?Imentionedinthelastchapterthatthereisnohintfromzirconsthattheearlyatmospherecontainedmuchmethane.Thatdoesn’tmeanitcouldn’thappeninprincipleonanotherplanet,though.Andifit’spossible,whyshouldn’t itpower life today?We’ll return to this in thenextchapter. I think it’sunlikely forotherreasons.

Consider the problem the other way around: what is good about the redox chemistry ofrespiration? Plenty, it seems.When I say respiration,we need to look beyond ourselves.We stripelectronsfromfoodandtransferthemdownourrespiratorychainstooxygen,butthecriticalpointhere is that thesourceand thesinkofelectronscanbothbechanged. It sohappens thatburningupfood in oxygen is as good as it gets in terms of energy yield, but the underlying principle isenormouslywiderandmoreversatile.Thereisnoneedtoeatorganicmatter,forexample.Hydrogengas,hydrogensulphideand ferrous ironareallelectrondonors,aswe’vealreadynoted.Theycanpass theirelectrons intoa respiratorychain, so longas theacceptorat theotherend isapowerfulenoughoxidanttopullthemthrough.Thatmeansbacteriacan‘eat’rocksormineralsorgases,usingbasicallythesameproteinequipmentthatweuseinrespiration.Nexttimeyouseeadiscolorationinaconcretewall,betrayingathrivingbacterialcolony,considerforamomentthat,howeveralientheymayseem,theyarelivingbyusingthesamebasicapparatusasyou.

There’snorequirementforoxygeneither.Lotsofotheroxidantscandothe jobnearlyaswell,suchasnitrateornitrite,sulphateorsulphite.Thelistgoesonandon.All theseoxidants(socalledbecausetheybehavealittlelikeoxygen)cansuckupelectronsfromfoodorothersources.Ineachcase,thetransferofelectronsfromanelectrondonortoanacceptorreleasesenergythatisstoredinthebondsofATP.Aninventoryofallknownelectrondonorsandelectronacceptorsusedbybacteriaandarchaea–so-called‘redoxcouples’–wouldextendoverseveralpages.Notonlydobacteria‘eat’rocks,buttheycan‘breathe’themtoo.Eukaryoticcellsarepatheticincomparison.Thereisaboutthesame metabolic versatility in the entire eukaryotic domain – all plants, animals, algae, fungi andprotists–asthereisinasinglebacterialcell.

Thisversatilityintheuseofelectrondonorsandacceptorsisaidedbythesluggishreactivityofmany of them. We noted earlier that all biochemistry occurs spontaneously, and must always bedrivenbyahighlyreactiveenvironment;butiftheenvironmentistooreactive,thenitwillgorightaheadandreact,andtherewillbenofreeenergyleftover topowerbiology.Anatmospherecouldnever be full of fluorine gas, for example, as it would immediately react with everything anddisappear. But many substances accumulate to levels that far exceed their natural thermodynamicequilibrium, because they react very slowly. If given a chance, oxygenwill react vigorouslywithorganicmatter, burning everything on the planet, but this propensity to violence is tempered by aluckychemicalquirkthatmakesitstableoveraeons.Gasessuchasmethaneandhydrogenwillreactevenmore vigorouslywith oxygen – just think of theHindenburg airship – but again, the kineticbarriertotheirreactionmeansthatallthesegasescancoexistintheairforyearsatatime,indynamicdisequilibrium.Thesameappliestomanyothersubstances,fromhydrogensulphidetonitrate.Theycan be coerced into reacting, andwhen they do they release a large amount of energy that can beharnessedby livingcells;butwithout the rightcatalysts,nothingmuchhappens.Lifeexploits thesekineticbarriers, and in sodoing increasesentropy faster thanwouldotherwisehappen.Someevendefine life in these terms, as anentropygenerator.Regardless: life existspreciselybecausekineticbarriers exist – it specialises tobreak themdown.Without the loopholeofgreat reactivitypentupbehindkineticbarriers,it’sdoubtfulthatlifecouldexistatall.

Thefactthatmanyelectrondonorsandacceptorsarebothsolubleandstable,enteringandexitingcells without much ado, means that the reactive environment required by thermodynamics can bebroughtsafelyinside,rightintothosecriticalmembranes.Thatmakesredoxchemistrymucheasier

todealwiththanheatormechanicalenergy,orUVradiationorlightning,asaformofbiologicallyusefulenergyflux.HealthandSafetywouldapprove.

Perhapsunexpectedly,respirationisalsothebasisofphotosynthesis.Recallthatthereareseveraltypesofphotosynthesis. Ineachcase, theenergyofsunlight (asphotons) isabsorbedbyapigment(usuallychlorophyll)whichexcitesanelectron,sending itoffdownachainof redoxcentres toanacceptor,inthiscasecarbondioxideitself.Thepigment,bereftofanelectron,acceptsonegratefullyfromthenearestdonor,whichcouldbewater,hydrogensulphide,orferrousiron.Asinrespiration,theidentityoftheelectrondonordoesn’tmatterinprinciple.‘Anoxygenic’formsofphotosynthesisusehydrogensulphideorironaselectrondonors,leavingbehindbrimstoneorrustyirondepositsaswaste.8Oxygenicphotosynthesisusesamuch tougherdonor,water, releasingoxygengasaswaste.Butthepointisthatallofthesedifferenttypesofphotosynthesisobviouslyderivefromrespiration.They use exactly the same respiratory proteins, the same types of redox centre, the same protongradientsovermembranes,thesameATPsynthase–allthesamekit.9Theonlyrealdifferenceistheinnovationofapigment,chlorophyll,whichisinanycasecloselyrelatedtothepigmenthaem,usedinmanyancient respiratoryproteins.Tapping into theenergyof the sunchanged theworld,but inmoleculartermsallitdidwassetelectronsflowingfasterdownrespiratorychains.

Thegreatadvantageofrespiration,then,isitsimmenseversatility.Essentiallyanyredoxcouple(any pair of electron donor and electron acceptor) can be used to set electrons flowing downrespiratorychains.Thespecificproteinsthatpickupelectronsfromammoniumareslightlydifferentfromthosethatpickupelectronsfromhydrogensulphide,buttheyareverycloselyrelatedvariationsonatheme.Likewise,attheotherendoftherespiratorychain,theproteinsthatpasselectronsontonitrate or nitrite differ from those that pass electrons on to oxygen, but all of them are related.They’re sufficiently similar to each other that one can be used in place of another. Because theseproteins are plugged into a common operating system, they can bemixed andmatched to fit anyenvironment.Theyarenotonly interchangeable inprinciple, but inpractice they’repassedaroundwith abandon.Over the past fewdecades,we’ve come to realise that lateral gene transfer (passingaround littlecassettesofgenes fromonecell toanother,as if sparechange) is rife inbacteriaandarchaea.Genesencoding respiratoryproteinsareamong thosemostcommonlyswappedby lateraltransfer. Together, they comprise what biochemist Wolfgang Nitschke calls the ‘redox proteinconstructionkit’.Didyoujustmovetoanenvironmentwherehydrogensulphideandoxygenarebothcommon,suchasadeep-seavent?Noproblem,helpyourselftotherequisitegenes,they’llworkjustfineforyou,sir.You’verunoutofoxygen?Trynitrite,ma’am!Don’tworry.Takeacopyofnitritereductaseandplugitin,you’llbefine!

Allthesefactorsmeanthatredoxchemistryshouldbeimportantforlifeelsewhereintheuniversetoo.Whilewecouldimagineotherformsofpower, therequirementforredoxchemistrytoreducecarbon,combinedwiththemanyadvantagesofrespiration,meansitishardlysurprisingthatlifeonearthisredoxpowered.Buttheactualmechanismofrespiration,protongradientsovermembranes,is anothermatter altogether.The fact that respiratoryproteins canbepassed roundby lateral genetransfer,andmixedandmatchedtoworkinanyenvironment,islargelydowntothefactthatthereisacommon operating system – chemiosmotic coupling. Yet there is no obvious reason why redoxchemistryshouldinvolveprotongradients.ThatlackofanintelligibleconnectionpartlyexplainstheresistancetoMitchell’sideas,andtheoxphoswars,all thoseyearsago.Overthepast50years,wehavelearnedalotabouthowlifeusesprotons;butuntilweknowwhylifeusesprotons,wewillnotbeabletopredictmuchelseaboutthepropertiesoflifehereoranywhereelseintheuniverse.

Lifeisallaboutprotons

Theevolutionofchemiosmoticcouplingisamystery.Thefactthatalllifeischemiosmoticimpliesthatchemiosmoticcouplingaroseveryearlyindeedinevolution.Haditarisenlateron,itwouldbedifficulttoexplainhowandwhyitbecameuniversal–whyprotongradientsdisplacedeverythingelsecompletely.Suchuniversalityissurprisinglyrare.Alllifesharesthegeneticcode(again,withafewminor exceptions, which prove the rule). Some fundamental informational processes are alsouniversallyconserved.Forexample,DNAistranscribedintoRNA,whichisphysicallytranslatedintoproteinsonnanomachinescalled ribosomes inall livingcells.But thedifferencesbetweenarchaeaandbacteria are really shocking.Recall that thebacteria andarchaeaare the twogreatdomainsofprokaryotes,cellsthatlackanucleusandindeedmostoftheparaphernaliaofcomplex(eukaryotic)cells.Intheirphysicalappearance,bacteriaandarchaeaarevirtuallyindistinguishable;butinmuchoftheirbiochemistryandgenetics,thetwodomainsareradicallydifferent.

TakeDNAreplication,whichwemightguesswouldbeasfundamentaltolifeasthegeneticcode.YetthedetailedmechanismsofDNAreplication,includingalmostalltheenzymesneeded,turnouttobetotallydifferentinbacteriaandarchaea.Likewise,thecellwall,therigidouterlayerthatprotectsthe flimsy cell inside, is chemically completely different in bacteria and archaea. So are thebiochemical pathways of fermentation. Even the cell membranes – strictly necessary forchemiosmoticcoupling,otherwiseknownasmembranebioenergetics–arebiochemicallydifferentinbacteria and archaea. In other words, the barriers between the inside and outside of cells and thereplication of hereditarymaterial are not deeply conserved.What could bemore important to thelivesofcellsthanthese!Inthefaceofallthatdivergence,chemiosmoticcouplingisuniversal.

These are profoundly deep differences, and lead to sobering questions about the commonancestorofbothgroups.Assumingthattraitsincommonwereinheritedfromasharedancestor,buttraitsthatdifferaroseindependentlyinthetwolines,whatmannerofacellcouldthatancestorhavebeen? Itdefies logic.At facevalue itwasaphantomofa cell, in someways likemoderncells, inotherways…well,whatexactly?IthadDNAtranscription,ribosomaltranslation,anATPsynthase,bitsandpiecesofaminoacidbiosynthesis,butbeyondthat,littleelsethatisconservedinbothgroups.

Consider themembrane problem.Membrane bioenergetics are universal – butmembranes arenot. One might imagine that the last common ancestor had a bacterial-type membrane, and thatarchaea replaced it for some adaptive reason, perhaps because archaeal membranes are better athigher temperatures. That is superficially plausible, but there are two big problems. First, mostarchaeaarenothyperthermophiles;manymoreliveintemperateconditions,inwhicharchaeallipidsoffer no obvious advantage; and conversely plenty of bacteria live happily in hot springs. Theirmembranes cope perfectly well with high temperatures. Bacteria and archaea live alongside eachotherinalmostallenvironments,frequentlyinveryclosesymbioses.Whywouldoneofthesegroupshavegonetotheserioustroubleofreplacingalltheirmembranelipids,onjustoneoccasion?Ifitispossibletoswitchmembranes,thenwhydon’tweseethewholesalereplacementofmembranelipidsonotheroccasions,ascellsadapt tonewenvironments?Thatshouldbemucheasier thaninventingnewonesfromscratch.Whydon’tsomebacterialivinginhotspringsacquirearchaeallipids?

Second,andmoretelling,amajordistinctionbetweenbacterialandarchaealmembranesseemstobepurelyrandom–bacteriauseonestereoisomer(mirrorform)ofglycerol,whilearchaeausetheother.10 Even if archaea really did replace all their lipids because theywere better adapted to hightemperatures, there isnoconceivableselectivereasontoreplaceglycerolwithglycerol.That’s justperverse.Yettheenzymethatmakestheleft-handedformofglycerolisnotevenremotelyrelatedtotheenzymethatmakestheright-handedtype.Toswitchfromoneisomertotheotherwouldrequirethe‘invention’ofanewenzyme(tomakethenewisomer)followedbythesystematiceliminationoftheold(butfullyfunctional)enzymeineachandeverycell,eventhoughthenewversionofferednoevolutionaryadvantage.Ijustdon’tbuythat.Butifonetypeoflipidwasnotphysicallyreplacedwith

another, thenwhat kind ofmembrane did the last common ancestor actually possess? Itmust havebeenverydifferentfromallmodernmembranes.Why?

Therearealsochallengingproblemswiththeideathatchemiosmoticcouplingaroseveryearlyinevolution.One is the sheer sophisticationof themechanism.Wehave alreadypaidourdues to thegiantrespiratorycomplexesandtheATPsynthase–incrediblemolecularmachineswithpistonsandrotarymotors.Couldthesereallybeaproductoftheearliestdaysofevolution,beforetheadventofDNAreplication?Surelynot!But that’sapurelyemotionalresponse.TheATPsynthase isnomorecomplex than a ribosome, and everyone is agreed that ribosomeshad to evolve early.The secondproblem is themembrane itself.Evenputting aside the question ofwhat type ofmembrane itwas,there is again the issueof disturbing early sophistication. Inmodern cells, chemiosmotic couplingonlyworks if themembrane is almost impermeable to protons.But all experimentswith plausibleearly membranes suggest that they would have been highly permeable to protons. It’s extremelydifficult to keep them out. The problem is that chemiosmotic coupling looks to be useless until anumberofsophisticatedproteinshavebeenembeddedinaproton-tightmembrane;andthen,butonlythen, does it serve a purpose. So how on earth did all the parts evolve in advance? It’s a classicchickenandeggproblem.What’sthepointoflearningtopumpprotonsifyouhavenowaytotapthegradient?Andwhat’sthepointoflearningtotapagradient,ifyouhavenowayofgeneratingone?I’llputforwardapossibleresolutioninChapter4.

Iclosedthefirstchapterwithsomebigquestionsabouttheevolutionoflifeonearth.Whydidlifearisesoearly?Whydiditstagnateinmorphologicalcomplexityforseveralbillionyears?Whydidcomplex,eukaryotic,cellsarisejustoncein4billionyears?Whydoalleukaryotesshareanumberofperplexing traits thatareneverfoundinbacteriaorarchaea, fromsexand twosexes toageing?HereIamaddingtwomorequestionsofanequallyunsettlingmagnitude:whydoesalllifeconserveenergyintheformofprotongradientsacrossmembranes?Andhow(andwhen)didthispeculiarbutfundamentalprocessevolve?

Ithinkthetwosetsofquestionsarelinked.Inthisbook,Iwillarguethatnaturalprotongradientsdrovetheoriginoflifeonearthinaveryparticularenvironment,butanenvironmentthatisalmostcertainlyubiquitousacrossthecosmos:theshoppinglistisjustrock,waterandCO2.Iwillarguethatchemiosmoticcouplingconstrained theevolutionof lifeonearth to thecomplexityofbacteriaandarchaeaforbillionsofyears.Asingularevent,inwhichonebacteriumsomehowgotinsideanotherone, overcame these endless energetic constraints on bacteria. That endosymbiosis gave rise toeukaryoteswithgenomesthatswelledoverordersofmagnitude,therawmaterialformorphologicalcomplexity.Theintimaterelationshipbetweenthehostcellanditsendosymbionts(whichwentontobecome mitochondria) was, I shall argue, behind many strange properties shared by eukaryotes.Evolutionshouldtendtoplayoutalongsimilarlines,guidedbysimilarconstraints,elsewhereintheuniverse.IfIamright(andIdon’tforamomentthinkIwillbeinallthedetails,butIhopethatthebiggerpictureiscorrect)thenthesearethebeginningsofamorepredictivebiology.Onedayitmaybepossibletopredictthepropertiesoflifeanywhereintheuniversefromthechemicalcompositionofthecosmos.

Footnotes1Theyalsoneedmineralssuchasnitrateandphosphate,ofcourse.Manycyanobacteria(thebacterialprecursorsofplants’photosyntheticorganelles,thechloroplasts)canfixnitrogen,whichistosay,theycanconverttherelativelyinertnitrogengas(N2)intheairintothemoreactiveandusableformammonia.Plantshavelostthisability,andrelyonthelargesseoftheirenvironment,sometimesintheformofsymbioticbacteriaintherootnodulesofleguminousplants,whichprovidetheiractivenitrogen.Withoutthatextraneousbiochemicalmachinery,plants,likeviruses,couldnotgroworreproduce.Parasites!2Somethingsimilarhappenswhenastarforms:here,thephysicalforceofgravitationactingbetweenmatteroffsetsthelocallossof

disorder,butthehugereleaseofheatproducedbynuclearfusionincreasesdisorderelsewhereinthesolarsystemandtheuniverse.3AmorehumanexampleistheVasa,aformidableseventeenth-centurySwedishwarshipthatsankinthebayoutsideStockholmonitsmaidenvoyagein1628,andwassalvagedin1961.IthadbeenwonderfullypreservedasthegrowingcityofStockholmpoureditssewageintothemarinebasin.Itwasliterallypreservedinshit,withthesewergashydrogensulphidepreventingoxygenfromattackingtheship’sexquisitewoodencarving.Eversinceraisingtheship,ithasbeenafighttokeepitintact.4That’saninterestingpointintermsoftheevolutionofendothermy,orhotblood.Whilethereisnonecessaryconnectionbetweenthegreaterheatlossofendothermsandgreatercomplexity,itisnonethelesstruethatgreatercomplexitymustultimatelybepaidforbygreaterheatloss.Thusendothermscouldinprinciple(eveniftheydon’tinfact)attaingreatercomplexitythanectotherms.Perhapsthesophisticatedbrainsofsomebirdsandmammalsareacaseinpoint.51ångström(Å)is10–10m,oroneten-billionthofametre.It’stechnicallyanoutmodedtermnow,generallyreplacedbythenanometre(nm),whichis10–9m,butitisstillveryusefulforconsideringdistancesacrossproteins.14Åis1.4nm.Mostoftheredoxcentresintherespiratorychainarebetween7and14Åapart,withafewstretchingoutto18Å.Tosaythattheyarebetween0.7and1.4nmapartisthesamething,butsomehowcompressesoursenseofthatrange.Theinnermitochondrialmembraneis60Åacross–adeepoceanoflipidscomparedwithaflimsy6nm!Unitsdoconditionoursenseofdistance.6NotonlyATP.Theprotongradientisanall-purposeforcefield,whichisusedtopowertherotationofthebacterial(butnotthearchaeal)flagellumandtheactivetransportofmoleculesinandoutofthecell,anddissipatedtogenerateheat.It’salsocentraltothelifeanddeathofcellsbyprogrammedcelldeath(apoptosis).We’llcometoallofthat.7IamprivilegedthatmyofficeisdownthecorridorfromPeterRich,whoheadedtheGlynnInstituteafterPeterMitchell’sretirement,andfinallybroughtittoUCLastheGlynnLaboratoryofBioenergetics.HeandhisgroupareworkingactivelyonthedynamicwaterchannelsthatconductprotonsthroughcomplexIV(cytochromeoxidase),thefinalrespiratorycomplexinwhichoxygenisreducedtowater.8Thisisoneofthedisadvantagesofanoxygenicphotosynthesis–cellsultimatelyencasethemselvesintheirownwasteproduct.Somebanded-ironformationsarepittedwithtinybacteria-sizedholes,presumablyreflectingjustthat.Incontrast,oxygen,thoughpotentiallytoxic,isamuchbetterwasteproductasitisagasthatsimplydiffusesaway.9Howcanwebesosureitwasthatwayround,ratherthanrespirationderivingfromphotosynthesis?Becauserespirationisuniversalacrossalllife,butphotosynthesisisrestrictedtojustafewgroupsofbacteria.Ifthelastuniversalcommonancestorwerephotosynthetic,thenmostgroupsofbacteriaandallarchaeamusthavelostthisvaluabletrait.That’snotparsimonious,tosaytheleast.10Lipidsarecomposedoftwoparts:ahydrophilichead-group,andtwoorthreehydrophobic‘tails’(fattyacidsinbacteriaandeukaryotes,andisoprenesinarchaea).Thesetwopartsenablelipidstoformbilayers,ratherthanfattydroplets.Thehead-groupinarchaeaandbacteriaisthesamemolecule,glycerol,buttheyeachusetheoppositemirrorimageform.Thisisaninterestingtangenttothecommonlycitedfactthatalllifeusesleft-handedaminoacidsandright-handedsugarsinDNA.Thischiralityisoftenexplainedintermsofsomesortofabioticprejudiceforoneisomerovertheother,ratherthanselectionatthelevelofbiologicalenzymes.Thefactthatarchaeaandbacteriausetheoppositestereoisomersofglycerolshowsthatchanceandselectionprobablyplayedalargerole.

PARTII

THEORIGINOFLIFE

M

3

ENERGYATLIFE’SORIGIN

edievalwatermillsandmodernhydroelectricpowerstationsarepoweredbythechannellingofwater.Funneltheflowintoaconfinedchannelanditsforceincreases.Nowitcandrivework

suchasturningawaterwheel.Conversely,allowtheflowtospreadoutacrossawiderbasin,andtheforcediminishes.Inariver,itbecomesapondoraford.Youmightattempttomakeacrossing,safeintheknowledgethatyouareunlikelytobesweptawaybytheforceofthecurrent.

Livingcellsworkinasimilarway.Ametabolicpathwayis likeawaterchannel,except that theflow isoforganiccarbon. Inametabolicpathway,a linear sequenceof reactions iscatalysedbyaseriesofenzymes,eachoneactingontheproductofthepreviousenzyme.Thisconstrainstheflowoforganiccarbon.Amoleculeentersapathway,undergoesasuccessionofchemicalmodifications,andexits as a differentmolecule. The succession of reactions can be repeated reliably, with the sameprecursorenteringand thesameproduct leavingeach time.With theirvariousmetabolicpathways,cellsare likenetworksofwatermills, inwhich the flow isalwaysconfinedwithin interconnectingchannels, alwaysmaximised.Such ingeniouschannellingmeans that cellsneed far less carbonandenergytogrowthantheywouldifflowwereunconstrained.Ratherthandissipatingtheforceateachstep–molecules‘escaping’toreactwithsomethingelse–enzymeskeepbiochemistryonthestraightand narrow. Cells don’t need a great river surging to the sea, but drive theirmills using smallerchannels.Fromanenergeticpointofview,thepowerofenzymesisnotsomuchthattheyspeedupreactions,butthattheychanneltheirforce,maximisingtheoutput.

Sowhathappenedattheoriginoflife,beforetherewereanyenzymes?Flowwasnecessarilylessconstrained.Togrow– tomakemoreorganicmolecules, todouble,ultimately to replicate–musthavecostmoreenergy,morecarbon,notless.Moderncellsminimisetheirenergyrequirements,butwe have already seen that they still get through colossal amounts of ATP, the standard energy‘currency’.Eventhesimplestcells,whichgrowfromthereactionofhydrogenwithcarbondioxide,produceabout40 timesasmuchwaste fromrespirationasnewbiomass. Inotherwords, foreverygram of new biomass produced, the energy-releasing reactions that support this production mustgenerateatleast40gramsofwaste.Lifeisasidereactionofamainenergy-releasingreaction.Thatremainsthecasetoday,after4billionyearsofevolutionaryrefinement.Ifmoderncellsproduce40times more waste than organic matter, just think how much the first primitive cells, without anyenzymes,wouldhavehad tomake!Enzymes speedup chemical reactionsbymillionsof times theunconstrained rate.Take away those enzymes, and throughputwouldneed to increaseby a similarfactor, sayamillionfold, toachieve thesame thing.Thefirstcellsmayhaveneeded toproduce40tonnesofwaste–literallyatruck-load–tomake1gramofcell!Intermsofenergyflow,thatdwarfs

evenariverinspate;it’smorelikeatsunami.Thesheerscaleofthisenergeticdemandhasconnotationsforallaspectsoftheoriginoflife,yet

is rarely considered explicitly.As an experimental discipline, the origin-of-life field dates back to1953 and the famousMiller–Urey experiment, published in the same year asWatson and Crick’sdoublehelixpaper.Bothpapershavehungoverthefieldeversince,castingashadowlikethewingsof two giant bats, in some respects rightly, in others regrettably. The Miller–Urey experiment,brilliantasitwas,bolsteredtheconceptionofaprimordialsoup,whichinmyviewhasblinkeredthefieldfortwogenerations.CrickandWatsonusheredinthehegemonyofDNAandinformation,whichis plainly of vital importance to the origin of life; but considering replication and the origins ofnatural selection in near isolation has distracted attention from the importance of other factors,notablyenergy.

In 1953, StanleyMillerwas an earnest young PhD student in the lab ofNobel laureateHaroldUrey. In his iconic experiment, Miller passed electrical discharges, simulating lightning, throughflaskscontainingwaterandamixtureofreduced(electron-rich)gasesreminiscentoftheatmosphereofJupiter.Atthetime,theJovianatmospherewasthoughttoreflectthatoftheearlyearth–bothwerepresumed to be rich in hydrogen, methane and ammonia.1 Amazingly, Miller succeeded insynthesisinganumberofaminoacids,whicharethebuildingblocksofproteins,theworkhorsesofcells.Suddenlytheoriginoflife lookedeasy!Intheearly1950stherewasfarmoreinterest inthisexperimentthaninWatsonandCrick’sstructure,whichinitiallycausedlittlestir.Miller,incontrast,featuredon the coverofTimemagazine in 1953.Hisworkwas seminal, stillworth recapitulating,becauseitwasthefirst to testanexplicithypothesisabout theoriginof life: thatboltsof lightning,passingthroughanatmosphereofreducedgases,couldproducethebuildingblocksofcells.Intheabsenceof existing life, theseprecursorswere taken to accumulate in theoceans,whichover timebecamearichbrothoforganicmolecules,theprimordialsoup.

IfWatsonandCrickmade lessofa stir in1953, the spellofDNAhasbeguiledbiologistseversince.Formanypeople,lifeisallabouttheinformationcopiedinDNA.Theoriginoflife,forthem,is the origin of information, without which, all are agreed, evolution by natural selection is notpossible. And the origin of information boils down to the origin of replication: how the firstmolecules thatmade copies of themselves – replicators – arose. DNA itself is too complex to becredible as the first replicator, but the simpler, more reactive precursor RNA fits the bill. RNA(ribonucleicacid)is,eventoday,thekeyintermediarybetweenDNAandproteins,servingasbothatemplateandacatalystinproteinsynthesis.BecauseRNAcanactasbothatemplate(likeDNA)andacatalyst(likeproteins),itcaninprincipleserveasasimplerforerunnerofbothproteinsandDNAinaprimordial ‘RNAworld’.Butwhere did all the nucleotide building blocks come from,which jointogether into chains to form RNA? The primordial soup, of course! There is no necessaryrelationship between the formation of RNA and a soup, but soup is nonetheless the simplestassumption,whichavoidsworryingaboutcomplicateddetailslikethermodynamicsorgeochemistry.Putall that tooneside,and thegene-jockscangetonwith the importantstuff.Andso, if therehasbeenaleitmotifdominatingorigin-of-liferesearchoverthelast60years,itisthataprimordialsoupgave rise toanRNAworld, inwhich these simple replicatorsgraduallyevolvedandbecamemorecomplex,begancodingformetabolism,andultimatelyspawnedtheworldofDNA,proteinsandcellsthatweknowtoday.Bythisview,lifeisinformationfromthebottomup.

What is missing here is energy. Of course, energy figures in the primordial soup – all thoseflashesoflightning.Ioncecalculatedthattosustainatinyprimitivebiosphere,equivalentinsizetothatbeforetheevolutionofphotosynthesis,bylightningalone,wouldrequirefourboltsoflightningpersecond,foreverysquarekilometreofocean.Andthat’sassumingamodernefficiencyofgrowth.There are just not all thatmany electrons in each bolt of lightning.A better alternative source of

energy is UV radiation, which can fashion reactive precursors like cyanide (and derivatives likecyanamide) from a mixture of atmospheric gases including methane and nitrogen. UV radiationstreamsinendlesslyontheearthandotherplanets.UVfluxwouldhavebeenstrongerintheabsenceof an ozone layer, andwith themore aggressive electromagnetic spectrum of the young sun. TheingeniousorganicchemistJohnSutherlandhasevensucceededinsynthesisingactivatednucleotidesunder so-called ‘plausible primordial conditions’ using UV radiation and cyanide.2 But there areseriousproblemsheretoo.Nolifeonearthusescyanideasasourceofcarbon;andnoknownlifeusesUVradiationasasourceofenergy.Quite thecontrary,bothareconsidereddangerouskillers.UV is too destructive, even for the sophisticated life forms of today, as it breaks down organicmoleculesrathermoreeffectivelythanitpromotestheirformation.Itismuchmorelikelytoscorchtheoceansthantofillthemwithlife.UVisablitz.Idoubtitwouldworkasadirectsourceofenergy,hereoranywhereelse.

TheadvocatesofUVradiationdon’t claim that itwouldworkasadirect sourceofpower,butrather that it would favour the formation of small stable organic molecules like cyanide, whichaccumulateovertime.Intermsofchemistry,cyanideisindeedagoodorganicprecursor.Itistoxictousbecauseitblockscellrespiration;butthatmightbeaquirkoflifeonearth,ratherthananydeeperprinciple. The real problem with cyanide is its concentration, which afflicts the whole idea ofprimordialsoup.Theoceansareextremelylargerelativetotherateofformationofcyanide,orforthatmatterofanyothersimpleorganicprecursor,evenassumingthatasuitablyreducingatmosphereexisted here or on any other planet. At any reasonable rate of formation, the steady-stateconcentrationofcyanideintheoceansat25°Cwouldhavebeenaroundtwo-millionthsofagramperlitre–notnearlyenoughtodrivetheoriginsofbiochemistry.Theonlywayoutofthisimpasseistoconcentrate the seawater somehow, and this has been the mainstay of prebiotic chemistry for ageneration.Eitherfreezingorevaporatingtodrynesscouldpotentiallyincreasetheconcentrationoforganics, but these are drasticmethods, hardly congruent with the physically stable state that is adefiningfeatureofalllivingcells.Oneexponentofcyanideoriginsturnswithwildeyestothegreatasteroidbombardment4billionyears ago: it couldhave concentrated cyanide (as ferricyanide) byevaporatingall the oceans!Tome, that smacks of desperation to defend anunworkable idea.3 Theproblem here is that these environments are too variable and unstable. A succession of drasticchanges in conditions are required to achieve the steps to life. In contrast, living cells are stableentities–theirfabriciscontinuallyreplaced,buttheoverallstructureisunchanging.

Heraclitustaughtthat‘nomaneverstepsinthesamerivertwice’;buthedidn’tmeanthattheriverhad evaporated or frozen (or been exploded into space) in themeantime.Aswater flows betweenunchangingbanks, at least onourhuman timescale, so life is continuously renewing itselfwithoutchangingitsform.Livingcellsremaincells,evenwhenalltheirconstituentpartsarereplacedinanunceasing turnover.Could itbeanyotherway?Idoubt it. In theabsenceof informationspecifyingstructure–asmustlogicallyhavebeenthecaseattheoriginoflife,beforetheadventofreplicators–structure isnotabsent,but itdoes requireacontinuous fluxofenergy.Energy fluxpromotes self-organisationofmatter.Weare all familiarwithwhat thegreatRussian-bornBelgianphysicist IlyaPrigoginecalled‘dissipativestructures’: just thinkofconvectioncurrentsinaboilingkettle,orforthatmatterwaterswirlingdownaplughole.Noinformationisrequired–justheatinthecaseofthekettle andangularmomentum for theplughole.Dissipative structures areproducedby the fluxofenergy and matter. Hurricanes, typhoons and whirlpools are all striking natural examples ofdissipativestructures.Wefindthemonavastscaleintheoceansandatmospheretoo,drivenbythedifferencesinenergyfluxfromthesunattheequatorrelativetothepoles.Reliableoceancurrents,suchastheGulfStream,andwinds,suchastheRoaringFortiesortheNorthAtlanticjetstream,arenotspecifiedbyinformation,butareasstableandcontinuousas theenergyfluxthatsustains them.

TheGreatRedSpotof Jupiter is ahuge storm, ananti-cyclone several times the sizeof theearth,whichhaspersistedforatleastafewhundredyears.Justastheconvectioncellsinakettlepersistforas long as the electric current keeps thewater boiling and steam evaporating, all these dissipativestructuresrequireacontinuousfluxofenergy.Inmoregeneralterms,theyarethevisibleproductsofsustained far-from-equilibrium conditions, inwhich energy fluxmaintains a structure indefinitely,untilatlast(afterbillionsofyearsinthecaseofstars)equilibriumisattainedandthestructurefinallycollapses. Themain point is that sustained and predictable physical structures can be produced byenergy flux.Thishasnothing todowith information,butwe’ll see that it cancreate environmentswheretheoriginofbiologicalinformation–replicationandselection–isfavoured.

Alllivingorganismsaresustainedbyfar-from-equilibriumconditionsintheirenvironment:we,too, aredissipative structures.Thecontinuous reactionof respirationprovides the freeenergy thatcells need to fix carbon, to grow, to form reactive intermediates, to join these building blockstogether into long-chainpolymerssuchascarbohydrates,RNA,DNAandproteins,and tomaintaintheir low-entropy state by increasing the entropy of the surroundings. In the absence of genes orinformation,certaincellstructures,suchasmembranesandpolypeptides,shouldformspontaneously,so longas there isacontinuoussupplyofreactiveprecursors–activatedaminoacids,nucleotides,fattyacids;so longas there isacontinuousfluxofenergyproviding therequisitebuildingblocks.Cellstructuresareforcedintoexistencebythefluxofenergyandmatter.Thepartscanbereplacedbut the structure is stable andwill persist for as long as the flux persists. This continuous flux ofenergyandmatter ispreciselywhat ismissingfromtheprimordialsoup.There isnothing insoupthatcandrivetheformationofthedissipativestructuresthatwecallcells,nothingtomakethesecellsgrowanddivide,andcomealive,all in theabsenceofenzymesthatchannelanddrivemetabolism.Thatsoundslikeatallorder.Istherereallyanenvironmentthatcandrivetheformationofthefirstprimitivecells?Theremostcertainlymusthavebeen.Butbeforeweexplorethatenvironment,let’sconsiderexactlywhatisneeded.

HowtomakeacellWhatdoesittaketomakeacell?Sixbasicpropertiesaresharedbyalllivingcellsonearth.Withoutwishingtosoundlikeatextbook,let’sjustenumeratethem.Allneed:

i. acontinuoussupplyofreactivecarbonforsynthesisingneworganics;ii. asupplyoffreeenergytodrivemetabolicbiochemistry–theformationofnewproteins,DNA,

andsoon;iii. catalyststospeedupandchannelthesemetabolicreactions;iv. excretionofwaste,topaythedebttothesecondlawofthermodynamicsanddrivechemical

reactionsinthecorrectdirection;v. compartmentalisation–acell-likestructurethatseparatestheinsidefromtheoutside;vi. hereditarymaterial–RNA,DNAoranequivalent,tospecifythedetailedformandfunction.

Everythingelse(thekindofthingyouwillfindinstandardmnemonicsforlife’sproperties,suchasmovementorsensitivity)arejustnice-to-haveaddedextrasfromthepointofviewofbacteria.

Itdoesn’ttakemuchreflectiontoappreciatethatallsixfactorsareprofoundlyinterdependent,andalmostcertainlyneededtobefromtheverybeginningtoo.Acontinuoussupplyoforganiccarbonisobviously central to growth, replication,… everything. At a simple level, even an ‘RNA world’involvesthereplicationofRNAmolecules.RNAisachainofnucleotidebuildingblocks,eachoneofwhich is an organicmolecule thatmust have come from somewhere.There is an old rift between

origin-of-life researchers about what came first, metabolism or replication. It’s a barren debate.Replication is doubling, which consumes building blocks in an exponential fashion. Unless thosebuildingblocksarereplenishedatasimilarrate,replicationswiftlyceases.

Oneconceivableescapeistoassumethatthefirstreplicatorswerenotorganicatall,butwereclayminerals or some such, as long and ingeniously argued byGrahamCairns-Smith. Yet that solveslittle, because minerals are too physically clumsy to encode anything even approaching an RNA-world level of complexity, although they are valuable catalysts. But if minerals are no use asreplicators, then we need to find the shortest and fastest route to get from inorganics to organicmoleculesthatdoworkasreplicators,likeRNA.Giventhatnucleotideshavebeensynthesisedfromcyanamide,it’spointlesstopositunknownandunnecessaryintermediates;it’sfarbettertocutstraightto the chase, to assume that some early environments on earth could have provided the organicbuildingblocks–activatednucleotides–neededforthebeginningsofreplication.4Evenifcyanamideis a poor starting point, the tendency to produce a strikingly similar spectrum of organics underdisparate conditions, fromelectrical discharges in a reducing atmosphere, to cosmic chemistryonasteroids,tohigh-pressurebombreactors,suggeststhatcertainmolecules,probablyincludingsomenucleotides, are favoured by thermodynamics. To a first approximation, then, the formation oforganic replicators requires a continuous supplyoforganic carbon in the sameenvironment.Thatrulesoutfreezingenvironments,incidentally–whilefreezingcanconcentrateorganicsbetweenicecrystals,thereisnomechanismtoreplenishthebuildingblocksneededtocontinuetheprocess.

What about energy? That is also needed in the same environment. Joining individual buildingblocks (amino acids or nucleotides) together to form long-chain polymers (proteins or RNA)requires first activating the building blocks. That in turn demands a source of energy – ATP, orsomething similar.Perhapsvery similar. In awaterworld, aswas the earth4billionyears ago, thesourceofenergyneedstobeofaratherspecifickind:itneedstodrivethepolymerisationoflong-chain molecules. That involves removing one molecule of water for each new bond formed, adehydrationreaction.Theproblemofdehydratingmoleculesinsolutionisabitliketryingtowringoutawetclothunderwater.SomeprominentresearchershavebeensodistractedbythisproblemthattheyhaveevencontendedthatlifemusthavestartedonMars,wheretherewasmuchlesswater.Lifethen hitch-hiked to earth on ameteorite,making us allMartians really.But of course life here onearthdoesperfectlywellinwater.Everylivingcellpullsoffthedehydrationtrickthousandsoftimesasecond.WedosobycouplingthedehydrationreactiontothesplittingofATP,whichtakesuponemoleculeofwatereachtimeitissplit.Couplingadehydrationtoa‘rehydration’reaction(technicallytermed‘hydrolysis’) ineffect just transfers thewater,whileat thesame timereleasingsomeof theenergy pent up in the bonds of ATP. That greatly simplifies the problem; all that is needed is acontinuoussupplyofATPorasimplerequivalent,suchasacetylphosphate.We’lladdresswherethismay have come from in the next chapter. For now, the point is that replication in water needs acontinuous and liberal supply of both organic carbon and somethingmuch likeATP, in the sameenvironment.

That’sthreeoutofsixfactors:replication,carbonandenergy.Whataboutcompartmentalisationintocells?Thisisagainamatterofconcentration.Biologicalmembranesaremadeoflipids,whicharethemselvescomposedoffattyacidsorisoprenes(joinedtoaglycerolhead-group,asnotedintheprevious chapter).When concentrated above a threshold level, fatty acids spontaneously form intocell-likevesiclesthatcangrowanddivideifcontinuously‘fed’withnewfattyacids.Hereagain,weneedacontinuoussupplyofbothorganiccarbonandenergytodrivetheformationofnewfattyacids.Forfattyacids,orforthatmatternucleotides,toaccumulatefasterthantheydissipate,theremustbesome sort of focusing: a physical funnelling or natural compartmentalisation that increases theirconcentrationlocally,enablingthemtoformlarger-scalestructures.Whensuchconditionsaremet,

the formation of vesicles is not magic: physically, this is the most stable state – overall entropyincreasesasaresult,aswesawinthepreviouschapter.

Ifreactivebuildingblocksareindeedsuppliedcontinuously,thensimplevesicleswillgrowanddividespontaneously,asaresultofsurface-area-to-volumeconstraints.Imagineasphericalvesicle–a simple ‘cell’ – enclosing various organic molecules. The vesicle grows by incorporating newmaterials:lipidsinthemembraneandotherorganicsinsidethecell.Nowlet’sdoubleinsize:doublethemembrane surface area, and double the organic contents.What happens?Doubling the surfaceareamore thandoubles thevolume,because thesurfacearea increasesby thesquareof the radius,whilethevolumeincreasesbyitscube.Butthecontentsonlydoubled.Unlessthecontentsincreaseatafasterratethanthemembranesurfacearea,thevesiclewillbuckleintoadumb-bell,whichisalreadyhalfwaytoformingtwonewvesicles.Inotherwords,arithmeticgrowthintroducesaninstabilitythatleadstodivisionanddoubling,ratherthansimplygettingbigger.It’sonlyamatteroftimebeforeagrowingspheredividesupintosmallerbubbles.Soacontinuousfluxofreactivecarbonprecursorsentailsnotonlyaprimitivecellformationbutalsoarudimentaryformofcelldivision.Suchbudding,incidentally,isalsohowL-formbacteria,whichlackacellwall,divide.

Theproblemof surface-area-to-volume ratiomust set a limit to the sizeofcells.This is just amatterofthesupplyofreactantsandtheremovalofwaste.Nietzscheonceobservedthathumanswillnot mistake ourselves for gods so long as we need to defecate. But in fact excretion is athermodynamicnecessity, binding even for thegodliest.For any reaction to continue in a forwarddirection,theendproductmustberemoved.Thisisnomoremysteriousthanthebuild-upofacrowdatarailwaystation.Ifpassengerscan’tgetontoatrainasfastasnewpeoplearrive,therewillsoonbeablockage.Inthecaseofcells,therateatwhichnewproteinsareformeddependsontheratesofdeliveryofreactiveprecursors(activatedaminoacids)andremovalofwaste(methane,water,CO2,ethanol–whatevertheenergy-releasingreactionmaybe).Ifthesewasteproductsarenotphysicallyremovedfromthecell,theypreventtheforwardreactionfromcontinuing.

The problemofwaste removal is another fundamental difficultywith the idea of a primordialsoup, inwhich reactants andwastemarinade together.There isno forwardmomentum,nodrivingforce for new chemistry.5 Likewise, the larger a cell becomes, the closer it approximates to soup.Becausethevolumeofacellrisesfasterthanitssurfacearea,therelativerateatwhichfreshcarboncanbedeliveredandwasteremovedacrossitsboundingmembranemustfallasthecellgetslarger.Acell on the scale of theAtlantic ocean, or even a football, could neverwork; it is just soup. (Youmightthinkthananostricheggisasbigasafootball,buttheyolksacismostlyjustafooddump–thedevelopingembryoitselfismuchsmaller.)Attheoriginoflife,naturalratesofcarbondeliveryandwasteremovalmusthavedictatedasmallcellvolume.Somesortofphysicalchannellingwouldalsoseemtobenecessary:acontinuousnaturalflowthatdeliversprecursorsandcarriesawaywaste.

That leaves us with catalysts. Today, life uses proteins – enzymes – but RNA also has somecatalytic capabilities. The trouble here is that RNA is already a sophisticated polymer, aswe haveseen. It iscomposedofmultiplenucleotidebuildingblocks,eachofwhichmustbesynthesisedandactivated to join together intoa longchain.Before thathappened,RNAcouldhardlyhavebeen thecatalyst.Whateverprocessgave rise toRNAmustalsohavedriven the formationofotherorganicmoleculesthatareeasiertomake,notablyaminoacidsandfattyacids.Thusanyearly‘RNAworld’musthavebeen‘dirty’–contaminatedwithmanyother typesofsmallorganicmolecules.The ideathatRNAsomehowinventedmetabolismbyitself isabsurd,evenifRNAdidplayakeyrole in theoriginsofreplicationandproteinsynthesis.Sowhatdidcatalysethebeginningsofbiochemistry?Theprobable answer is inorganic complexes, such as metal sulphides (in particular iron, nickel andmolybdenum). These are still found as cofactors in several ancient, and universally conserved,proteins.While we tend to think of the protein as the catalyst, in fact the protein only speeds up

reactions thathappenanyway– thecofactordetermines thenatureof the reaction.Strippedof theirproteincontext, cofactorsarenotveryeffectiveor specificcatalysts,but theyaremuchbetter thannothing.Howeffective theyaredepends,yetagain,on the throughput.The first inorganiccatalystsjustbeganthechannellingofcarbonandenergyinthedirectionoforganics,buttheycuttheneedforatsunamibackdowntoamereriver.

Andthesesimpleorganics(notablyaminoacidsandnucleotides)alsohavesomecatalyticactivityoftheirown.Inthepresenceofacetylphosphate,aminoacidscanevenjointogether,toformshort‘polypeptides’– little stringsofaminoacids.The stabilityof suchpolypeptidesdepends inpartontheirinteractionswithothermolecules.Hydrophobicaminoacidsorpolypeptidesthatassociatewithfattyacidsshouldpersistlonger;andchargedpolypeptidesthatbindtoinorganicclusterssuchasFeSminerals could also be more stable. Natural associations between short polypeptides and mineralclusters may enhance the catalytic properties of minerals, and could be ‘selected’ for by simplephysicalsurvival.Imagineamineralcatalystthatpromotesorganicsynthesis.Someoftheproductsbind to the mineral catalyst, prolonging their own survival, while at once improving (or at leastvarying)thecatalyticpropertiesofthemineral.Suchasystemcouldinprinciplegiverisetoricherandmorecomplexorganicchemistry.

Sohowcouldacellbebuilt fromscratch?Theremustbea continuouslyhigh fluxof reactivecarbon and usable chemical energy, flowing past rudimentary catalysts that convert a modestproportionofthatfluxintoneworganics.Thiscontinuousfluxmustbeconstrainedinsomewaythatenablestheaccumulationofhighconcentrationsoforganics,includingfattyacids,aminoacidsandnucleotides,withoutcompromisingtheoutflowofwaste.Suchafocusingofflowcouldbeachievedby a natural channelling or compartmentalisation,which has the same effect as the channelling offlowinawatermill–itincreasestheforceofagivenfluxintheabsenceofenzymes,soloweringthetotalamountofcarbonandenergyrequired.Onlyifthesynthesisofneworganicsexceedstheirrateof loss into the outsideworld, enabling their concentration,will they self-assemble into structuressuchascell-likevesicles,RNAandproteins.6

Plainlythisisnomorethanthebeginningsofacell–necessary,butfarfromsufficient.Butlet’sput aside the details for now, and focus on just this one point.Without a high flux of carbon andenergythatisphysicallychannelledoverinorganiccatalysts,thereisnopossibilityofevolvingcells.Iwouldratethisasanecessityanywhereintheuniverse:giventherequirementforcarbonchemistrythatwediscussedinthelastchapter,thermodynamicsdictatesacontinuousflowofcarbonandenergyovernaturalcatalysts.Discountingspecialpleading,thatrulesoutalmostallenvironmentsthathavebeentoutedaspossiblesettingsfortheoriginoflife:warmponds(sadlyDarwinwaswrongonthat),primordialsoup,microporouspumicestones,beaches,panspermia,younameit.Butitdoesnotruleouthydrothermalvents;onthecontrary,itrulesthemin.Hydrothermalventsareexactlythekindofdissipativestructuresthatweseek–continuousflow,far-from-equilibriumelectrochemicalreactors.

HydrothermalventsasflowreactorsTheGrandPrismaticSpring inYellowstoneNationalPark remindsmeof theEyeofSauron in itsmalevolent yellows, oranges and greens. These remarkably vivid colours are the photosyntheticpigmentsofbacteriathatusehydrogen(orhydrogensulphide)emanatingfromthevolcanicspringsasanelectrondonor.Beingphotosynthetic, theYellowstonebacteriagive little real insight into theoriginoflife,buttheydogiveasenseoftheprimalpowerofvolcanicsprings.Theseareplainlyhotspots for bacteria, in otherwise meagre environments. Go back 4 billion years, strip away thesurrounding vegetation to the bare rocks, and it’s easy to imagine such a primal place as thebirthplaceoflife.

Exceptthatitwasn’t.Backthen,theearthwasawaterworld.Perhapstherewereafewterrestrialhot springson small volcanic islandsprotrudingabove tempestuousglobaloceans,butmostventsweresubmergedbeneath thewaves indeep-seahydrothermalsystems.Thediscoveryofsubmarineventsinthelate1970scameasashock,notbecausetheirpresencewasunsuspected(plumesofwarmwater had betrayed their presence) but because nobody anticipated the brutal dynamism of ‘blacksmokers’, or the overwhelming abundance of life clinging precariously to their sides. The deepocean floor ismostly a desert, nearly destitute of life.Yet these tottering chimneys, billowing outblacksmokeasiftheirlivesdependedonit,werehometopeculiarandhithertounknownanimals–gianttubewormslackingamouthandanus,clamsasbigasdinnerplates,andeyelessshrimp–allliving at a density equivalent to tropical rain forests. This was a seminal moment, not only forbiologistsandoceanographers,butperhapsevenmoreforthoseinterestedintheoriginoflife,asthemicrobiologistJohnBarosswasquick toappreciate.Since then,Barossmore thananyonehaskeptattention focusedon theextraordinaryvigourofchemicaldisequilibria inventsdown in thebible-blackoceandepths,farawayfromthesun.

Yetthesevents,too,aremisleading.Theyarenotreallycutofffromthesun.Theanimalsthatlivehererelyonsymbioticrelationshipswithbacteriathatoxidisethehydrogensulphidegasemanatingfrom the smokers. That is the principal source of disequilibrium: hydrogen sulphide (H2S) is areducedgasthatreactswithoxygentoreleaseenergy.Recallthemechanicsofrespirationfromthepreviouschapter.BacteriauseH2Sasanelectrondonorforrespiration,andoxygenas theelectronacceptor,todriveATPsynthesis.Butoxygenisaside-productofphotosynthesis,andwasnotpresenton the early earth, before the evolution of oxygenic photosynthesis. The stunning eruption of lifearoundtheseblacksmokerventsisthereforecompletely,albeitindirectly,dependentonthesun.Andthatmeanstheseventsmusthavebeenverydifferent4billionyearsago.

Take away the oxygen and what is left? Well, black smokers are produced by the directinteractionsofseawaterwithmagmaat thetectonicspreadingcentresofmid-oceanridgesorothervolcanicallyactiveplaces.Waterpercolatesdownthroughtheseafloortothemagmachambersnotfar below, where it is heated instantaneously to hundreds of degrees, and charged with dissolvedmetalsandsulphides,makingthewaterstronglyacidic.Asthesuperheatedwaterblastsbackupintothe ocean above, burstingwith explosive power, it cools abruptly.Tiny particles of iron sulphidessuchaspyrites(fool’sgold)precipitateimmediately–thisistheblacksmokethatgivestheseangryvolcanicventstheirname.Mostofthatwouldhavebeenthesame4billionyearsago,butnoneofthisvolcanic fury is available to life. Only the chemical gradients matter; and there’s the rub. Thechemical boost providedbyoxygenwouldhavebeenmissing.Trying toget hydrogen sulphide toreactwithCO2 toformorganicsismuchharder,especiallyathightemperatures.Inasuccessionofgroundbreaking papers from the late 1980s onwards, the revolutionary and notoriously irascibleGermanchemistandpatentattorneyGünterWächtershäuser redrew the landscape.7Heproposed ingreatdetailawayofreducingCO2toorganicmoleculesonthesurfaceofthemineralironpyrites,whichhetermed‘pyritespulling’.Morebroadly,Wächtershäusertalkedofan‘iron–sulphurworld’,inwhich iron–sulphur(FeS)mineralscatalysed theformationoforganicmolecules.Suchmineralsaretypicallycomposedofrepeatinglatticesofferrousiron(Fe2+)andsulphide(S2–).Littlemineralclusters of ferrous iron and sulphide, known as FeS clusters, are still found at the heart ofmanyenzymestoday,includingthoseinvolvedinrespiration.Theirstructureisessentiallyidenticaltothelattice structure of FeSminerals such asmackinawite and greigite (Figure11; see alsoFigure 8),lending credence to the idea that these minerals could have catalysed the first steps of life.Nonetheless, even though FeS minerals are good catalysts, Wächtershäuser ’s own experimentsshowedthatpyritespulling,asheoriginallyconceivedit,doesnotwork.Onlywhenusingthemore

reactivegascarbonmonoxide(CO)couldWächtershäuserproduceanyorganicmolecules.Thefactthatnoknownlifegrowsby‘pyritespulling’suggeststhatthefailuretomakeitworkinthelabisnoaccident;itreallydoesn’twork.

Figure11Iron–sulphurmineralsandiron–sulphurclustersTheclosesimilaritybetweeniron–sulphurmineralsandiron–sulphurclustersembeddedinmodernenzymes,asdepictedbyBillMartinandMikeRussellin2004.Thecentralpanelshowsarepeatingcrystallineunitfromthemineralgreigite;thisstructureisrepeatedtomakeupalatticeofmultipleunits.Thesurroundingpanelsshowiron–sulphurclustersembeddedinproteins,withstructuressimilartogreigiteandrelatedmineralssuchasmackinawite.Theshadedareasrepresenttheroughshapeandsizeoftheproteinnamedineachcase.Eachproteintypicallycontainsafewiron–sulphurclusters,withorwithoutnickel.

WhileCOisfoundinblacksmokervents,itsconcentrationisvanishinglylow–thereisfartoolittletodriveanyseriousorganicchemistry.(ConcentrationsofCOare1,000–1,000,000-foldlowerthanCO2.) There are other grave problems too. Black smoker vents are excessively hot; the ventfluidsemergeat250–400°C,butarepreventedfromboilingbytheextremepressureatthebottomoftheocean.Atthesetemperatures,themoststablecarboncompoundisCO2.Thismeansthatorganicsynthesiscan’ttakeplace;onthecontrary,anyorganicsthatdoformshouldbeswiftlydegradedbackto CO2. The idea of organic chemistry catalysed by the surface of minerals is problematic too.Organicseitherremainboundto thesurface, inwhichcaseeverythingeventuallygumsup,or theydissociate,inwhichcasetheyareflushedoutintotheopenoceanswithunseemlyhaste,throughthebillowingchimneysofthevents.Blacksmokersarealsoveryunstable,growingandcollapsingovera few decades atmost. That’s not long to ‘invent’ life.While they are truly far-from-equilibriumdissipativestructures,andcertainlysolvesomeoftheproblemsofsoup,thesevolcanicsystemsaretooextremeandunstabletonurturethegentlecarbonchemistryneededfortheoriginoflife.Whatthey did do, which was indispensable, was charge the early oceans with catalytic metals such asferrousiron(Fe2+)andnickel(Ni2+)derivedfrommagma.

The beneficiary of all thesemetals dissolved in the oceanwas another type of vent known as

alkalinehydrothermalvents(Figure12).Inmyview,theseresolvealltheproblemsofblacksmokers.Alkaline hydrothermal vents are not volcanic at all, and lack the drama and excitement of blacksmokers; but they do have other properties that fit them out much better as electrochemical flowreactors. Their relevance to the origin of lifewas first signalled by the revolutionary geochemistMikeRussell,inashortlettertoNaturein1988,anddevelopedinaseriesofidiosyncratictheoreticalpapersthroughthe1990s.Lateron,BillMartinbroughthisinimitablemicrobiologicalperspectivetobearontheventworld,thepairpointingoutmanyunexpectedparallelsbetweenventsandlivingcells.Russell andMartin, likeWächtershäuser, argue that life started from the ‘bottom up’, through thereaction of simple molecules such as H2 andCO2, in much the same way as autotrophic bacteria(whichsynthesisealltheirorganicmoleculesfromsimpleinorganicprecursors).RussellandMartinlikewisealways stressed the importanceof iron–sulphur (FeS)mineralsasearlycatalysts.The factthatRussell,MartinandWächtershäuseralltalkofhydrothermalvents,FeSmineralsandautotrophicoriginsmeansthattheirideasareeasilyconflated.Inreality,thedifferencesareasblackandwhite.

Figure12Deep-seahydrothermalventsComparisonofanactivealkalinehydrothermalventatLostCity(A)withablacksmoker(B).Thescalebarisonemetreinbothcases:alkalineventscanstandasmuchas60mtall,equivalenttoa20-storeybuilding.Thewhitearrowatthetopmarksaprobefixedtothetopofthealkalinevent.Thewhiterregionsofalkalineventsarethemostactive,butunlikeblacksmokers,thesehydrothermalfluidsdonotprecipitateas‘smoke’.Thesenseofabandonment,thoughmisleading,influencedthechoiceofthenameLostCity.

Alkalineventsarenotproducedbytheinteractionsofwaterwithmagmabutbyamuchgentlerprocess–achemicalreactionbetweensolidrockandwater.Rocksderivedfromthemantle,richinmineralssuchasolivine,reactwithwatertobecomethehydratedmineralserpentinite.Thismineralhas a beautiful mottled green appearance, which resembles the scales of a serpent. Polishedserpentinite is commonly used as an ornamental stone, like green marble, in public buildings,includingtheUnitedNationsinNewYork.Thechemicalreactionthatformstherockhasacquiredtheforbidding name of ‘serpentinisation’, but all thismeans is that olivine reacts withwater to formserpentinite.Thewasteproductsofthisreactionarekeytotheoriginoflife.

Olivineisrichinferrousironandmagnesium.Theferrousironisoxidisedbywatertotherustyferric oxide form. The reaction is exothermic (releasing heat), and generates a large amount ofhydrogengas,dissolvedinwarmalkalinefluidscontainingmagnesiumhydroxides.Becauseolivineiscommonin theearth’smantle, this reactionoccurs largelyon theseafloor,close to the tectonicspreading centres,where freshmantle rocks are exposed to oceanwaters.Mantle rocks are rarelyexposed directly – water percolates down beneath the sea floor, sometimes to depths of severalkilometres,whereitreactswitholivine.Thewarm,alkaline,hydrogen-richfluidsproducedaremorebuoyantthanthedescendingcoldoceanwater,andbubblebackuptotheseafloor.Theretheycool,andreactwithsaltsdissolvedintheocean,precipitatingintolargeventsontheseafloor.

Unlike black smokers, alkaline vents have nothing to do with magma, and so are not founddirectlyabovethemagmachambersatthespreadingcentres,buttypicallysomemilesaway.Theyarenot superheated, butwarm,with temperatures of 60 to 90°C.They are not open chimneys, ventingdirectly into the sea, but riddled with a labyrinth of interconnected micropores. And they are notacidic, but strongly alkaline.Or at least, these are theproperties thatRussell predicted in the early1990sonthebasisofhistheory.Hiswasaloneandimpassionedvoiceatconferences,arguingthatscientistsweremesmerised by the dramatic vigour of black smokers, and overlooking the quietervirtuesofalkalinevents.Notuntilthediscoveryofthefirstknownsubmarinealkalineventintheyear2000, dubbedLostCity, did researchers really begin to listen. LostCity, remarkably, conforms toalmostallofRussell’spredictions,rightdowntoits location,some10milesfromthemid-AtlanticRidge.Asithappens,thiswasthetimethatIfirstbeganthinkingandwritingaboutbioenergeticsinrelationtotheoriginsoflife(mybookOxygenwaspublishedin2002).Theseideaswereimmediatelyappealing: for me, the wonderful reach of Russell’s hypothesis is that, uniquely, it ties in naturalprotongradientstotheoriginoflife.Thequestionis:how,exactly?

TheimportanceofbeingalkalineAlkalinehydrothermalventsprovideexactlytheconditionsrequiredfortheoriginoflife:ahighfluxofcarbonandenergythatisphysicallychannelledoverinorganiccatalysts,andconstrainedinawaythatpermitstheaccumulationofhighconcentrationsoforganics.Thehydrothermalfluidsarerichindissolvedhydrogen,with lesserquantitiesofother reducedgases includingmethane,ammoniaandsulphide.LostCityandotherknownalkalineventsaremicroporous–thereisnocentralchimney,butthe rock itself is like a mineralised sponge, with thin walls separating interconnected pores,

micrometrestomillimetresinscale,altogetherformingavast labyrinththroughwhichthealkalinehydrothermalfluidspercolate(Figure13).Becausethesefluidsarenotsuperheatedbymagma,theirtemperaturesfavournotonlythesynthesisoforganicmolecules(moreonthissoon)butalsoslowerratesofflow.Ratherthanbeingpumpedoutatafuriousspeed,thefluidswendtheirwaygentlyacrosscatalyticsurfaces.Andtheventspersistformillennia,atleast100,000yearsinthecaseofLostCity.AsMikeRussell points out, that’s 1017microseconds, amoremeaningful time unit formeasuringchemistry.Timeaplenty.

Thermal currents through microporous labyrinths have a remarkable capacity to concentrateorganicmolecules(includingaminoacids,fattyacidsandnucleotides)toextremelevels, thousandsorevenmillionsoftimesthestartingconcentration,bywayofaprocessknownasthermophoresis.Thisisalittlelikethetendencyofsmallitemsoflaundrytoaccumulateinsideaduvetcoverinthewashing machine. It all depends on kinetic energy. At higher temperatures, small molecules (andsmall items of laundry) dance around, with some freedom to move in all directions. As thehydrothermal fluids mix and cool, the kinetic energy of the organic molecules falls, and theirfreedom todancearounddiminishes (which iswhathappens to socks inside theduvetcover).Thatmeans theyare less likely to leaveagain,andso theyaccumulate in these regionsof lowerkineticenergy (Figure 13). The power of thermophoresis depends in part on molecular size: largemolecules, such as nucleotides, are retained better than smaller ones. Small end products, such asmethane,areeasilylostfromthevent.Allinall,continuoushydrothermalflowthroughmicroporousventsshouldactivelyconcentrateorganicsbyadynamicprocess thatdoesnotalter thesteady-stateconditions(unlikefreezingorevaporation)butactuallyisthesteadystate.Betterstill,thermophoresisdrives the formationofdissipative structureswithinventpores,bypromoting interactionsbetweenorganics. These can spontaneously precipitate fatty acids into vesicles, and possibly polymeriseaminoacidsandnucleotidesintoproteinsandRNA.Suchinteractionsareamatterofconcentration:anyprocessthatincreasesconcentrationpromoteschemicalinteractionsbetweenmolecules.

Thismaysound toogood tobe true, and inone sense it is.ThealkalinehydrothermalventsatLostCityarehometoplentyoflifetoday,albeitmostlyratherundramaticbacteriaandarchaea.Theyalso produce low concentrations of organics, including methane and trace amounts of otherhydrocarbons.Buttheseventsarecertainlynotgivingrisetonewlifeformstoday,norevenformingarichmilieuoforganicsbythermophoresis.That’spartlybecausethebacteriaalreadylivingtherehooverupanyresourcesveryeffectively;buttherearealsomorefundamentalreasons.

Figure13ExtremeconcentrationoforganicsbythermophoresisAAsectionofanalkalinehydrothermalventfromLostCity,showingtheporousstructureofthewalls–thereisnocentralchimneybutaninterconnectedlabyrinthofpores,micrometrestomillimetresindiameter.BOrganicssuchasnucleotidescantheoreticallyconcentrateuptomorethan1000timestheirstartingconcentrationbythermophoresis,drivenbyconvectioncurrentsandthermaldiffusionintheventpores,illustratedinC.DAnexampleofexperimentalthermophoresisfromourreactoratUniversityCollegeLondon,showing5000-foldconcentrationofafluorescentorganicdye(fluorescein)inamicroporousceramicfoam(diameter:9cm).EEvengreaterconcentrationofthefluorescentmoleculequinine,atleast1million-foldinthiscase.

Just as black smoker vents were not exactly the same 4 billion years ago, so alkalinehydrothermalventsmusthavebeendifferentintheirchemistry.Certainaspectswouldhavebeenverysimilar. The process of serpentinisation itself should not have been any different: the samewarm,hydrogenrich,alkalinefluidsoughttohavebubbleduptotheseafloor.Butoceanchemistrywasverydifferentthenandthatshouldhavealteredthemineralcompositionofalkalinevents.Today,LostCityis composedmostly of carbonates (aragonite),while other similar vents discoveredmore recently(suchasStrýtan, innorthern Iceland)arecomposedofclays.Back in theHadeanoceans,4billionyears ago,we can’t be surewhat kind of structureswould have formed, but therewere twomaindifferencesthatmusthavehadabigeffect:oxygenwasabsent,andCO2concentrationintheairandocean was much greater. These differences should have made ancient alkaline vents far moreeffectiveasflowreactors.

Intheabsenceofoxygen,irondissolvesinitsferrousform.Weknowthattheearlyoceanswerefullofdissolvediron,becauselateronitallprecipitatedoutasvastbandedironformations,asnoted

inChapter1.Muchof thisdissolved ironcamefromblacksmoker (volcanic)vents.Wealsoknowthatironwouldhaveprecipitatedoutinalkalinehydrothermalvents–notbecausewehaveseenit,butbecausetherulesofchemistrydictateit;andwecansimulateitinthelab.Inthiscase,theironwouldhaveprecipitated as ironhydroxides and iron sulphides,which formcatalytic clusters that are stillfound in enzymes driving carbon and energymetabolism today – proteins like ferredoxin. In theabsence of oxygen, then, the mineral walls of alkaline vents would have contained catalytic ironminerals,likelydopedwithotherreactivemetalssuchasnickelandmolybdenum(whichdissolvesinalkaline fluids). Now we are getting close to a real flow reactor: hydrogen-rich fluids circulatethrough a labyrinth of micropores with catalytic walls that concentrate and retain products whileventingwaste.

Butwhatexactlyisreacting?Herewearereachingthecruxofthematter.ThisiswherethehighCO2 levels enter the equation. The alkaline hydrothermal vents of today are relatively starved ofcarbon,becausemuchoftheavailableinorganiccarbonprecipitatesoutascarbonate(aragonite)inthe vent walls. Back inHadean times, 4 billion years ago, our best guess is that CO2 levels weresubstantially higher, perhaps 100–1,000 times greater than today. Beyond relieving the carbonlimitationofprimordialvents,highCO2levelswouldalsohavemadetheoceansmoreacidic,inturnmakingithardertoprecipitatecalciumcarbonate.(Thisisthreateningcoralreefstoday,asrisingCO2

begins to acidifymodern oceans.) The pH ofmodern oceans is around 8, slightly alkaline. In theHadean, the oceans are likely to have been neutral ormildly acidic, perhaps pH5–7, although theactual value is practically unconstrained by geochemical proxies. The combination of high CO2,mildlyacidicoceans,alkalinefluids,andthin,FeS-bearingventwallsiscrucial,becauseitpromoteschemistrythatwouldotherwisenothappeneasily.

Two broad principles govern chemistry: thermodynamics and kinetics. Thermodynamicsdetermineswhichstatesofmatteraremorestable–whichmoleculeswillform,givenunlimitedtime.Kineticsrelatestospeed–whichproductswillforminalimitedtime.Intermsofthermodynamics,CO2willreactwithhydrogen(H2)toformmethane(CH4).Thisisanexothermicreaction,meaningthat it releases heat. That in turn increases the entropy of the surroundings, at least under certainconditions, favouring the reaction. Given the opportunity, it should occur spontaneously. Theconditions required include moderate temperatures and an absence of oxygen. If the temperatureclimbstoohigh,CO2ismorestablethanmethane,asalreadynoted.Likewise,ifoxygenispresent,itwill react preferentially with hydrogen to form water. Four billion years ago, the moderatetemperaturesandanoxicconditionsinalkalineventsshouldhavefavouredthereactionofCO2withH2 to form CH4. Even today, with some oxygen present, Lost City produces a small amount ofmethane.ThegeochemistsJanAmendandTomMcCollomhavegoneevenfurtherandcalculatedthatthe formation of organic matter fromH2 and CO2 is thermodynamically favoured under alkalinehydrothermalconditions,solongasoxygenisexcluded.That’sremarkable.Undertheseconditions,between25and125°C, theformationof totalcellbiomass(aminoacids,fattyacids,carbohydrates,nucleotidesandsoon)fromH2andCO2isactuallyexergonic.ThismeansthatorganicmattershouldformspontaneouslyfromH2andCO2undertheseconditions.Theformationofcellsreleasesenergyandincreasesoverallentropy!

But–andthisisabigbut–H2doesnoteasilyreactwithCO2.Thereisakineticbarrier,meaningthat although thermodynamics says they should react spontaneously, some other obstacle stops itfromhappening rightaway.H2 andCO2 are practically indifferent to eachother.To force them toreact together requires an input of energy – a firecracker to break the ice. Now they will react,initiallytoformpartiallyreducedcompounds.CO2canonlyacceptelectronsinpairs.Additionoftwo

electronsgivesformate(HCOO–);twomoregiveformaldehyde(CH2O);anothertwogivemethanol(CH3OH); and a final pair gives the fully reducedmethane (CH4). Life, of course, is notmade ofmethane,butitisonlypartiallyreducedcarbon,roughlyequivalentinitsredoxstatetoamixtureofformaldehyde and methanol. This means there are two important kinetic barriers relating to theoriginsoflifefromCO2andH2.Thefirstneedstobeovercome,togettoformaldehydeormethanol.Thesecondmustnotbeovercome!HavingcoaxedH2andCO2intoawarmembrace,thelastthingacell needs is for the reaction to run straight through to methane. Everything would dissipate anddisperseasagas,andthatwouldbethat.Life,itseems,knowsexactlyhowtolowerthefirstbarrierandexactlyhowtokeep thesecondbarrier raised(onlydropping itwhen itneeds theenergy).Butwhathappenedatthebeginning?

This is the stumblingpoint. If itwere easy togetCO2 to reactwithH2 economically –withoutputtinginmoreenergythanwegetout–thenwewouldhavedoneitbynow.Thatwouldbeahugestep to solving the world’s energy problems. Imagine it: mimic photosynthesis to split water,releasingH2andO2.That’sbeendone,andcouldpotentiallydriveahydrogeneconomy.Buttherearepracticaldrawbackstoahydrogeneconomy.HowmuchbettertoreacttheH2withCO2fromtheairtomake natural gas or even synthetic gasoline! Thenwe can go right on burning gas in our powerstations.ThatwouldbalanceCO2 emissionswithCO2 capture, halting the rise in atmosphericCO2

levels and relieving our dependence on fossil fuels. Energy security. The returns could hardly begreater, and yetwe have still not succeeded in driving this simple reaction economically.Well…that’swhatthesimplestlivingcellsdoallthetime.Methanogens,forexample,getalltheenergyandallthecarbonneededtogrowfromreactingH2withCO2.Butmoredifficult:howcouldithavebeendonebeforetherewereanylivingcells?Wächtershäuserdismissedthisasimpossible:lifecouldnothavestartedfromthereactionofCO2andH2,hesaid,theysimplywon’treact.8EvenrampingupthepressuretotheintensepressuresfoundseveralkilometresdowninhydrothermalventsatthebottomoftheoceansdoesnotforceH2toreactwithCO2.That’swhyWächtershäusercameupwiththeideaof‘pyritespulling’inthefirstplace.

Butthereisonepossibleway.

ProtonpowerRedoxreactionsinvolvethetransferofelectronsfromadonor(H2inthiscase)toanacceptor(CO2).Thewillingnessofamoleculetotransferitselectronsisconnotedintheterm‘reductionpotential’.Theconventionisnothelpful,butiseasyenoughtounderstand.Ifamolecule‘wants’toberidofitselectrons, it isassignedanegativevalue; themore that itwants tobe ridof itselectrons, themorenegativeisthereductionpotential.Conversely,ifanatomormoleculecraveselectronsandwillpickthemupfromalmostanywhere,itisassignedapositivevalue(youcouldthinkofitasthepowerofattractionfornegativelychargedelectrons).Oxygen‘wants’tograbelectrons(oxidisingwhateverittakesthemfrom),givingitaverypositivereductionpotential.Allthesetermsareinfactrelativetotheso-calledstandardhydrogenelectrode,butwedon’tneedtoworryaboutthathere.9Thepointisthatamoleculewithanegativereductionpotentialwilltendtogetridofitselectrons,passingthemontoanymoleculewithamorepositivereductionpotential,butnottheotherwayaround.

That’s the problem with H2 and CO2. At neutral pH (7.0), the reduction potential of H2 istechnically –414 mV. If H2 gives up its two electrons, that leaves behind two protons, 2H+. Thereduction potential for hydrogen reflects this dynamic balance – the tendency of H2 to lose itselectrons,becomingH+,andthetendencyof2H+topickupelectronstoformH2.IfCO2weretopick

upthoseelectrons,itwouldbecomeformate.Butformatehasareductionpotentialof–430mV.Thatmeans itwill tend topasselectronson toH+ to formCO2andH2.Formaldehyde isevenworse. Itsreductionpotential is about–580mV. It is extremely reluctant tohangon to its electrons, andwilleasilypassthemontoprotonstoformH2.ThuswhenconsideringpH7,Wächtershäuseriscorrect:thereisnowaythatH2canreduceCO2.Butofcoursesomebacteriaandarchaealivefromexactlythisreaction,soitmustbepossible.We’lllookintothedetailsofhowtheydothatinthenextchapter,astheyaremorerelevant to thenextstageofourstory.Fornow,allweneedtoknowis thatbacteriagrowingfromH2andCO2canonlygrowwhenpoweredbyaprotongradientacrossamembrane.Andthat’sahelluvaclue.

The reduction potential of a molecule often depends on pH, which is to say on protonconcentration.Thereasonissimpleenough.Transferofanelectrontransfersanegativecharge.Ifthemoleculethat isreducedcanalsoacceptaproton, theproductbecomesmorestable,as thepositivecharge of the proton balances the negative charge of the electron. Themore protons available tobalancecharges,themoreeasilyanelectrontransferwillproceed.Thatmakesthereductionpotentialmore positive – it becomes easier to accept a pair of electrons. In fact, the reduction potentialincreasesbyabout59mVforeachpHunitofacidity.Themoreacidicthesolution,theeasieritistotransfer electronson toCO2 to produce formate or formaldehyde.Unfortunately, exactly the sameappliestohydrogen.Themoreacidicthesolution,theeasieritistotransferelectronsontoprotonstoformH2gas.SimplychangingpHthereforehasnoeffectatall. It remains impossible to reduceCO2withH2.

Butnowthinkofaprotongradientacrossamembrane.Theprotonconcentration–theacidity–isdifferentonoppositesidesofthemembrane.Exactlythesamedifferenceisfoundinalkalinevents.Alkaline hydrothermal fluids wend their way through the labyrinth of micropores. So do mildlyacidic ocean waters. In some places there is a juxtaposition of fluids, with acidic ocean waterssaturated in CO2 separated from alkaline fluids rich in H2 by a thin inorganic wall, containingsemiconducting FeS minerals. The reduction potential of H2 is lower in alkaline conditions: itdesperately ‘wants’ to be rid of its electrons, so the left-over H+ can pair up with the OH– in thealkaline fluids to form water, oh so stable. At pH 10, the reduction potential of H2 is –584 mV:strongly reducing. Conversely, at pH 6, the reduction potential for formate is –370m V, and forformaldehyde it is–520mv. In otherwords, given this difference in pH, it is quite easy forH2 toreduceCO2 tomake formaldehyde. The only question is: how are electrons physically transferredfromH2toCO2?Theanswerisinthestructure.FeSmineralsinthethininorganicdividingwallsofmicroporousventsconductelectrons.Theydon’tdoitnearlyaswellasacopperwire,buttheydoit,nonetheless.Andso in theory, thephysicalstructureofalkalineventsshoulddrive thereductionofCO2byH2,toformorganics(Figure14).Fantastic!

Figure14HowtomakeorganicsfromH2andCO2

ATheeffectofpHonreductionpotential.Themorenegativethereductionpotential,themorelikelyacompoundistotransferoneormoreelectrons;themorepositive,themorelikelyitistoacceptelectrons.NotethatthescaleontheYaxisbecomesmorenegativewithheight.AtpH7,H2can’ttransferelectronstoCO2toproduceformaldehyde(CH2O);thereactionwouldratherproceedintheoppositedirection.However,ifH2isatpH10,asinalkalinehydrothermalvents,andCO2isatpH6,asinearlyoceans,thereductionofCO2toCH2Oistheoreticallypossible.BInamicroporousvent,fluidsatpH10andpH6couldbejuxtaposedacrossathinsemiconductingbarriercontainingFeSminerals,facilitatingthereductionofCO2toCH2O.FeSishereactingasacatalyst,asitstilldoesinourownrespiration,transferringelectronsfromH2toCO2.

Butisittrue?Hereisthebeautyofscience.Thisisasimpletestablequestion.That’snottosayitiseasy to test; I’vebeen trying todo so in the lab,with thechemistBarryHerschyandPhDstudentsAlexandraWhicherandEloiCamprubi,forawhilenow.WithfundingfromtheLeverhulmeTrust,we’ve built a small benchtop reactor to try to drive these reactions. Precipitating these thin,semiconducting FeS walls in the lab is not straightforward. There’s also the problem thatformaldehydeisnotstable–it‘wants’topassitselectronsbackontoprotons,toformH2andCO2

once again, and it will do that more easily in acidic conditions. The exact pH and hydrogenconcentrationiscritical.Andplainlyit’snoteasytosimulatethecolossalscaleofrealventsinthelab–tensofmetreshigh,operatingatintensepressures(whichpermitsamuchhigherconcentrationofgasessuchashydrogen).Yetdespitealltheseissues,theexperimentissimpleinthesensethatitisacircumscribed,testablequestion,ananswertowhichcouldtellusagreatdealabouttheoriginoflife.Andwehave indeedproduced formate, formaldehydeandother simpleorganics (including ribose

anddeoxyribose).Fornow,let’stakethetheoryatfacevalue,andassumethatthereactionwillindeedtakeplaceas

predicted.Whatwillhappen?Thereshouldbeaslowbutsustainedsynthesisoforganicmolecules.We’lldiscusswhichones,andhowexactlytheyshouldbeformed,inthenextchapter;fornowlet’sjust note that this is another simple testable prediction. Once formed, these organics should beconcentrated to thousands of times their starting concentration by thermophoresis, as discussedearlier, promoting the formation of vesicles and perhaps polymers like proteins. Once again, thepredictionsthatorganicswillconcentrateandthenpolymerisearetestabledirectlyinthelab,andwearetryingtodoso.Firststepsareencouraging:thefluorescentdyefluorescein,similarinsizetoanucleotide,concentratesatleast5000-foldinourthrough-flowreactor,andquininemayconcentrateevenmore(Figure13).

Sowhatdoesallthisstuffaboutreductionpotentialsreallymean?Itatonceconstrainsandopenswide theconditionsunderwhich life shouldevolve in theuniverse.This isoneof the reasons thatscientistsoften lookas if theyare in theirown littleworld, lost in abstract thought about themostarcanedetails.CantherepossiblybeanymightyimportaboutthefactthatthereductionpotentialofhydrogenfallswithpH?Yes!Yes!Yes!Underalkalinehydrothermalconditions,H2shouldreactwithCO2toformorganicmolecules.Underalmostanyotherconditions,itwillnot.Inthischapter,Ihavealreadyruledoutvirtuallyallotherenvironmentsasworkablesettingsfortheoriginoflife.Wehaveestablishedonthermodynamicgroundsthattomakeacellfromscratchrequiresacontinuousflowofreactive carbon and chemical energy across rudimentary catalysts in a constrained through-flowsystem. Only hydrothermal vents provide the requisite conditions, and only a subset of vents –alkalinehydrothermalvents–matchall theconditionsneeded.Butalkalineventscomewithbothaseriousproblemandabeautifulanswer to theproblem.Theseriousproblemis that theseventsarerichinhydrogengas,buthydrogenwillnotreactwithCO2toformorganics.Thebeautifulansweristhat the physical structure of alkaline vents – natural proton gradients across thin semiconductingwalls–will(theoretically)drivetheformationoforganics.Andthenconcentratethem.Tomymind,atleast,allthismakesagreatdealofsense.Addtothisthefactthatalllifeonearthuses(stilluses!)protongradientsacrossmembranestodrivebothcarbonandenergymetabolism,andI’mtemptedtocry,withthephysicistJohnArchibaldWheeler,‘Oh,howcouldithavebeenotherwise!Howcouldweallhavebeensoblindforsolong!’

Let’scalmdownandfinish.Isaidthatreductionpotentialsbothconstrainandopentheconditionsunderwhichlifeshouldevolve.Bythisanalysis,theconditionsthatbestencouragetheoriginsoflifearefoundinalkalinevents.Perhapsyourheartissinking…whynarrowdowntheoptionssotightly?Surelytheremustbeotherways!Well,maybe.Inaninfiniteuniverse,anything ispossible;but thatdoesn’tmake it probable.Alkalinevents areprobable.Theyare formed, remember,bya chemicalreactionbetweenwaterandthemineralolivine.Rock.Infact,oneofthemostabundantmineralsintheuniverse,amajorpartofinterstellardustandtheaccretiondiscsfromwhichplanets,includingtheearth,areformed.Serpentinisationofolivinemayevenoccurinspace,hydratingtheinterstellardust.Whenourplanetaccreted,thiswaterwasdrivenoffbytherisingtemperaturesandpressures,givingrise, some say, to the earth’s oceans.However thatmaybe, olivine andwater are twoof themostabundantsubstancesintheuniverse.AnotherisCO2.Thisisacommongasintheatmosphereofmostplanetsinthesolarsystem,andhasevenbeendetectedintheatmosphereofexoplanetsinotherstellarsystems.

Rock,water andCO2: the shopping list for life.Wewill find themonpractically allwet rockyplanets.By the rules of chemistry and geology, theywill formwarm alkaline hydrothermal vents,with proton gradients across thin-walled catalyticmicropores.We can count on that. Perhaps their

chemistryisnotalwaysconducivetolife.Yetthisisanexperimentgoingonrightnow,onasmanyas40billionearth-likeplanetsintheMilkyWayalone.Weliveinacosmicculturedish.Howoftentheseperfectconditionsgiverisetolifedependsonwhathappensnext.

Footnotes1Basedonthechemistryofzirconcrystalsandtheearliestrocks,theearlyearthisnowbelievedtohavehadarelativelyneutralatmosphere,reflectingvolcanicdegassing,andcomposedmostlyofcarbondioxide,nitrogen,andwatervapour.2Thatinnocuousphrase‘plausibleprimordialconditions’actuallyconcealsamultitudeofsins.Onthefaceofit,itmeanssimplythatthecompoundsandconditionsusedcouldreasonablyhavebeenfoundontheearlyearth.ItisindeedplausiblethattherewassomecyanideintheHadeanoceans;alsothattemperaturescouldrangebetweenseveralhundreddegrees(inhydrothermalvents)andfreezingontheearlyearth.Thetroubleisthatrealisticconcentrationsoforganicsinasouparefarlowerthantendtobeusedinthelab;anditishardlyfeasibletohavebothheatingandfreezinginoneandthesameenvironment.Soyes:alltheseconditionsmayhaveexistedsomewhereontheplanet,buttheycouldonlydriveprebioticchemistryifthewholeplanetistakenasasingleunit,engagedinacoherentsetofexperimentsasifitwereasyntheticchemist’slab.Thatisimplausibleintheextreme.3Ihavediscussedsoupasifitwas‘madeonearth’bylightningorUVradiation.Analternativesourceoforganicsisdeliveryfromspacebychemicalpanspermia.Thereisnodoubtthatorganicmoleculesareabundantinspaceandonasteroids;andtherecertainlywasasteadydeliveryoforganicstoearthonmeteorites.Butoncehere,theseorganicsmusthavedissolvedintheoceans,atbeststockingaprimordialsoup.Thatmeansthatchemicalpanspermiaisnoanswertotheoriginoflife:itsuffersfromthesameintractableproblemsassoup.Thedeliveryofwholecells,asadvocatedbyFredHoyle,FrancisCrick,andothers,islikewisenosolution:itsimplypushestheproblemsomewhereelse.Wemayneverbeabletosayexactlyhowlifeoriginatedonearth,butwecanexploretheprinciplesthatmustgoverntheemergenceoflivingcellshereoranywhereelse.Panspermiafailsutterlytoaddressthoseprinciples,andsoisirrelevant.4ThisisanappealtoOccam’srazor,thephilosophicalbasisofallscience:assumethesimplestnaturalcause.Thatanswermightturnoutnottobecorrect,butweshouldnotresorttomorecomplexreasoningunlessitisshowntobenecessary.Wemightultimatelyneedtoinvokecelestialmachinationstoexplaintheoriginofreplication,whenallotherpossibilitieshavebeendisproved(thoughIdoubtit);butuntilthenweshouldnotmultiplycauses.Thisissimplyawayofapproachingaproblem;buttheremarkablesuccessofscienceshowsthatitisaveryeffectiveapproach.5Afamiliarexampleisthealcoholcontentofwine,whichcannotriseaboveabout15%byalcoholicfermentationalone.Asalcoholbuildsup,itblockstheforwardreaction(fermentation),preventingtheformationofanymorealcohol.Unlessthealcoholisremoved,fermentationgrindstoahalt:thewinehasreachedthermodynamicequilibrium(ithasbecomesoup).Spiritssuchasbrandyareproducedbydistillingwine,therebyconcentratingthealcoholfurther;Ibelievewearetheonlylifeformthathasperfecteddistillation.6Idon’treallymeanproteins,Imeanpolypeptides.Thesequenceofaminoacidsinaproteinisspecifiedbyagene,inDNA.Apolypeptideisastringofaminoacidsjoinedtogetherbythesametypeofbond,butisusuallymuchshorter(perhapsjustafewaminoacids)andtheirsequencedoesnotneedtobespecifiedbyagene.Shortpolypeptideswillformspontaneouslyfromaminoacids,inthepresenceofachemical‘dehydrating’agentsuchaspyrophosphateoracetylphosphate,whichareplausibleabioticprecursorsofATP.7Wächtershäusertransformedperceptionsabouttheoriginoflife.Hedismissedprimordialsoupinnouncertainterms,beginningaprolongedandbitterargumentinthejournalswithStanleyMiller.Here’sonebroadsidefromWächtershäuser,foranyonewhothinksthatscienceisinsomesensedispassionate:‘Theprebioticbroththeoryhasreceiveddevastatingcriticismforbeinglogicallyparadoxical,incompatiblewiththermodynamics,chemicallyandgeochemicallyimplausible,discontinuouswithbiologyandbiochemistry,andexperimentallyrefuted.’8I’msadtosaythatthisisnowtheconsideredviewofMikeRusselltoo.HehastriedandfailedtoforceCO2toreactwithH2toproduceformaldehydeandmethanol,andnolongerbelievesitispossible.IncollaborationwithWolfgangNitschke,henowcallsonothermolecules,notablymethane(producedinvents)andnitricoxide(arguablypresentinearlyoceans)todrivetheoriginoflife,viaaprocessanalogoustomodernmethanotrophicbacteria.BillMartinandIdisagreewiththem,forreasonsthatIwon’tdiscussherebut,ifyouarereallyinterested,youwillfindinSousaetal.,giveninFurtherReading.Thisisnotatrivialquestion,asitdependsontheoxidationstateoftheearlyoceans,butitisamenabletoexperimentaltesting.Amajoradvanceoverthepastdecadeorsoispreciselythatthealkalineventtheoryisnowbeingconsideredveryseriouslybyawideninggroupofscientists,whoareformulatingspecificanddistincttestablehypotheseswithinasimilaroverallframework,andsettingouttotestthemexperimentally.Thisishowscienceshouldwork,andIdon’tdoubtthatallofuswouldbehappytobeprovedwrongondetails,whilehoping(naturally)thattheoverallframeworkstandsrobust.9OK,soyouareworried…Reductionpotentialismeasuredinmillivolts.Imagineanelectrodemadeofmagnesiuminsertedintoabeakerofmagnesiumsulphatesolution.Magnesiumhasastrongtendencytoionise,releasingmoreMg2+ionsintothesolution,andleavingelectronsbehindontheelectrode.Thatimpartsanegativecharge,whichcanbequantifiedrelativetoastandard‘hydrogenelectrode’.Thisisaninertplatinumelectrode,inanatmosphereofhydrogen,whichisinsertedintoasolutionofprotonsatpH0(1gramofprotonsperlitre)and25°C.Ifthemagnesiumandstandardhydrogenelectrodesareconnectedbyawire,electronswillflowfromthenegativemagnesiumelectrodetotherelativelypositive(infactit’sjustlessnegative)hydrogenelectrode,toformhydrogengas,byabstractingprotonsfromtheacid.Magnesiumactuallyhasaverynegativereductionpotential(–2.37volts,tobeprecise)comparedwiththestandardhydrogenelectrode.NoticethatallthesevaluesareatpH0,bytheway.InthemaintextIsaythatthereductionpotentialofhydrogenis–414mVatpH7.That’sbecausethereductionpotentialgetsmorenegativebyabout–59mVforeachpHunitincrease(see

maintext).

‘I

4

THEEMERGENCEOFCELLS

think’,wroteDarwin:justthosetwowords,scrawlednexttoasketchofabranchingtreeoflife,inanotebookfrom1837.ThatwasonlyayearafterreturningfromthevoyageoftheBeagle.Twenty-twoyearslater,amoreartfullydrawntreewastheonlyillustrationinTheOriginofSpecies.TheideaofatreeoflifewassocentraltoDarwin’sthinking,andtothecurrencyofevolutionarybiologyeversince,thatit’sshockingtobetolditiswrong,asNewScientistdidinlargelettersontheirfrontcoverin2009, 150years after thepublicationofDarwin’sOrigin. The cover flirted shamelesslywith anextendedreadership,butthearticleitselfwasmoremoderateintoneandmadeaspecificpoint.Toadegree that is very hard to define, the tree of life is indeedwrong. That does notmeanDarwin’smajorcontributiontoscience,evolutionbynaturalselection,isalsowrong:itmerelyshowsthathisknowledgeofhereditywas limited.That’snotnews. It iswellknown thatDarwinknewnothingofDNA,or genes, orMendel’s laws, let alone the transfer of genes betweenbacteria, so his viewofhereditywas through a glass, darkly.None of that discreditsDarwin’s theory of natural selection;hencethecoverwascorrectinanarrowtechnicalsense,butgrosslymisleadinginadeepersense.

Whatthecoverdiddo,though,wasbringaseriousissuetotheforeground.Theideaofatreeoflife assumes ‘vertical’ inheritance, inwhich parents pass on copies of genes to their offspring bysexualreproduction.Overgenerations,genesarepassedonmostlywithinaspecies,withrelativelylittle intercourse between species. Populations that become reproductively isolated diverge slowlyovertime,astheinteractionsbetweenthemdecline,andultimatelyformnewspecies.Thisgivesrisetothebranchingtreeoflife.Bacteriaaremoreequivocal.Theydon’thavesexintheeukaryoticway,sotheydon’tformniceneatspeciesinthesamewayeither.Definingtheterm‘species’inbacteriahasalwaysbeenproblematic.Buttherealdifficultywithbacteriaisthattheyspreadtheirgenesaroundby‘lateral’genetransfer,passinghandfulsofgenesfromonetoanother likesmallchange,aswellasbequeathingacopyoftheirfullgenometodaughtercells.Noneofthisunderminesnaturalselectioninanysense–itisstilldescentwithmodification;it’sjustthatthe‘modification’isachievedinmorewaysthanweoncethought.

Theprevalenceoflateralgenetransferinbacteriaposesadeepquestionaboutwhatwecanknow–aquestionasfundamentalinitsownwayasthecelebrated‘uncertaintyprinciple’inphysics.Almostanytreeoflifeyoulookatfromthemoderneraofmoleculargeneticswillbebasedonasinglegene,chosencarefullybythepioneerofmolecularphylogenetics,CarlWoese–thegeneforsmallsubunitribosomalRNA.1Woeseargued(withsomejustification)thatthisgeneisuniversalacrosslife,andisrarely, if ever, transferred by lateral gene transfer. It therefore supposedly indicates the ‘one truephylogeny’ofcells(Figure15).Inthelimitedsensethatonecellgivesrisetodaughtercells,andthat

thesedaughtercellsarealwayslikelytosharetheribosomalRNAoftheirparent,thisistrue.Butwhathappens if, overmany generations, other genes are replaced by lateral gene transfer? In complexmulticellularorganisms,thatrarelyhappens.WecansequencetheribosomalRNAofaneagle,anditwilltellusthatthisisabird.Wecaninferthatithasabeak,feathers,claws,wings,layseggs,andsoon.That’s because vertical inheritance ensures that there is always a good correlation between theribosomal ‘genotype’ and the overall ‘phenotype’: the genes encoding all these birdlike traits arefellow travellers; they sail down the generations together, beingmodifiedover time, certainly, butrarelyinadramaticfashion.

Figure15Thefamousbutmisleadingthree-domainstreeoflifeThetreeoflifeasportrayedbyCarlWoesein1990.Thetreeisbasedonasinglehighlyconservedgene(forsmallsubunitribosomalRNA)andisrootedusingthedivergencebetweenpairsofgenesfoundinallcells(whichmustthereforehavealreadybeenduplicatedinthelastuniversalcommonancestor,LUCA).Thisrootingsuggeststhatthearchaeaandtheeukaryotesaremorecloselyrelatedtoeachotherthaneithergroupistothebacteria.However,whilethatisgenerallytrueforacoreofinformationalgenes,itisnottrueforthemajorityofgenesineukaryotes,whicharemorecloselyrelatedtobacteriathanarchaea.Thisiconictreeisthereforeprofoundlymisleading,andshouldbeseenstrictlyasatreeofonegeneonly:itismostcertainlynotatreeoflife!

Butnowimaginethatlateralgenetransferpredominates.SowesequencetheribosomalRNA,andittellsuswearedealingwithabird.Onlynowdowelookatthis‘bird’.Itturnsouttopossessatrunk,sixlegs,eyesonitsknees,fur;itproduceseggslikefrogspawn,lackswingsandhowlslikeahyena.Yes,ofcoursethatisabsurd;butthisispreciselytheproblemthatwefacewithbacteria.Monstrouschimerasregularlystareusintheface;butbecausebacteriaaretypicallysmallandmorphologicallysimple,wedon’tscream.Nonetheless,intheirgenesbacteriaarealmostalwayschimeric,andsomearerealmonsters,geneticallyasmangledasmy‘bird’.Phylogeneticistsreallyoughttoscream.Wecan’tinferwhatacellmighthavelookedlike,orhowitmighthavelivedinthepast,onthebasisofitsribosomalgenotype.

What is theuseofsequencingasinglegene if itcan tellusnothingabout thecell that itcomesfrom?Itcanbeuseful,dependingonthetimescaleandtherateofgenetransfer.Iftherateoflateralgenetransferislow(asinplantsandanimals,manyprotists,andsomebacteria)thentherewillbeagoodcorrelationbetweenribosomalgenotypeandphenotype,solongaswearecarefulnottorangetoofarbackintothepast.Butiftherateofgenetransferisfast,thatcorrelationcanbewipedoutveryquickly.ThedifferencebetweenpathogenicvariantsofE.coli andharmlesscommonstrains isnotreflectedintheribosomalRNA,butintheacquisitionofothergenesthatconferaggressivegrowth–asmuchas30%ofthegenomecanvaryindifferentstrainsofE.coli–that’s10timesthevariation

betweenusandchimpanzees,yetwestillcallthemthesamespecies!AribosomalRNAphylogenyisthelastthingyouneedtoknowaboutthesekillerbugs.Conversely,prolongedperiodsoftimewillwipeoutacorrelationeveniftheratesoflateralgenetransferareverylow.Thatmeansitisalmostimpossible toknowhowabacteriumearned its living3billionyearsago,given thatslowratesoftransfercouldhavereplacedessentiallyallofitsgenesmanytimesoverinthatperiod.

And so the conceit behind the tree of life is wrong. The hope is that it may be possible toreconstructtheonetruephylogenyofallcells,toinferhowonespeciesarosefromanother,totracerelatednessbacktothebeginning,ultimatelyallowingustoinferthegeneticmake-upofthecommonancestorof all life on earth. Ifwe could indeeddo that,wewouldknoweverything about that lastancestralcell,fromitsmembranecomposition,totheenvironmentinwhichitlived,tothemoleculesthatfuelleditsgrowth.Butwecannotknowthesethingswiththatprecision.AstrikingtestwaslaidoutbyBillMartin,inavisualparadoxwhichhecallsthe‘amazingdisappearingtree’.Heconsidered48genesthatareuniversallyconservedacrossalllife,andbuiltagenetreeforeachofthesegenes,toshowtherelationshipbetween50bacteriaand50archaea(Figure16).2Atthetipsofthistree,all48genes recovered exactly the same relationship between all 100 species of bacteria and archaea.Likewiseat theverybase:nearlyall48genes ‘agreed’ that thedeepestbranch in the treeof life isbetween the bacteria and the archaea. In other words, the last universal common ancestor, fondlyknownasLUCA,wasthecommonancestorofbacteriaandarchaea.Butwhenitcomestoelucidatingthedeepbrancheswithineitherbacteriaorarchaea,notasinglegenetreecouldagree.All48genesgaveadifferenttree!Theproblemcouldbetechnical(thesignaliserodedbysheerdistance)ortheresult of lateral gene transfer – patterns of vertical descent are destroyed if individual genes areswapped around at random.We don’t knowwhich possibility is true, and at themoment it seemsimpossibletosay.

What does that mean? In essence, it means we can’t determine which species of bacteria orarchaeaarethemostancient.Ifonegenetreesaysthatmethanogensarethemostancientarchaea,thenext tree says theyarenot, so it is practically impossible to reconstructwhichproperties themostancientcellsmighthavehad.Evenifbysomeingeniousmeanswecouldprovethatmethanogensareindeedthemostancientarchaea,westillcouldnotbesurethattheyalwayslivedbymakingmethane,as domodernmethanogens. Lumping genes together to enhance the signal strength does not helpmuchaseachgenemayhavehadadifferenthistory,makinganycompositesignalafabrication.

Figure16The‘amazingdisappearingtree’Thetreecomparesthebranchingof48universallyconservedgenesin50bacteriaand50archaea.All48genesareconcatenatedintoasinglesequence,givinggreaterstatisticalpower(acommonpracticeinphylogenetics);this‘supergene’sequenceisnextusedtobuildatreeshowinghowthe100speciesrelatetoeachother.Eachindividualgeneisthenusedtobuildaseparatetree,andeachofthesetreesiscomparedwiththe‘supergene’treebuiltfromtheconcatenatedgenes.Thestrengthofshadingdenotesthenumberofindividualgenetreesthatcorrespondtotheconcatenatedtreeforeachbranch.Atthebaseofthetree,nearlyallthe48genesrecoverthesametreeastheconcatenatedsequence,clearlyindicatingthatthearchaeaandthebacteriaaregenuinelydeeplydivided.Atthetipsofthebranches,mostindividualgenetreesalsoagreewiththeconcatenatedtree.Butthedeeperbrancheswithinbothgroupshavevanished:notasingleindividualgenetreerecoversthesamebranchingorderastheconcatenatedsequence.Thisproblemcouldbearesultoflateralgenetransfersconfoundingbranchingpatterns,orsimplytheerosionofastatisticallyrobustsignaloveranunimaginable4billionyearsofevolution.

Butthefactthatall48ofBillMartin’suniversalgenesagreethatthedeepestdivergenceinthetreeoflifeisbetweenbacteriaandarchaeaholdsoutsomehope.Ifwecanfigureoutwhichpropertiesareshared by all bacteria and archaea, and which are distinct, presumably arising later in particulargroups,thenwecanputtogethera‘photofit’ofLUCA.Yethereagainwequicklyrunintotrouble:genesfoundinbotharchaeaandbacteriacouldhaveariseninonegroupandbeentransferredintotheothergroupbylateralgenetransfer.Transfersofgenesacrossentiredomainsarewellknown.Ifsuchtransfersoccurredearlyinevolution–intheblankbitsoftheamazingdisappearingtree–thenthesegeneswouldappear todescendvertically fromacommonancestor, even though theydidnot.Themoreusefulthegene,themorelikelyitistohavebeentransferredwidely,earlyoninevolution.Todiscount suchwidespread lateral gene transfer,we are obliged to fall backon genuinely universalgenes,whicharesharedbyrepresentativesofessentiallyeverygroupofbacteriaandarchaea.Thatatleastminimisesthepossibilitythatthesegeneswerepassedaroundbyearlylateralgenetransfer.Theproblemnowisthattherearefewerthan100suchuniversalgenes,aremarkablysmallnumber,andtheypaintaverypeculiarpictureofLUCA.

We’vealreadynoted this strangeportrait inChapter2.Takenat facevalue,LUCAhadproteinsandDNA:theuniversalgeneticcodewasalreadyinoperation,DNAwasreadoffintotranscriptsofRNA, and then translated into proteins on ribosomes, thosemightymolecular factories that buildproteinsinallknowncells.TheremarkablemolecularmachineryneededforreadingoffDNA,andfor protein synthesis, is composed of scores of proteins andRNAs common to both bacteria andarchaea.Fromtheir structuresandsequences thesemachinesappear tohavedivergedveryearly inevolution,andhavenotbeenswappedaroundmuchbylateralgenetransfer.Sofarsogood.Equally,bacteria and archaea are all chemiosmotic, driving ATP synthesis using proton gradients acrossmembranes.TheATPsynthaseenzymeisanotherextraordinarymolecularmachine,onaparwiththeribosome itself and apparently sharing its antiquity. Like the ribosome, the ATP synthase isuniversally conserved across all life, but differs in a few details of its structure in bacteria andarchaea,suggestingthat itdivergedfromacommonancestor inLUCA,withoutmuchconfoundinglateral gene transfer later on.So theATP synthase, like ribosomes,DNAandRNA, seems to havebeenpresentinLUCA.Andthenthereareafewbitsandpiecesofcorebiochemistry,suchasaminoacid biosynthesis and parts of the Krebs cycle, which share common pathways in bacteria andarchaea,againimplyingtheywerepresentinLUCA;butthereisremarkablylittleelse.

What’s different? An astonishing parade. Most of the enzymes used for DNA replication aredistinct in bacteria and archaea. What could be more fundamental than that! Possibly only themembrane–yetit,too,isdistinctinbacteriaandarchaea.Soisthecellwall.Thatmeansbothofthebarriersthatseparatelivingcellsfromtheirenvironmentareutterlydifferentinbacteriaandarchaea.It isalmost impossible toguessexactlywhat theircommonancestormighthavepossessed instead.The listgoeson,but thatwilldo.Of thesix fundamentalprocessesof livingcellsdiscussed in the

previous chapter – carbon flux, energy flux, catalysis,DNA replication, compartmentalisation andexcretion–onlythefirstthreeshareanydeepsimilarity,andeventhenonlyincertainrespects,asweshallsee.

Thereare severalpossibleexplanations.LUCAcouldhavepossessed twocopiesofeverything,andlostonecopyinbacteria,andtheothercopyinarchaea.Thatsoundsinherentlydaft,butitcan’tbe ruled out easily. For example,we know thatmixtures of bacterial and archaeal lipids domakestablemembranes; perhapsLUCAhadboth typesof lipid, andherdescendants later specialisedbylosingoneortheother.Thatmightconceivablybetrueforsometraits,butisnotgeneralisabletoall,as it runs into a problem known as ‘the genome of Eden’. If LUCA had everything, and herdescendants streamlined later on, then shemust have started outwith an enormous genome,muchlarger than anymodernprokaryote.That seems tome to put the cart before the horses –wehavecomplexitybeforesimplicity,and twosolutions toeveryproblem.Andwhydidall thedescendantsloseoneofeverything?Idon’tbuyit;rollonthesecondoption.

ThenextpossibilityisthatLUCAwasaperfectlynormalbacterium,withabacterialmembrane,cell wall and DNA replication. At some later point, one group of descendants, the first archaea,replacedallthesetraitsastheyadaptedtoextremeconditionssuchashightemperaturesinhotvents.Thisisprobablythemostwidelyacceptedexplanation,butittooishardlypersuasive.Ifitistrue,whyare the processes of DNA transcription and translation into proteins so similar in bacteria andarchaea, yet DNA replication so different? Why, if archaeal cell membranes and cell walls helparchaeaadapttohydrothermalenvironments,didextremophilebacterialivinginthesameventsnotreplace theirownmembranesandwallswith thearchaealversions,or somethingsimilar?Whydoarchaea living in the soil or open oceans not replace their membranes and walls with bacterialversions? Bacteria and archaea share the same environments across the world, yet remainfundamentallydifferent in theirgeneticsandbiochemistry inall theseenvironments,despite lateralgenetransferbetweenthetwodomains.It’sjustnotcrediblethatalltheseprofounddifferencescouldreflect adaptation to one extreme environment, and yet then remain fixed in archaea, withoutexception,regardlessofhowinappropriatetheywereforallotherenvironments.

Thatleavesuswiththefinalbarefacedoption.Theapparentparadoxisnotaparadoxatall:LUCAreallywaschemiosmotic,withanATPsynthase,butreallydidnothaveamodernmembrane,oranyofthelargerespiratorycomplexesthatmoderncellsusetopumpprotons.ShereallydidhaveDNA,andtheuniversalgeneticcode,transcription,translationandribosomes,butreallyhadnotevolvedamodernmethodofDNAreplication.Thisstrangephantomcellmakesnosenseinanopenocean,butbeginstoaddupwhenconsideredintheenvironmentofalkalinehydrothermalventsdiscussedinthepreviouschapter.Theclueliesinhowbacteriaandarchaealiveinthesevents–someofthem,atleast,by an apparently primordial process called the acetyl CoA pathway, which bears an uncannyresemblancetothegeochemistryofvents.

TherockyroadtoLUCARight across the entire living world, there are only six different ways of fixing carbon – ofconverting inorganic molecules such as carbon dioxide into organic molecules. Five of thesepathwaysarequitecomplexandrequireaninputofenergytodrivethemforwards,fromthesuninphotosynthesis for example. Photosynthesis is a good example for another reason too: the ‘Calvincycle’,abiochemicalpathwaythat trapscarbondioxideandconverts it intoorganicmoleculeslikesugars, is found only in photosynthetic bacteria (and plants, which acquired these bacteria aschloroplasts).Thismeans that theCalvincycle isunlikely tobeancestral.HadphotosynthesisbeenpresentinLUCA,itmusthavebeenlostsystematicallyfromallarchaea,plainlyafoolishthingtodo

forsuchausefultrick.It isfarmorelikelythattheCalvincyclearoselateron,at thesametimeasphotosynthesis,inthebacteriaalone.Muchthesamegoesforallbutoneoftheotherpathwaystoo.Only one pathway of carbon fixation is found in both the bacteria and archaea, meaning that itplausiblyaroseintheircommonancestor–theacetylCoApathway.

Eventhatclaimisnotquitetrue.TherearesomestrangedifferencesbetweenbacteriaandarchaeaintheacetylCoApathway,whichwewilladdresslaterinthischapter.Fornow,let’sbrieflyconsiderthereasonswhythispathwayhasagoodclaimtobeingancestral,eventhoughthephylogeneticsaretooambiguous tosupportanearlyorigin(neitherdo theydiscount it).Thearchaea that liveby theacetyl CoA pathway are calledmethanogens, the bacteria acetogens. Some trees of life depict themethanogensasbranchingverydeep;othersdepicttheacetogensasbranchingverydeep;andsomedepict both groups as evolving somewhat later on, with their simplicity purportedly reflectingspecialisationandstreamliningrather thananancestral state. Ifwestick tophylogeneticsalone,wemayneverbeanythewiser.Luckily,wedon’thaveto.

TheacetylCoApathwaystartswithhydrogenandcarbondioxide–thesametwomoleculesthatwediscussedinthelastchapterasbeingplentifulinalkalinehydrothermalvents.Aswenotedthen,thereactionbetweenCO2andH2toformorganicsisexergonic,whichistosaythatitreleasesenergy:inprinciplethereactionshouldtakeplacespontaneously.Inpractice,thereisanenergeticbarrierthatpreventsH2andCO2 fromreactingquickly.Methanogensuse theprotongradient toovercome thisbarrier,whichIshallarguewastheancestralstate.Bethatasitmay,methanogensandacetogensbothpowertheirgrowththroughthereactionofH2andCO2alone: that reactionprovidesall thecarbonand all the energy needed for growth. This sets the acetylCoA pathway apart from the other fivepathwaysofcarbonfixation.ThegeochemistEverettShocksummeditupmemorablyas‘afreelunchthatyou’repaidtoeat’.Itmaybeameagrelunch,butintheventsit’sservedallday.

That’snotall.Unlike theotherpathways, theacetylCoApathway is shortand linear.Therearefewerstepsneededtogetfromsimpleinorganicmoleculestothehubofmetabolisminallcells,thesmallbutreactivemoleculeacetylCoA.Don’tbeafraidofwords.CoAstandsforcoenzymeA,whichis an important and universal chemical ‘hook’ to hang small molecules on, so that they can beprocessedbyenzymes.Theimportantbitisnotsomuchthehookaswhathangsfromit,inthiscasetheacetylgroup.‘Acetyl’hasthesamerootasaceticacid,vinegar,asimpletwo-carbonmoleculethatisatthecentreofbiochemistryinallcells.WhenattachedtocoenzymeA,theacetylgroupisinanactivatedstate(oftencalled‘activatedacetate’–ineffect,reactivevinegar)whichenablesit toreactreadilywithotherorganicmolecules,therebydrivingbiosynthesis.

Thus theacetylCoApathwaygeneratessmall reactiveorganicmolecules fromCO2andH2,viajustafewsteps,whileatoncereleasingenoughenergytodrivenotjusttheformationofnucleotidesandothermolecules,butalsotheirpolymerisationintolongchains–DNA,RNA,proteins,andsoon.Theenzymes thatcatalyse thefirst fewstepscontain inorganicclustersof iron,nickelandsulphur,whicharephysicallyresponsiblefortransferringelectronsontoCO2toformreactiveacetylgroups.Theseinorganicclustersarebasicallyminerals–rocks!–moreorlessidenticalintheirstructuretotheiron–sulphurmineralsthatprecipitateinhydrothermalvents(seeFigure11).Thefitbetweenthegeochemistryofalkalineventsand thebiochemistryofmethanogensandacetogens issoclose thatthe word analogous does not do it justice. Analogy implies similarity, which is potentially onlysuperficial.Infact,thesimilarityhereissoclosethatitmightbetterbeseenastruehomology–oneform physically gave rise to the other. So geochemistry gives rise to biochemistry in a seamlesstransitionfromtheinorganictotheorganic.AsthechemistDavidGarnerputit:‘Itistheinorganicelementsthatbringorganicchemistrytolife.’3

ButperhapsthegreatestboonofacetylCoAisthatitsitsatthecrossroadsofcarbonandenergy

metabolism.TherelevanceofacetylCoAtotheoriginoflifewaspointedoutintheearly1990sbythe distinguishedBelgian biochemistChristian deDuve, albeit in the context of soup, not alkalinevents.AcetylCoAnotonlydrivesorganicsyntheses,butitcanalsoreactdirectlywithphosphatetoformacetylphosphate.WhilenotasimportantanenergycurrencyasATPtoday,acetylphosphateisstillwidelyusedacrosslife,andcandomuchthesamejobasATP.Asnotedinthelastchapter,ATPdoesmorethansimplyreleaseenergy;italsodrivesdehydrationreactions,inwhichonemoleculeofwaterisextractedfromtwoaminoacidsorotherbuildingblocks,therebylinkingthemtogetherinachain.Theproblemofdehydratingaminoacidsinsolution,wenoted,isequivalenttowringingoutawet cloth under water; but that is exactly what ATP does. We have shown in the lab that acetylphosphatecandoexactlythesamejob,asitschemistryisbasicallyequivalent.Thismeansthatearlycarbonandenergymetabolismcouldbedrivenbythesamesimplethioester,acetylCoA.

Simple? I hear you say. The two-carbon acetyl group may be simple, but coenzyme A is acomplex molecule, undoubtedly the product of natural selection, and therefore a later product ofevolution. So is this whole argument circular? No, because there are genuinely simple ‘abiotic’equivalentstoacetylCoA.ThereactivityofacetylCoAliesinitsso-called‘thioesterbond’,whichisnomorethanasulphuratomboundtocarbon,boundinturntooxygen.Itcanbedepictedas:

R–S–CO–CH3

inwhich‘R’standsforthe‘rest’ofthemolecule,CoAinthiscase,andCH3isamethylgroup.ButtheRneedn’tstandforCoA;itcouldstandforsomethingassimpleasanotherCH3group,givingasmallmoleculecalledmethylthioacetate:

CH3–S–CO–CH3

Thisisareactivethioester,equivalentinitschemistrytoacetylCoAitself,butsimpleenoughtobeformedfromH2andCO2 inalkalinehydrothermalvents– indeed ithasbeenproducedbyClaudiaHuberandGünterWächtershäuser fromCOandCH3SHalone.Evenbetter,methyl thioacetate, likeacetyl CoA, should be able to react directlywith phosphate to form acetyl phosphate.And so thisreactivethioestercouldinprincipledrivethesynthesisofneworganicmoleculesdirectly,aswellastheirpolymerisation intomorecomplexchainssuchasproteinsandRNA,viaacetylphosphate–ahypothesis thatwe’re testing in our benchtop reactor in the lab (in factwe have just succeeded inproducingacetylphosphate,albeitatlowconcentration).

AprimordialversionoftheacetylCoApathwaycouldinprinciplepowereverythingneededforthe evolution of primitive cells within the micropores of alkaline hydrothermal vents. I wouldenvisage three stages. In the first stage, proton gradients across thin inorganic barriers containingcatalyticiron–sulphurmineralsdrovetheformationofsmallorganicmolecules(Figure14).Theseorganicswereconcentrated in thecoolerventporesby thermophoresis, and in turnactedasbettercatalysts, as we discussed in Chapter 3. These were the origins of biochemistry – the continuousformationandconcentrationofreactiveprecursors,fosteringinteractionsbetweenmoleculesandtheformationofsimplepolymers.

Thesecondstagewastheformationofsimpleorganicprotocellswithintheporesofthevents,asa natural outcome of the physical interactions between organics – simple dissipative cell-likestructures, formed by the self-organisation ofmatter, but as yet without any genetic basis or realcomplexity.Iwouldseethesesimpleprotocellsasdependingontheprotongradienttodriveorganicsynthesis,butnowacrosstheirownorganicmembranes(lipidbilayersformedspontaneouslyfromfattyacids,forexample)ratherthantheinorganicwallsoftheventitself.Noproteinsareneededfor

this. The proton gradient could drive the formation ofmethyl thioacetate and acetyl phosphate asdiscussed above, driving both carbon and energy metabolism. There’s one key difference at thisstage: new organic matter is now formed within the protocell itself, driven by natural protongradients across organic membranes. Reading this back I’m struck by my overuse of the word‘drive’.Itmightbepoorliterarystyle,butthereisn’tabetterword.Ineedtogetacrossthatthisisnotpassivechemistrybutitisforced,pushed,drivenbythecontinuousfluxofcarbon,energy,protons.Thesereactionsneed tohappen, theyare theonlywayofdissipating theunstabledisequilibriumofreduced,hydrogen-rich,alkalinefluidsenteringanoxidised,acidic,metal-richocean.Theonlywayofreachingblessedthermodynamicequilibrium.

Thethirdstageistheoriginofthegeneticcode,trueheredity,finallyenablingprotocellstomakemoreor lessexactcopiesof themselves.Theearliest formsofselection,basedonrelativeratesofsynthesisanddegradation,gavewaytopropernaturalselection, inwhichpopulationsofprotocellswithgenesandproteinsbegantocompeteforsurvivalwithinventpores.Thestandardmechanismsofevolutioneventuallyproducedsophisticatedproteinsinearlycells,includingribosomesandtheATPsynthase, proteins conserved universally across life today. I envisage that LUCA, the commonancestor of bacteria and archaea, livedwithin themicroporesof alkalinehydrothermal vents.ThatmeansallthreestagesfromabioticoriginstoLUCAtakeplacewithintheventpores.Allaredrivenbyproton gradients across inorganicwalls or organicmembranes; but the advent of sophisticatedproteinssuchastheATPsynthaseisalatesteponthisrockyroadtoLUCA.

I amnot concerned in thisbookwith thedetailsofprimordial biochemistry:where thegeneticcode came from, and other equally difficult problems. These are real problems, and there areingeniousresearchersaddressingthem.Wedon’tyetknowtheanswers.Butalltheseideasassumeaplentifulsupplyofreactiveprecursors.Just togiveasingleexample,abeautiful ideafromShelleyCopley, Eric Smith and Harold Morowitz on the origin of the genetic code posits that catalyticdinucleotides(twonucleotidesjoinedtogether)couldgenerateaminoacidsfromsimplerprecursors,such as pyruvate. Their clever scheme shows how the genetic code may have arisen fromdeterministicchemistry.Forthosewhoareinterested,IwroteachapterontheoriginsofDNAinLifeAscending, which touched on some of these questions. But what all of these hypotheses take forgranted is a steady supply of nucleotides, pyruvate, and other precursors. The question we areaddressinghere is:whatwere thedriving forces that impelled theoriginof lifeonearth?Andmymain point is simply that there is no conceptual difficulty aboutwhere all the carbon, energy andcatalystscamefromthatdrovetheformationofcomplexbiologicalmolecules,rightuptotheadventofgenesandproteins,andLUCA.

Theventscenariooutlinedherehasabeautifulcontinuitywiththebiochemistryofmethanogens,thearchaea that livefromH2andCO2bywayof theacetylCoApathway.Theseapparentlyancientcellsgenerateaprotongradientacrossamembrane(we’llcometohowtheydo that), reproducingexactlywhatalkalinehydrothermalventsprovideforfree.TheprotongradientdrivestheacetylCoApathway,bywayofaniron–sulphurproteinembeddedwithinthemembrane–theenergy-convertinghydrogenase, orEch for short. This protein funnels protons through themembrane on to anotheriron–sulphurprotein,calledferredoxin,whichinturnreducesCO2.Inthelastchapter,IsuggestedthatnaturalprotongradientsacrossthinFeSwallsinventscouldreduceCO2bychangingthereductionpotentials ofH2 andCO2. I suspect this iswhatEch is doingon a nanometre scale.Enzymesoftencontroltheprecisephysicalconditions(suchasprotonconcentration)withincleftsintheprotein,justafewångströmsacross,andEchmightbedoingthistoo.Ifso,therecouldbeanunbrokencontinuitybetween a primordial state, in which short polypeptides are stabilised by binding to FeSmineralsembeddedinfatty-acidprotocells,andthemodernstate,inwhichthegeneticallyencodedmembrane

proteinEchpowerscarbonmetabolisminmodernmethanogens.Bethatasitmay,thefactisthattoday,intheworldofgenesandproteins,Echdrawsontheproton

gradient generated bymethane synthesis to drive the reduction of CO2.Methanogens also use theprotongradienttodriveATPsynthesisdirectly,viatheATPsynthase.Thusbothcarbonandenergymetabolism are driven by proton gradients, exactlywhat the vents provided for free. The earliestprotocellslivinginalkalineventsmayhavepoweredcarbonandenergymetabolisminpreciselythisway.Thatsoundsplausibleenough,butinfactrelyingonnaturalgradientsbringsitsownproblems.Intriguinglyseriousproblems.BillMartinandIrealisedtheremaybeonlyonepossiblesolutiontotheseproblems–anditgivesatantalisinginsightintowhyarchaeaandbacteriadifferinfundamentalways.

TheproblemofmembranepermeabilityInsideourownmitochondria,themembranesarealmostimpermeabletoprotons.That’snecessary.Itis no good pumping protons across amembrane if they come rushing straight back at you, as ifthroughinnumerablelittleholes.Youmightaswelltrytopumpwaterintoatankwithasieveforabase. Inourmitochondria, then,wehave an electrical circuit, inwhich themembraneworks as aninsulator: we pump protons across the membrane, and most of them return through proteins thatbehaveas turbines,drivingwork. In thecaseof theATPsynthase, the flowofprotons through thisnanoscopic rotatingmotordrivesATPsynthesis.Butnote that thiswhole systemdependsonactivepumping.Blockthepumpsandeverythinggrindstoahalt.That’swhathappensifwetakeacyanidepill:itjamsupthefinalprotonpumpoftherespiratorychaininourmitochondria.Iftherespiratorypumpsareimpededinthisway,protonscancontinuetoflowinthroughtheATPsynthaseforafewsecondsbeforetheprotonconcentrationequilibratesacrossthemembrane,andnetflowceases.Itisalmost as hard to define death as life, but the irrevocable collapse of membrane potential comesprettyclose.

So how could a natural proton gradient drive ATP synthesis? It faces the ‘cyanide’ problem.Imagineaprotocellsittinginaporewithinavent,poweredbyanaturalprotongradient.Onesideofthe cell is exposed to a continuous flow of ocean water, the other to a constant flux of alkalinehydrothermalflow(Figure17).Fourbillionyearsago,theoceanswereprobablymildlyacidic(pH5–7), while the hydrothermal fluids were equivalent to today, with a pH of about 9–11. Sharp pHgradientscould thereforehavebeenasmuchas3–5pHunits inmagnitude,whichis tosay that thedifference inprotonconcentrationcouldhavebeen1,000–100,000-fold.4For the sakeofargument,imaginethattheprotonconcentrationinsidethecellissimilartothatoftheventfluids.Thatgivesadifferenceinprotonconcentrationbetweenthe insideandtheoutside,soprotonswill flowinwardsdown the concentration gradient.Within a few seconds, though, the influx should grind to a halt,unless the protons that flow in can be removed again. There are two reasons for this. First, theconcentration difference swiftly evens out. And second, there is an issue with electrical charge.Protons (H+) are positively charged, but in seawater their positive charge is counterbalanced bynegatively charged atoms such as chloride ions (Cl–). The problem is that protons cross themembranemuchfasterthanchlorideions,sothereisaninfluxofpositivechargethatisnotoffsetbyaninfluxofnegativecharge.Theinsideofthecellthereforebecomespositivelychargedrelativetotheoutside,andthatopposestheinfluxofanymoreH+.Inshort,unlessthereisapumpthatcangetridofprotons from inside thecell,naturalprotongradientscan’tdriveanything.Theyequilibrate,andequilibriumisdeath.

But there is an exception. If the membrane is nearly impermeable to protons, the influx mustindeedcease.Protonsenter thecellbutcan’t leaveagain.But if themembrane isvery leaky, it isa

different story.Protonscontinue toenter thecell, asbefore,butnow theycan leave it again, albeitpassively, through the leakymembrane on the other side of the cell. In effect, a leakymembraneimposeslessofabarriertoflow.Betterstill,hydroxideions(OH–)fromthealkalinefluidscrossthemembraneataboutthesamerateasprotons.Whentheymeet,H+andOH–reacttoformwater(H2O),eliminating the proton with its positive charge in one fell swoop. Using the classic equations ofelectrochemistry, it’s possible to calculate the rates at which protons enter and exit a hypothetical(computational)cellasafunctionofmembranepermeability.VictorSojo,achemistinterestedinbigproblemsinbiology,whoisdoingaPhDwithmeandAndrewPomiankowski,hasdoneexactlythis.Bytrackingthesteady-statedifferenceinprotonconcentration,wecouldcalculatethefreeenergy(G) available from a pH gradient alone. The results are just beautiful. The driving force availabledependson the leakinessof themembrane toprotons. If themembrane isextremely leaky,protonscomerushinginlikefools,buttheyalsodisappearagainquickly,eliminatedbyarapidinfluxofOH–

ions.Evenwithveryleakymembranes,wefoundthatprotonswillstillenterfasterthroughmembraneproteins(liketheATPsynthase)thanthroughthelipidsthemselves.Thismeansthatprotonfluxcandrive ATP synthesis or carbon reduction via the membrane protein Ech. Taking concentrationdifferencesandchargeintoconsideration,aswellastheoperationofproteinsliketheATPsynthase,we showed that only cells with very leaky membranes can use natural proton gradients to powercarbon and energymetabolism. Remarkably, these leaky cells theoretically glean asmuch energyfromanaturalprotongradientof3pHunitsasmoderncellsgainfromrespiration.

Figure17AcellpoweredbyanaturalprotongradientAcellsitsinthemiddleoftheframe,enclosedbyamembranethatisleakytoprotons.Thecellis‘wedged’inasmallbreakinaninorganicbarrierthatseparatestwophaseswithinamicroporousvent.Inthetopphase,mildlyacidicoceanwaterpercolatesalonganelongatedpore,atapHof5–7(generallytakentobepH7inthemodel).Inthebottomphase,alkalinehydrothermalfluidspercolatealonganunconnectedpore,atapHofabout10.Laminarflowindicatesalackofturbulenceandmixing,whichischaracteristicoffluidsflowinginsmallconfinedspaces.Protons(H+)canflowdirectlythroughthelipidmembrane,orthroughproteinsembeddedinthemembrane(triangularshape),downaconcentrationgradientfromtheacidicoceantothealkalinehydrothermalfluid.Hydroxideions(OH–)flowintheoppositedirection,fromthealkalinehydrothermalfluidtotheacidocean,butonlythroughthemembrane.TheoverallrateofprotonfluxdependsonthepermeabilityofthemembranetoH+,neutralisationbyOH–(toformH2O);thenumberofmembraneproteins;thesizeofthecell;andthechargeacrossthemembraneaccruedbythemovementofionsfromonephasetotheother.

Actually, theycouldgaina lotmore.Thinkagainaboutmethanogens.Theyspendmostof theirtime generating methane, hence their name. On average, methanogens produce about 40 times asmuch waste (methane and water) as organic matter. All the energy derived from the synthesis ofmethaneisusedtopumpprotons(Figure18).That’sit.Methanogensspendpractically98%oftheirenergybudgetongeneratingprotongradientsbymethanogenesis,andlittlemorethan2%producingnew organic matter. With natural proton gradients and leaky membranes, none of that excessiveenergyspendisneeded.Thepoweravailableisexactlythesamebuttheoverheadsarecutbyatleast40-fold, a very substantial advantage. Just imagine having 40 timesmore energy!Evenmy youngsonsdon’toutdomethatmuch.Inthepreviouschapter,Imentionedthatprimitivecellswouldhaveneededmorecarbonandenergyfluxthanmoderncells;havingzeroneedtopumpgivesthemalotmorecarbonandenergy.

Figure18GeneratingpowerbymakingmethaneAsimplifiedviewofmethanogenesis.InAtheenergyfromthereactionbetweenH2andCO2powerstheextrusionofprotons(H

+)acrossthecellmembrane.Ahydrogenaseenzyme(Hdr)catalysesthesimultaneousreductionofferredoxin(Fd)andadisulphidebond(–S–S–),usingthetwoelectronsfromH2.FerredoxininturnreducesCO2,ultimatelytoamethyl(–CH3)groupboundtoacofactordesignatedR.Themethylgroupisthentransferredtoasecondcofactor(R’),andthisstepreleasesenoughenergytopumptwoH+(orNa+)acrossthemembrane.Inthefinalstage,the–CH3groupisreducedtomethane(CH4)bytheHS–group.Overall,someoftheenergyreleasedbytheformationofmethane(CH4)fromH2andCO2isconservedasanH+(orNa+)gradientacrossthecellmembrane.InBtheH+gradientisuseddirectlythroughtwodistinctmembraneproteinstodrivecarbonandenergymetabolism.Theenergy-convertinghydrogenase(Ech)reducesferredoxin(Fd)directly,whichagainpassesitselectronsontoCO2

toformamethyl(–CH3)group,whichisreactedwithCOtoformacetylCoA,thelinchpinofmetabolism.Likewise,H+fluxthrough

theATPsynthasedrivesATPsynthesis,andsoenergymetabolism.

Consider a leakycell, sitting in anaturalprotongradient.Rememberwearenow in the eraof

genesandproteins,whichare themselves theproductofnaturalselectionactingonprotocells.Ourleaky cell can use the continuous flux of protons to drive carbonmetabolism, byway ofEch, theenergy-converting hydrogenase thatwe discussed earlier. This protein enables the cell to reactH2

withCO2,toformacetylCoA,andthenceonwardstoallthebuildingblocksoflife.Itcanalsousetheproton gradient to drive ATP synthesis, using theATP synthase. And of course it can useATP topolymeriseaminoacidsandnucleotidestomakenewproteins,RNAandDNA,andultimatelycopiesofitself.Importantly,ourleakycellhasnoneedtowasteenergyonpumpingprotons,andsoitshouldgrowwell, even allowing for inefficient early enzymes that hadnot yet beenhonedbybillions ofyearsofevolution.

Butsuchleakycellsarealsostuckrightwheretheyare,utterlydependentonhydrothermalflowandunable to survive anywhere else.When that flowceases or shifts elsewhere, they are doomed.Evenworse,theyappeartobeinanunevolvablestate.Thereisnobenefittoimprovingthepropertiesofthemembrane;onthecontrary,lessleakymembranesswiftlycollapsetheprotongradient,asthereisnolongeranywayofgettingridofprotonsfrominsidethecell.Soanyvariantcellsthatproducedamore ‘modern’ impermeablemembranewould be eliminated by selection. Unless they learnt topump, of course; but that is equally problematic.We have seen that there is no point in pumpingprotons across a leakymembrane.Our study confirms that pumping offers no benefit, even if thepermeabilityofthemembraneisdecreasedbyawhoppingthreeordersofmagnitude.

Letme spell that out. A leaky cell in a proton gradient has plenty of energy, enough to drivecarbonandenergymetabolism.Ifbysomeevolutionarysleightofhand,afullyfunctionalpumpisplaced in themembrane, it offersnobenefitwhatsoever in termsof energyavailability: thepoweravailable remainsexactly the sameas in its absence.That’sbecausepumpingprotonsover a leakymembrane is pointless – they come straight back through. Decrease membrane permeability by afactorof10,andtryagain;stillzerobenefit.Decreasepermeabilitybyafactorof100;stillnobenefit.Decreasepermeabilitybyafactorof1,000;stillnobenefit.Whynot?Thereisabalanceofforces.Decreasingmembrane permeability helps pumping, but also collapses the natural proton gradient,undermining thecell’spower supply.Only if largeamountsofpumpareplasteredacrossanearlyimpermeablemembrane(equivalenttothatinourowncells)isthereanybenefittopumping.Thatisaserious problem. There is no selective driving force for the evolution of either modern lipidmembranesormodernprotonpumps.Without adriving force they shouldnot evolve;but theydoexist,nonetheless.Sowhatarewemissing?

Hereisanexampleoftheserendipityofscience.BillMartinandIwereponderingoverexactlythatquestion,andwemusedthatmethanogensuseaproteincalledanantiporter.Themethanogensinquestionactuallypumpoutsodiumions(Na+),notprotons(H+),but theystillhavea fewproblemswithprotonsaccumulatinginside.TheantiporterswapsanNa+ foranH+,as if itwereastrict two-wayturnstile,orrevolvingdoor.ForeachNa+passing into thecelldownaconcentrationgradient,oneH+ is forcedout. It isaprotonpumppoweredbya sodiumgradient.Butantiportersareprettyundiscriminating.Theydon’tcarewhichwayroundtheywork.IfacellpumpedH+ratherthanNa+,thentheantiporterwouldsimplygointoreverse.ForeveryH+ thatentered,oneNa+wouldthenbeforcedout.Ha!Suddenlywehadit!IfourleakycellsittinginthealkalinehydrothermalventevolvedanNa+/H+antiporter, itwouldactasaproton-poweredNa+pump!ForeachH+ thatenteredthecellthrough theantiporter,oneNa+wouldbe forced to leave! In theory, theantiportercouldconvertanaturalprotongradientintoabiochemicalsodiumgradient.

Howwouldthathelpexactly?Ishouldstressthatthisisathoughtexperiment,basedontheknownpropertiesof theprotein;butbyourcalculations, itcouldmakeasurprisingdifference.Ingeneral,lipidmembranesarearoundsixordersofmagnitudelesspermeabletoNa+thanH+.Soamembranethat isextremelypermeable toprotons is fairly impermeable to sodium.Pumpoutaproton,and it

will comestraightbackatyou;pumpouta sodiumacross the samemembrane,and itwon’t comebacknearlyasfast.Thismeansthatanantiportercanbedrivenbyanaturalprotongradient:foreveryH+thatcomesin,oneNa+isextruded.Solongasthemembraneisleakytoprotons,asbefore,protonfluxthroughtheantiporterwillcontinueunabated,drivingNa+extrusion.Because themembrane islesspermeabletoNa+,theextrudedNa+ismorelikelytostayout;ormorespecifically,itshouldre-enter thecellviamembraneproteins, rather thancomingstraightback through the lipids.And thatimprovesthecouplingofNa+influxtoworkdone.

Ofcourse,that’sonlyanyuseifthemembraneproteinsthatdrivecarbonandenergymetabolism–EchandtheATPsynthase–can’tdiscriminatebetweenNa+andH+.Thatsoundspreposterous,butrather surprisingly itmaywellbe true.Somemethanogens turnout tohaveATPsynthaseenzymesthatcanbepoweredbyeitherH+orNa+,withroughlyequal facility.Even theprosaic languageofchemistrydeclares themtobe ‘promiscuous’.The reasoncould relate to theequivalentchargeandvery similar radii of the two ions.AlthoughH+ ismuch smaller thanNa+, protons rarely exist inisolation.Whendissolved,theybindtowatertoformH3O+,whichhasaradiusnearlyidenticaltoNa+.Othermembraneproteins includingEcharealsopromiscuousforH+andNa+,presumablyfor thesame reasons. The bottom line is that pumping Na+ is by no means pointless.When powered bynaturalprotongradients, there is essentiallyno cost to extrudingNa+; andonce a sodiumgradientexists, Na+ ions aremore likely to re-enter the cell viamembrane proteins such asEch and ATPsynthase than throughmembrane lipids. Themembrane is nowbetter ‘coupled’,meaning that it isbetterinsulated,andthereforelesslikelytoshort-circuit.Asaresult,moreionsarenowavailabletodrivecarbonandenergymetabolism,givingbetterpaybackforeachionpumpedout.

There are several surprising ramifications of this simple invention. One is almost incidental:pumping sodiumout of the cell lowers the concentration of sodiumwithin the cell.We know thatmany core enzymes found in both bacteria and archaea (those responsible for transcription andtranslation,forexample)havebeenoptimisedbyselectiontoworkatlowNa+concentration,despitemostprobablyevolvingintheoceans,wheretheNa+concentrationseemstohavebeenhigheven4billion years ago. The early operation of an antiporter could potentially explainwhy all cells areoptimisedtolowsodium,despiteevolvinginahigh-sodiumenvironment.5

Moresignificantlyforourimmediatepurposes,theantiportereffectivelyaddsanNa+gradienttoan existingH+ gradient. The cell is still powered by the natural proton gradient, so still requiresproton-permeablemembranes;butitnowhasanNa+gradienttoo,whichbyourcalculationsgivesthecellabout60%morepowerthanithadbefore,whenrelyingonprotonsalone.Thatgivescellstwobigadvantages.First,cellswithanantiporterhavemorepower,andsocangrowandreplicatefasterthancellswithout– anobvious selective advantage.Second, cells could surviveon smallerprotongradients.Inourstudy,cellswithleakymembranesgrowwellwithaprotongradientofabout3pHunits,which is to say that theproton concentrationof theoceans (aroundpH7) is threeorders ofmagnitudegreater thantheprotonconcentrationofalkalinefluids(aboutpH10).Byincreasingthepowerofanaturalprotongradient,cellswithanantiportercouldsurvivewithapHgradientoflessthan 2 pH units, allowing them to spread and colonisewider areas of the vent or contiguous ventsystems. Cells with an antiporter would therefore tend to outcompete other cells, and would alsospreadanddivergeinthevents.Butbecausetheystilldependtotallyonthenaturalprotongradient,theycouldnotleavethevents.Onemorestepwasrequired.

Thatbringsustothecrucialpoint.Withanantiporter,cellsmightnotbeabletoleavethevent,buttheyarenowprimedtodoso.Intheparlance,anantiporterisa‘preadaptation’–anecessaryfirststepthat facilitatesa laterevolutionarydevelopment.The reason isunexpected,orat least itwas tome.For the first time, an antiporter favours the evolution of active pumping. Imentioned there is nobenefit topumpingprotonsacross a leakymembrane,because theycomestraightbackatyou.But

with an antiporter there is an advantage.When protons are pumped out, some of them return notthroughtheleakylipidsbutthroughtheantiporter,whichextrudesNa+ionsintheirplace.BecausethemembraneisbetterinsulatedtoNa+,moreoftheenergythathadbeenspentonpumpingoutprotonsis retained as an ion gradient across themembrane. For every ion pumped out, there is a slightlyhigherchance itwill stayout.And thatmeans there isnowa small advantage topumpingprotons,whereasbeforetherehadbeennoadvantage.Pumpingonlypayswithanantiporter.

That’snotall.Onceaprotonpumphasevolved,thereisnow,forthefirsttime,anadvantagetoimproving themembrane. I reiterate: in a natural proton gradient it is strictly necessary to have aleakymembrane.Pumpingprotonsacrossaleakymembraneisnouseatall.Anantiporterimprovesthesituationbecauseitincreasesthepoweravailablefromanaturalprotongradient,butitdoesnotcutoffthecellfromitsdependenceonthenaturalgradient.Yetinthepresenceofanantiporter,itnowpaystopumpprotons,meaningthereislessdependenceonthenaturalgradient.Andnow–onlynow!– is itbetter tohavea lesspermeablemembrane.Making themembraneslightly less leakygivesaslightadvantagetopumping.Improvingitalittlebitmoregivesaslightlybiggeradvantage,andsoon, all the way up to a modern proton-tight membrane. For the first time, we have a sustainedselective driving force for the evolution of both proton pumps and modern lipid membranes.Ultimately,cellscouldcut theirumbilical link tonaturalprotongradients: theywerefinallyfree toescapefromthevents,andsubsistinthegreatemptyworld.6

Figure19TheoriginofbacteriaandarchaeaApossiblescenarioforthedivergenceofbacteriaandarchaea,basedonamathematicalmodelofenergyavailabilityinnaturalprotongradients.ThefigureshowsonlytheATPsynthaseforsimplicity,butthesameprincipleappliestoothermembraneproteinssuchasEch.Anaturalh+gradientinaventcandriveATPsynthesissolongasthemembraneisleaky(bottom),butthereisnobenefittoimprovingthemembrane,asthatcollapsesthenaturalgradient.Asodium-protonantiporter(SPAP)addsabiochemicalsodiumgradienttothegeochemicalprotongradient,enablingsurvivalonsmallerh+gradients,facilitatingspreadanddivergenceofpopulationsinthevent.TheextrapowerprovidedbySPAPmeansthatpumpingh+offersabenefitforthefirsttime.Withapump,thereisabenefittoloweringmembranepermeabilitytoh+.Whenthemembraneh+permeabilityapproachesmodernvalues,cellsfinallybecomeindependentofnaturalgradients,andcanleavethevent.Bacteriaandarchaeaaredepictedescapingfromtheventindependently.

This isabeautifulsetofphysicalconstraints.Unlikephylogenetics,whichcan tellusvery littlewith certainty, these physical constraints put an order on the possible succession of evolutionarysteps,beginningwithadependenceonnaturalprotongradientsandendingwithessentiallymoderncells,whichgenerate theirownprotongradientsacross impermeablemembranes(Figure19). Andbetterstill,theseconstraintscouldexplainthedeepdivergenceofbacteriaandarchaea.BothgenerateATPusingprotongradientsacrossmembranes,yetthosemembranesarefundamentallydifferentinthetwodomains,alongwithothertraitsthatincludethemembranepumpsthemselves,thecellwall,andDNAreplication.Letmeexplain.

WhybacteriaandarchaeaarefundamentallydifferentHereisabriefsummaryofthestorysofar.Inthepreviouschapterweconsidered,fromanenergeticpointofview,thepossibleenvironmentsontheearlyearththatwereconducivetotheoriginoflife.Wenarrowed thesedowntoalkalinehydrothermalvents,whereasteadycarbonandenergyflux iscombinedwithmineralcatalystsandnaturalcompartmentalisation.Buttheseventsfaceaproblem:thecarbonandenergyfluxcomesintheformofH2andCO2,whichdonotreacttogethereasily.Wesawthatgeochemicalprotongradientsacrossthinsemiconductingbarriersinventporescouldpotentiallybreak down the energy barrier to their reaction. By producing reactive thioesters such as methylthioacetate (functionallyequivalent toacetylCoA)protongradientscoulddrive theoriginsofbothcarbonandenergymetabolism,leadingtotheaccumulationoforganicmoleculeswithinventpores,while facilitating ‘dehydration’ reactions that form complex polymers including DNA, RNA andproteins.Iwasevasiveondetailssuchashowthegeneticcodearose,butfocusedontheconceptualargument that these conditionscould theoreticallyhaveproduced rudimentarycellswithgenesandproteins.Populationsofcellsweresubjecttoperfectlynormalnaturalselection.Isuggestedthatthelastcommonancestorofbacteriaandarchaea,LUCA,mayhavebeentheproductofselectionactingonsuchpopulationsofsimplecellslivingintheporesofalkalinehydrothermalventsanddependentonnaturalprotongradients.Selectiongaverise tosophisticatedproteins, includingribosomes,EchandtheATPsynthase–allofwhichareuniversallyconserved.

In principle,LUCAcould have powered all of her carbon and energymetabolismwith naturalproton gradients, via the ATP synthase and Ech, but to do so needed extremely permeable cellmembranes.Shecouldnothaveevolved‘modern’impermeablemembranesequivalenttoeitherthebacteriaorarchaea,becausethatwouldhavecollapsedthenaturalprotongradients.Butanantiporterwould have helped, by converting natural proton gradients into biochemical sodium gradients,increasing thepoweravailableandsopermittingcells to surviveonsmallergradients.Thiswouldhaveenabledcells tospreadandcolonisepreviouslyuntenableregionsofvents, in turnfacilitatingdivergenceofpopulations.Beingabletosurviveunderawiderrangeofconditionscouldevenhaveenabledcellsto‘infect’contiguousventsystems,potentiallyspreadingwidelyacrosstheoceanfloor

oftheearlyearth,muchofwhichmayhavebeenpronetoserpentinisation.Butanantiporteralsogaveanadvantagetopumping,forthefirsttime.Finallywecometothose

strange differences in the acetyl CoA pathway in methanogens and acetogens. These differencessuggestthatactivepumpingaroseindependentlyintwodistinctpopulations,whichhaddivergedfromacommonancestralpopulationwiththehelpofanantiporter.Recall thatmethanogensarearchaea,whileacetogensarebacteria–representativesof the twogreatdomainsofprokaryotes, thedeepestbranches of the ‘tree of life’. We have noted that bacteria and archaea are similar in their DNAtranscriptionandtranslation,ribosomes,proteinsynthesis,andsoon,butdifferinotherfundamentalrespects, including cell membrane composition. I mentioned that they also differ in details of theacetylCoApathway,while claiming that this pathway is nonetheless ancestral.The similarities anddifferencesarerevealing.

Likemethanogens, acetogens reactH2withCO2 to formacetylCoA, via a series of analogoussteps. Both groups use a clever trick known as electron bifurcation to power pumping. Electronbifurcationwas discovered only recently by the distinguishedmicrobiologist Rolf Thauer and hiscolleaguesinGermany,inwhatcouldbethebiggestbreakthroughinbioenergeticsofrecentdecades.Thauer has now formally retired, but his findingswere the culmination of decades spent puzzlingover theenergeticsofobscuremicrobes,whichkeepgrowingwhenthestoichiometriccalculationssay theyshouldnot.Evolution,assooften, iscleverer thanweare. Inessence,electronbifurcationamountstoashort-termenergyloan,madeonthepromiseofpromptrepayment.Aswe’venoted,thereactionofH2withCO2isexergonicoverall(releasingenergy)butthefirstfewstepsareendergonic(requiringanenergyinput).Electronbifurcationcontrivestousesomeoftheenergythatisreleasedinthelater,exergonic,stepsofCO2reductiontopayforthedifficultfirststeps.7Asmoreenergyisreleased in the last few steps than needs to be spent in the first few steps, some energy can beconservedasaprotongradientacrossamembrane(Figure18).Overall, theenergyreleasedbythereactionofH2andCO2powerstheextrusionofprotonsacrossamembrane.

Thepuzzleisthatthe‘wiring’ofelectronbifurcationdiffersinmethanogensandacetogens.Bothdependonrathersimilariron–nickel–sulphurproteins;buttheexactmechanismdiffers,asdomanyoftheproteinsneeded.Likemethanogens,acetogensconservetheenergyreleasedbythereactionofH2andCO2asanH+orNa+gradientacrossamembrane.Inbothcases,thegradientisusedtopowercarbonandenergymetabolism.Likemethanogens,acetogenshaveanATPsynthaseandEch.Unlikemethanogens,however, acetogensdonotuse theEch topowercarbonmetabolismdirectly.On thecontrary,someofthemuseitinreverseasanH+orNa+pump.Andtheexactpathwaythattheyusetodrivecarbonmetabolismisverydifferent.Thesedifferencesseemtobefundamental,tothepointthatsome experts believe the similarities to be the product of convergent evolution or lateral genetransfer,ratherthancommonancestry.

Yet the similarities and differences begin to make sense if we assume that LUCA did indeeddependonnaturalprotongradients.Ifso,thekeytopumpingcouldlieinthedirectionofprotonfluxthroughEch–whetherthenaturalflowofprotonsintothecelldrivescarbonfixation,orwhetherthisfluxisreversed,withtheproteinnowactingasamembranepump,pumpingprotonsoutofthecell(Figure20).Intheancestralpopulation,IsuggestthatthenormalinwardfluxofprotonsviaEchwasused to reduce ferredoxin, in turn driving CO2 reduction. Two separate populations then inventedpumping independently.Onepopulation,whichultimatelybecameacetogens, reversed thedirectionofEch,nowoxidisingferredoxinandusingtheenergyreleasedtopumpprotonsoutofthecell.Thisis nice and simple but created an immediate problem. The ferredoxin previously used to reducecarbonisnowusedtopumpprotons.Acetogenshadtocomeupwithanewwayofreducingcarbonthatdidnotrelyonferredoxin.Theirancestorsfoundaway–theclevertrickofelectronbifurcation,

whichenabledthemtoreduceCO2indirectly.Thebasicbiochemistryofacetogensarguablyfollowsfrom that simple premise – the direction of proton flux through Ech was reversed, giving theacetogensafunctionalpump,butleavingthemwithaspecificsetofproblemstosolve.

Figure20PossibleevolutionofactivepumpingHypotheticaloriginsofpumpinginbacteriaandarchaea,basedonthedirectionofH+fluxthroughthemembraneproteinEch.ATheancestralstate,inwhichnaturalprotongradientsdrivecarbonandenergymetabolismviaEchandtheATPsynthase(ATPase).Thiscanonlyworksolongasthemembraneisleakytoprotons.BMethanogens(postulatedtobetheancestralarchaea).ThesecellscontinuetouseEchandtheATPasetodrivecarbonandenergymetabolism,butwithH+-tightmembranescouldnolongerrelyonnaturalprotongradients.Theyhadto‘invent’anewbiochemicalpathwayandnewpump(methyltransferase,Mtr)togeneratetheirownH+(orNa+)gradient(dottedlines).NotethatthispanelisequivalenttoFigure18AandBcombined.CAcetogens(postulatedtobetheancestralbacteria).ThedirectionofH+fluxthroughEchisherereversed,andisnowpoweredbytheoxidationofferredoxin.Acetogensdidnotneedto‘invent’apump,buthadtofindanewwayofreducingCO2toorganics;thisisdoneusingNADHandATP(dottedlines).ThispostulatedscenariocouldexplainboththesimilaritiesanddifferencesintheacetylCoApathwaybetweenmethanogensandacetogens.

The second population, which became methanogens, found an alternative way. Like theirancestors, they continued to use proton gradients to reduce ferredoxin, and then used the reducedferredoxin to fix carbon.But they then had to ‘invent’ a pump from scratch.Well, not quite fromscratch;theymayhaverepurposedanexistingprotein.Itseemstheymodifiedanantiportertobecomeastraightpump.Thatisnotintrinsicallydifficulttodo,butitgaverisetoadifferentproblem:howtopowerthepump?Methanogenscameupwithadifferentformofelectronbifurcation,usingsomeofthe same proteins as acetogens, but hooked up quite differently, as their own requirements weredistinct and wired up to a different pump. The carbon and energy metabolism of each of thesedomainsarguablystemsfromthedirectionofprotonflowthroughEch.It’sabinarychoice,andthemethanogensandacetogensmadedifferentdecisions(Figure20).

Onceeachgrouphadactivepumps,therewasfinallyanadvantagetoimprovingthemembrane.For all the steps until now, there had never been any benefit to evolving a ‘modern’ membrane,replete with phospholipids – that would have been actively detrimental. But as soon as cells hadantiporters and ion pumps, there was now a benefit to incorporating glycerol head-groups onmembranelipids.Andthetwodomainsappeartohavedonesoindependently,witharchaeausingonestereoisomerofglycerol,andbacteriausingitsmirrorimage(seeChapter2).

Nowcellshadevolvedactiveionpumpsandmodernmembranes,andwerefinallyfreetoleavethevents,escapingintotheopenoceans.Fromacommonancestorthatlivedfromprotongradientsinvents,thefirstfree-livingcells,bacteriaandarchaea,emergedindependently.It’snotsurprisingthatbacteriaandarchaeashouldhavecomeupwithdistinctcellwalls toprotect themagainst thesenewshocks, nor indeed that they should have ‘invented’DNA replication independently.Bacteria attachtheir DNA to the cell membrane during cell division, at a site called the replicon; the attachmentenables eachdaughter cell to receivea copyof thegenome.Themolecularmachinery required toattachDNAtothemembrane,andmanydetailsofDNAreplication,mustdependatleastpartlyonthemechanicsofthatattachment.Thefact thatcellmembranesevolvedindependentlybeginstoexplainwhyDNAreplicationshouldbesodifferent inbacteriaandarchaea.Much the sameapplies tocellwalls,allthecomponentsofwhichmustbeexportedfrominsidethecellthroughspecificmembranepores–hencethesynthesisof thecellwalldependsonthepropertiesof themembrane,andshoulddifferinbacteriaandarchaea.

And sowe draw to a close.While bioenergetics do not predict from first principles that thereshouldbefundamentaldifferencesbetweenbacteriaandarchaea,theseconsiderationsdoexplainhowand why they could have arisen in the first place. The deep differences between the prokaryoticdomainshadnothingtodowithadaptationtoextremeenvironments,suchashightemperatures,butrather the divergence of cellswithmembranes thatwere obliged to remain leaky for bioenergeticreasons.Whilethedivergenceofarchaeaandbacteriamightnotbepredictablefromfirstprinciples,thefactthatbothgroupsarechemiosmotic(dependingonprotongradientsacrossmembranes)does

follow from the physical principles discussed in these last two chapters. The environment mostrealisticallycapableofgivingrisetolife,whetherhereoranywhereelseintheuniverse,isalkalinehydrothermalvents.Suchventsconstraincellstomakeuseofnaturalprotongradients,andultimatelyto generate their own. In this context it’s no mystery that all cells here on earth should bechemiosmotic.Iwouldexpectthatcellsacrosstheuniversewillbechemiosmotictoo.Andthatmeanstheywill faceexactly thesameproblemsthat lifeonearthdoes. In thenextpart,we’llseewhythisuniversalrequirementforprotonpowerpredictsthatcomplexlifewillberareintheuniverse.

Footnotes1SeetheIntroduction.Theribosomesaretheprotein-buildingfactoriesfoundinallcells.Theselargemolecularcomplexeshavetwomajorsubunits(largeandsmall),whicharethemselvescomposedofamixtureofproteinsandRNA.The‘smallsubunitribosomalRNA’iswhatWoesesequenced,inpartbecauseitwasfairlyeasytoextract(therearethousandsofribosomesinanyonecell);andinpartbecauseproteinsynthesisisfundamentaltolife,andsoisuniversallyconservedwithonlytrivialdifferencesbetweenhumansandhydrothermalbacteria.Itisnevereasytoreplacethefoundationstonesofanybuildingordiscipline;andformuchthesamereasons,ribosomesarerarelytransferredbetweencells.2Recallthatthebacteriaandarchaeaarethetwogreatdomainsofprokaryotes,whichareverysimilarintheirmorphologicalappearancebutdifferfundamentallyinaspectsoftheirbiochemistryandgenetics.3Andthesameinorganicelementsstillbringorganicchemistrytolife.Moreorlessidenticaliron–sulphurclustersarefoundinourownmitochondria,morethanadozenofthemineachrespiratorychain(seeFigure8forcomplexIalone),meaningtensofthousandsofthemineverymitochondrion.Withoutthem,respirationcouldnotworkandwewouldbedeadinminutes.4BecausethepHscaleislogarithmic,1pHunitrepresentsa10-folddifferenceinprotonconcentration.Differencesofthismagnitudeinsuchasmallspacemayseemunfeasible,butinfactarepossiblebecauseofthenatureoffluidflowthroughporesonthescaleofmicrometresindiameter.Flowinthesecircumstancescanbe‘laminar’,withlittleturbulenceandmixing.Theporesizesinalkalinehydrothermalventstendtocombinebothlaminarandturbulentflow.5ThefactthatancientenzymesareoptimisedtoalowNa+/highK+content,giventhatthefirstmembraneswereleakytotheseions,canonlymeanthatcellswereoptimisedtotheionicbalanceofthesurroundingmedium,accordingtoRussianbioenergeticistArmenMulkidjanian.AstheearlyoceanswerehighinNa+,lowinK+,hebelievesthatlifecan’thavebegunintheoceans.Ifhe’sright,thenImustbewrong.MulkidjanianpointstoterrestrialgeothermalsystemswithhighK+,lowNa+,althoughthesehaveproblemsoftheirown(hehasorganicsynthesisdrivenbyzincsulphidephotosynthesis,unknowninreallife).Butisitreallyimpossiblefornaturalselectiontooptimiseproteinsover4billionyears,orarewetobelievethattheprimordialionbalancewasperfectforeveryenzyme?Ifit’spossibletooptimiseenzymefunction,howcouldthatbedone,givenleakyearlymembranes?Theuseofantiportersinnaturalprotongradientsoffersasatisfyingresolution.6Thealertreadermaybewonderingwhycellsdon’tjustpumpNa+?ItisindeedbettertopumpNa+acrossaleakymembranethantopumpH+,butasthemembranebecomeslesspermeable,thatadvantageislost.Thereasonisesoteric.Thepoweravailabletoacelldependsontheconcentrationdifferencebetweenthetwosidesofthemembrane,notontheabsoluteconcentrationsofions.BecauseNa+concentrationissohighintheoceans,tomaintainanequivalentthreeordersofmagnitudedifferencebetweentheinsideandoutsideofthecellrequirespumpingalotmoreNa+thanH+,underminingtheadvantageofpumpingNa+ifthemembraneisrelativelyimpermeabletobothions.Intriguingly,cellsthatliveinvents,suchasmethanogensandacetogens,oftendopumpNa+.Onepossiblereasonisthathighconcentrationsoforganicacids,suchasaceticacid,increasethepermeabilityofthemembranetoH+,makingitmoreprofitabletopumpNa+.7Forthosewhowanttoknowmoreaboutthiscuriousprocessofelectronbifurcation:twoseparatereactionsarecoupledtogether,sothatthedifficult(endergonic)stepisdrivenbyamorefavourable(exergonic)reaction.OfthetwoelectronsinH2,onereactsimmediatelywithan‘easy’target,forcingtheothertoaccomplishamoredifficultstep,thereductionofCO2toorganicmolecules.Theproteinmachinerythatcarriesoutelectronbifurcationcontainsmanyiron–nickel–sulphurclusters.Inmethanogens,theseessentiallymineralstructuressplitupthepairsofelectronsfromH2,ultimatelyfeedinghalfofthemontoCO2,toformorganics,andtheotherhalfontosulphuratoms–the‘easier’targetthatdrivesthewholeprocess.Theelectronsarefinallyreunitedonmethane(CH4),whichisreleasedintotheworldaswaste,bequeathingmethanogenstheirname.Inotherwords,theprocessofelectronbifurcationisquitestaggeringlycircular.TheelectronsfromH2areseparatedforalittlewhile,butintheendallofthemaretransferredontoCO2,reducingittomethane,whichisswiftlydiscarded.TheonlythingconservedissomeoftheenergyreleasedintheexergonicstepsofCO2reduction,intheformofanH+gradientacrossamembrane(actually,inmethanogensthegradientistypicallyNa+,butH+andNa+areeasilyinterchangeableviatheantiporter).Insum,electronbifurcationpumpsprotons,regeneratingwhatventsprovideforfree.

PARTIII

COMPLEXITY

T

5

THEORIGINOFCOMPLEXCELLS

here’safamousline,deliveredbyOrsonWelles,inthe1940sfilmnoirTheThirdMan:‘InItaly,for thirty years under the Borgias, they had warfare, terror, murder and bloodshed, but they

producedMichelangelo,LeonardodaVinciandtheRenaissance. InSwitzerland, theyhadbrotherlylove,theyhadfivehundredyearsofdemocracyandpeace–andwhatdidthatproduce?Thecuckooclock.’Wellesissaidtohavewrittenthatlinehimself.TheSwissgovernmentreputedlysenthimanangry letter, inwhich theywrote ‘Wedon’tmakecuckooclocks.’ Idon’thaveanythingagainst theSwiss(orOrsonWelles);Itellthisstoryonlybecause,tomyownmind,itechoesevolution.Sincethefirst complexeukaryoticcellsarose, some1.5 to2billionyearsago,wehavehadwarfare, terror,murder andbloodshed:nature, red in tooth andclaw.But in theprecedingaeons,wehad2billionyears of peace and symbiosis, bacterial love (and not only love), and what did this infinity ofprokaryotescomeupwith?Certainlynothingaslargeoroutwardlycomplexasacuckooclock.Intherealmofmorphologicalcomplexity,neitherbacterianorarchaeabegintocomparewithevensingle-celledeukaryotes.

It’sworthemphasisingthispoint.Thetwogreatdomainsofprokaryotes,thebacteriaandarchaea,haveextraordinarygeneticandbiochemicalversatility.Intheirmetabolism,theyputtheeukaryotestoshame:asinglebacteriumcanhavemoremetabolicversatilitythantheentireeukaryoticdomain.Yetfor some reason, neither bacteria nor archaea ever gave rise directly to structural complexity onanythingliketheeukaryoticscale.Intheircellvolume,prokaryotesaretypicallyabout15,000timessmallerthaneukaryotes(althoughtherearesomerevealingexceptions,whichwe’llcometo).Whilethere is some overlap in genome size, the largest known bacterial genomes contain about 12megabases of DNA. In comparison, humans have about 3,000 megabases, and some eukaryoticgenomesrangeupto100,000megabasesormore.Mostcompellingly,thebacteriaandarchaeahavebarelychangedin4billionyearsofevolution.Therehavebeenmassiveenvironmentalupheavalsinthattime.Theriseofoxygenintheairandoceanstransformedenvironmentalopportunities,butthebacteria remained unchanged. Glaciations on a global scale (snowball earths) must have pushedecosystems to the brink of collapse, yet bacteria remained unchanged. The Cambrian explosionconjuredupanimals–pasturesnewforbacteriatoexploit.Throughourhumanprism,wetendtoseebacteria mainly as pathogens, even though the agents of disease are a mere tip of prokaryoticdiversity.Yetthroughouttheseshifts,thebacteriaremainedresolutelybacterial.Neverdidtheygiverisetosomethingaslargeandcomplexasaflea.Nothingismoreconservativethanabacterium.

InChapter1,Iarguedthatthesefactsarebestexplainedintermsofastructuralconstraint.Thereissomethingaboutthephysicalstructureofeukaryotesthatisfundamentallydifferentfromboththe

bacteriaandarchaea.Overcomingthisstructuralconstraintenabledtheeukaryotesalonetoexploretherealmofmorphologicalvariation.Inthebroadestofterms,prokaryotesexploredthepossibilitiesofmetabolism,findingingenioussolutionstothemostarcanechemicalchallenges,whileeukaryotesturned theirbackon thischemicalcleverness,andexplored instead theuntappedpotentialof largersizeandgreaterstructuralcomplexity.

There is nothing radical about the idea of structural constraints, but of course there is noconsensus on what those constraints might be. Many ideas have been put forward, from thecatastrophiclossofthecellwalltothenoveltyofstraightchromosomes.Lossofthecellwallcanbeacatastrophe,aswithout that rigidexternal scaffold,cellseasilyswellandburst.At thesame time,however, a straitjacket prevents cells from physically changing their shape, crawling around andengulfingother cellsbyphagocytosis.A rare successful lossof thecellwallmight thereforehavepermitted theevolutionofphagocytosis–an innovation thatOxfordbiologistTomCavalier-Smithhas long arguedwas key to the evolution of eukaryotes. It is true that the loss of the cell wall isnecessaryforphagocytosis,butmanybacterialosetheircellwallanditisoftenfarfromcatastrophic–so-calledL-formbacteriadoperfectlywellwithoutacellwall,butshownosignofevolvingintodynamic phagocytes. And quite a few archaea do not have a cell wall at all, but likewise do notbecomephagocytes.Toclaimthatthecumbersomecellwallistheconstraintthatpreventedboththebacteriaandarchaeafromevolvinggreatercomplexityhardlystandsuptoscrutinyifmanybacteriaandarchaealosetheircellwallbutdon’tbecomemorecomplex,whereasmanyeukaryotes,includingplants and fungi, have a cellwall (albeit different fromprokaryoticwalls) but are nonetheless farmorecomplexthanprokaryotes.Atellingexampleiseukaryoticalgaecomparedwithcyano-bacteria:both have similar lifestyles, living by photosynthesis, both have cellwalls; but algal genomes aretypically several orders ofmagnitude larger, encompassing far greater cell volume and structuralcomplexity.

Straight chromosomes suffer from a similar problem. Prokaryotic chromosomes are usuallycircular,andDNAreplicationbeginsataparticularpointonthatring(thereplicon).However,DNAreplication is often slower than cell division, and a cell can’t finish dividing in two until it hascompleted copying its DNA. This means a single replicon limits the maximal size of a bacterialchromosome,becausecellswithsmallerchromosomeswilltendtoreplicatefasterthancellswithalarger chromosome. If a cell loses anyunnecessarygenes, it candivide faster.Over time,bacteriawith smaller chromosomes will tend to prevail, especially if they can regain any genes theypreviouslylost,butnowneedagain,bylateralgenetransfer.Incontrast,eukaryotestypicallyhaveanumberof straightchromosomes,eachoneofwhichhasmultiple replicons.Thismeans thatDNAreplicationoperatesinparallelineukaryotes,butinseriesinbacteria.Yethereagain,thisconstrainthardly explains why prokaryotes could not evolve multiple straight chromosomes; indeed, somebacteria and archaeadonow turnout to have straight chromosomes and ‘parallel processing’, butevensotheyhavenotexpandedtheirgenomesizeinthemannerofeukaryotes.Somethingelsemustbestoppingthem.

Practicallyallthestructuralconstraintspositedtoexplainwhybacteriadon’tkeepongivingrisetoeukaryoticcomplexitysufferfromexactlythesameproblem:thereareplentyofexceptionstoeachpurported ‘rule’. As the celebrated evolutionary biologist John Maynard Smith used to say, withcrushingpoliteness,theseexplanationssimplywillnotdo.

Sowhatwilldo?Wehaveseenthatphylogeneticsdonotofferaneasyanswer.Thelastcommonancestor of eukaryotes was a complex cell that already had straight chromosomes, a membrane-bound nucleus, mitochondria, various specialised ‘organelles’ and other membrane structures, adynamic cytoskeleton, and traits like sex. Itwas recognisably a ‘modern’ eukaryotic cell.None ofthese traits exist in bacteria in anything resembling the eukaryotic state. This phylogenetic ‘event

horizon’means that the evolution of eukaryotic traits can’t be traced back in time beyond the lasteukaryoticcommonancestor.It’sas ifeverysingleinventionofmodernsociety–houses,hygiene,roads, division of labour, farming, courts of law, standing armies, universities, governments, youname it – all these inventions could be traced back to ancient Rome; but before Rome, therewasnothing but primitive hunter-gatherer societies. No remains of ancient Greece, China, Egypt, theLevant, Persia, or any other civilisation; just abundant traces of hunter-gatherers everywhere youlook. Here’s the rub. Imagine that experts have spent decades scrutinising the archaeology of theworld tounearth the remainsof earlier cities, civilisations thatpre-dated theRomans,whichcouldgivesomeinsightintohowRomewasbuilt.Hundredsofexampleswerediscovered,yeteachone,oncloserinspection,turnedouttopost-dateRome.Alltheseoutwardlyancientandprimitivecitieswereactually founded in the ‘dark ages’ by progenitors who could trace their own ancestors back toancientRome.Ineffect,allroadsleadtoRome,andRomereallywasbuiltinaday.

Thatmayseemanabsurdfantasy,butitisclosetothesituationthatconfrontsusinbiologyatthemoment.There really are no intermediate ‘civilisations’ betweenbacteria and eukaryotes.The fewthatmasquerade as intermediates (the ‘archezoa’ that we discussed in Chapter 1) were oncemoreglorious,liketheshellofByzantiumastheempireshrankinuponthecitywallsinitslastcenturies.Howcanwemakeheadortailof thisscandalousstateofaffairs?Phylogeneticsdo, infact,offeraclue,acluethatnecessarilyevadedstudiesofsinglegenes,buthasbeenunmaskedinthemoderneraoffullgenomecomparisons.

ThechimericoriginofcomplexityTheproblemwithreconstructingevolutionfromasinglegene(evenoneashighlyconservedasthecommonlyusedribosomalRNAgene)isthat,bydefinition,asinglegeneproducesabranchingtree.Asinglegenecannothavetwodistincthistoriesinthesameorganism–itcannotbechimeric.1Inanidealworld(forphylogeneticists)eachgenewouldproduceasimilartree,reflectingasharedhistory,butwehaveseenthatthisrarelyhappensinthedeepevolutionarypast.Theusualapproachistofallbackonthefewgenesthatdoshareahistory–literallyafewdozenatthemost–andclaimthatthisisthe ‘one true phylogenetic tree’. If thatwere the case, then eukaryoteswould be closely related toarchaea.Thisisthestandard‘textbook’treeoflife(Figure15).Preciselyhowtheeukaryotesrelatetoarchaea isdisputed (differentmethodsandgenesgivedifferentanswers)buteukaryoteswere foralongtimeclaimedtobea‘sister ’grouptothearchaea.Iliketoshowthisstandardtreeoflifewhengivinglectures.Thebranchlengthsindicategeneticdistance.Plainlythereisasmuchgenevariationamong the bacteria and archaea as there is in eukaryotes – sowhat happened in that long branchseparatingthearchaeafromtheeukaryotes?Nohintofaclueishiddeninthistree.

Takewholegenomes,though,andacompletelydifferentpatternemerges.Manyeukaryoticgenesdonothaveanyequivalentsinbacteriaorarchaea,althoughthisproportionisshrinkingasmethodsbecomemorepowerful.Theseuniquegenesareknownaseukaryotic‘signature’genes.Butevenbystandardmethods,roughlyone-thirdofeukaryoticgenesdohaveequivalents inprokaryotes.Thesegenesmustshareacommonancestorwiththeirprokaryoticcousins;they’resaidtobehomologous.Here’swhat’sinteresting.Differentgenesinthesameeukaryoticorganismdonotallsharethesameancestor. Around three-quarters of eukaryotic genes that have prokaryotic homologues apparentlyhavebacterialancestry,whereas the remainingquarter seem toderive fromarchaea.That’s trueofhumans,butwearenotalone.Yeastsareremarkablysimilar;so tooarefruit flies,seaurchinsandcycads.Atthelevelofourgenomes,itseemsthatalleukaryotesaremonstrouschimeras.

Thatmuchisincontestable.Whatitmeansisbitterlycontested.Eukaryotic‘signature’genes,forexample, donot share sequence similaritieswithprokaryotic genes.Whynot?Well, they couldbe

ancient, dating back to the origin of life –whatwemight call the venerable eukaryote hypothesis.Thesegenesdivergedfromacommonancestorsolongagothatanyresemblancehasbeenlostinthemistsof time.If thatwerethecase, theneukaryotesmusthavepickedupvariousprokaryoticgenesmuchmorerecently,forexamplewhentheyacquiredmitochondria.

Thishoaryoldidearetainsanemotionalappealtothosewhovenerateeukaryotes.Emotionsandpersonalityplayasurprisinglybigrole inscience.Someresearchersnaturallyembrace the ideaofabrupt catastrophic changes, whereas others prefer to emphasise continual small modifications –evolutionbyjerksversusevolutionbycreeps,astheoldjokehasit.Bothhappen.Inthecaseoftheeukaryotes, the problem seems to be a case of anthropocentric dignity.We are eukaryotes, and itoffendsourdignitytoseeourselvesasJohnny-come-latelygeneticmongrels.Somescientistsliketoviewtheeukaryotesasdescendingfromtheverybaseofthetreeoflife,forwhatIseeasbasicallyemotionalreasons.Itishardtoprovethatviewwrong;butifitistrue,thenwhydidittakesolongforeukaryotesto‘takeoff’,tobecomelargeandcomplex?Thedelaywas2.5billionyears.Whydoweseenotracesofancienteukaryotesinthefossilrecord(despiteseeingplentyofprokaryotes)?Andifeukaryotes were successful for so long, why are there no surviving early eukaryotes from thisextended period before the acquisition of mitochondria?We have seen that there is no reason tosuppose that theywere outcompeted to extinction, as the existence of the archezoa (seeChapter 1)proves that morphologically simple eukaryotes can survive for possibly hundreds of millions ofyearsalongsidebacteriaandmorecomplexeukaryotes.

Analternative explanation for the eukaryotic signaturegenes is simply that they evolved fasterthantheothergenes,andhencelostanyformersequencesimilarity.Whywouldtheyevolvesomuchfaster? Theywould do so if they had been selected for different functions from their prokaryoticancestors.That sounds entirely reasonable tomy ears.Weknow that eukaryotes have lots of genefamilies, in which scores of duplicated genes specialise to perform different tasks. Becauseeukaryotesexploredamorphologicalrealmthatisbarredtoprokaryotes,forwhateverreason,itishardlysurprisingthattheirgenesshouldhaveadaptedtocarryoutcompletelynewtasks,losingtheirerstwhilesimilaritytotheirprokaryoticancestors.Thepredictionisthatthesegenesdoinfacthaveancestorsamongbacterialorarchaealgenes,butthatadaptationtonewtasksexpungedtheirearlierhistory.Iwillarguelateronthatthisisindeedthecase.Fornow,let’sjustnotethattheexistenceofeukaryotic‘signature’genesdoesnotexcludethepossibilitythattheeukaryoticcellisfundamentallychimeric–theproductofsomesortofmergerbetweenprokaryotes.

So what about the eukaryotic genes that do have identifiable prokaryotic homologues? Whyshouldsomeofthemcomefrombacteriaandsomefromarchaea?Thisiswhollyconsistentwithachimericorigin,obviously.The realquestionconcerns thenumberof sources.Take the ‘bacterial’genes in eukaryotes. By comparing whole eukaryotic genomes with bacteria, the pioneeringphylogeneticist JamesMcInerney has shown that bacterial genes in eukaryotes are associatedwithmanydifferentbacterialgroups.Whendepictedonaphylogenetic tree, they‘branch’withdifferentgroups.Bynomeansdoall thebacterialgenes found ineukaryotesbranchwitha singlegroupofmodern bacteria such as the α-proteobacteria, as might be supposed if they all derived from thebacterialancestorsofmitochondria.Quitethereverse:atleast25differentgroupsofmodernbacteriaappear to have contributed genes to eukaryotes.Much the same goes for archaea, although fewerarchaealgroupslooktohavecontributed.Whatismorecuriousisthatallthesebacterialandarchaealgenesbranch togetherwithin theeukaryotic tree,asshownbyBillMartin(Figure21).Plainly theywereacquiredbytheeukaryotesearlyoninevolution,andhavesharedacommonhistoryeversince.That rules out a steady flow of lateral gene transfer over thewhole course of eukaryotic history.Something odd seems to have happened at the very origin of eukaryotes. It looks like the firsteukaryotes picked up thousands of genes from prokaryotes, but then ceased to ply any trade in

prokaryoticgenes.Thesimplestexplanationforthispictureisnotbacterial-stylelateralgenetransfer,buteukaryotic-styleendosymbiosis.

Onthefaceofit,therecouldhavebeenscoresofendosymbioses,asindeedpredictedbytheserialendosymbiosistheory.Yetitisbarelycrediblethattherecouldhavebeen25differentbacteriaand7or8archaeaallcontributingtoanearlyorgyofendosymbioses,acellularlove-fest;andthennothingfortherestofeukaryotichistory.Butifnotthat,thenwhatelsecouldexplainthispattern?Thereisaverysimpleexplanation–lateralgenetransfer.I’mnotcontradictingmyself.Therecouldhavebeenasingle endo-symbiosis at the origin of eukaryotes, and then almost no further exchange of genesbetweenbacteria and eukaryotes; but plentyof lateral gene transfer over the entireperiodbetweenvariousgroupsofbacteria.Whywouldeukaryoticgenesbranchwith25differentgroupsofbacteria?They would do so if eukaryotes acquired a large number of genes from a single population ofbacteria–apopulationthatsubsequentlychangedovertime.Takearandomassortmentofgenesfromthe25differentgroupsofbacteriaandplacethemall together inasinglepopulation.Let’ssaythatthesebacteriaweretheancestorsofmitochondria,andtheylivedaround1.5billionyearsago.Therearenocellsquitelikethemtoday,butgiventheprevalenceoflateralgenetransferinbacteria,whyshouldtherebe?Someofthispopulationofbacteriawereacquiredbyendosymbiosis,whereasothersretainedtheirfreedomasbacteria,andspentthenext1.5billionyearsswappingtheirgenesbylateraltransfer, asmodernbacteriado.So theancestralhandofgeneswasdealt across scoresofmoderngroups.

Thesamegoesforthehostcell.Takethegenesfromthe7or8groupsofarchaeathatcontributedtoeukaryotes,andplacetheminanancestralpopulationthatlived1.5billionyearsago.Again,someofthesecellsacquiredendosymbionts–whichultimatelyevolvedintomitochondria–whiletherestjust kept doing what archaea do, swapping genes around by lateral gene transfer. Notice that thisscenario is reverse engineering, and assumes nomore thanwhatwe already know to be true: thatlateralgenetransferiscommoninbacteriaandarchaea,andmuchlesscommonineukaryotes.Italsoassumesthatoneprokaryote(anarchaeon,whichbydefinitionisnotcapableofengulfingothercellsby phagocytosis) could acquire endosymbionts by someothermechanism.We’ll put that aside fornowandreturntoitlater.

Figure21TheremarkablechimerismofeukaryotesManyeukaryoticgeneshaveequivalentsinbacteriaorarchaea,buttherangeofapparentsourcesisstartling,asseeninthistreebyBillMartinandcolleagues.Thetreedepictstheclosestmatchestospecificbacterialorarchaealgroupsforeukaryoticgeneswithclearprokaryoticancestry.Thickerlinesindicatethatmoregenesapparentlyderivefromthatsource.Forexample,alargeproportionofgenesappearstoderivefromtheEuryarchaeota.Therangeofsourcescouldbeinterpretedasmultipleendosymbiosesorlateralgenetransfers,butthereisnomorphologicalevidenceforthis,anditisdifficulttoexplainwhyalloftheseprokaryoticgenesbranchtogetherwithintheeukaryotes;thatimpliestherewasashortevolutionarywindowearlyineukaryoticevolutionwhengenetictransferswererife,followedbynexttonothingforthefollowing1.5billionyears.Asimplerandmorerealisticexplanationisthattherewasasingleendosymbiosisbetweenanarchaeonandabacterium,neitherofwhichhadagenomeequivalenttoanymoderngroup;andsubsequentlateralgenebetweenthedescendantsofthesecellsandotherprokaryotesgaverisetomoderngroupswithanassortmentofgenes.

Figure22Two,notthree,primarydomainsoflifeSeminalworkbyMartinEmbleyandcolleaguesshowsthateukaryotesderivefromarchaea.Ashowsaconventionalthree-domainstree,inwhicheachdomainismonophyletic(unmixed):theeukaryotesareatthetop,thebacteriaatthebottom,andthearchaeaareshownsplitintoseverallargegroupsthataremorecloselyrelatedtoeachotherthantoeitherthebacteriaoreukaryotes.Bshowsamorerecentandstronglysupportedalternativetree,basedonfarwidersamplingandalargernumberofinformationalgenesinvolvedintranscriptionandtranslation.Theinformationalgenesofeukaryotesherebranchwithinthearchaea,closetoaspecificgroupknownaseocytes,hencethenameofthehypothesis.Theimplicationisthatthehostcellthatacquiredabacterialendosymbiontattheoriginoftheeukaryoticdomainwasabonafidearchaeon,somethinglikeaneocyte,andwasthereforenotsomesortof‘primitivephagocyte’.TACKstandsforthesuperpylumcomprisingThaumarchaeota,Aigarcheota,CrenarchaeotaandKorarchaeota.

This is thesimplestpossiblescenario for theoriginofeukaryotes: therewasa singlechimericeventbetweenanarchaealhostcellandabacterialendosymbiont.Idonotexpectyoutobelievemeatthispoint. I amarguing simply that this scenario is compatiblewitheverythingweknowabout thephylogenetichistoryofeukaryotes,asareseveralotherpossiblescenarios.IfavourthisviewonthebasisofOccam’srazoralone(itisthesimplestexplanationofthedata),thoughthereisincreasinglypowerfulphylogeneticevidencefromMartinEmbleyandcolleaguesinNewcastlethatthisisexactlywhathappened(Figure22).But given that eukaryotic phylogenetics remains controversial, can thequestion be resolved in some other way? I think so. If the eukaryotes arose in an endosymbiosisbetween two prokaryotes, an archaeal host cell and a bacterial endosymbiont, which went on tobecomemitochondria,thenwecanexplorethequestionfromamoreconceptualpointofview.Canwethinkofagoodreasonwhyonecellgettinginsideanothercellshouldtransformtheprospectsofprokaryotes,unleashing thepotentialofeukaryoticcomplexity?Yes.There isacompellingreason,anditrelatestoenergy.

WhybacteriaarestillbacteriaThekeytoitall is thatprokaryotes–bothbacteriaandarchaea–arechemiosmotic.Wesawinthepreviouschapterhowthefirstcellsmighthavearisenwithintherockywallsofhydrothermalvents,hownatural protongradients couldhavedrivenboth carbonand energymetabolism, andwhy thisreliance on proton gradients could have forced the deep split between bacteria and archaea.Theseconsiderationscouldindeedexplainhowchemiosmoticcouplingfirstarose,buttheydonotexplainwhyitpersistedforevermoreinallbacteria,allarchaea,andalleukaryotes.Wasitnotpossibleforsomegroupstolosechemiosmoticcoupling,toreplaceitwithsomethingelse,somethingbetter?

Somegroupsdid.Yeasts,forexample,spendmuchoftheirtimefermenting,asdoafewbacteria.TheprocessoffermentationgeneratesenergyintheformofATP,butalthoughfaster,fermentationisan inefficient use of resources. Strict fermenters soon pollute their environment, preventingthemselvesfromgrowing,whiletheirwastefulendproducts,suchasethanolorlactate,arefuelsforothercells.Chemiosmoticcellscanburnthesewasteproductswithoxygenorothersubstances,suchasnitrate, toglean farmoreenergy,permitting them tokeepongrowing for longer.Fermentationworkswell as part of amix inwhich other cells burn up the end products, but is very limited byitself.2There is strongevidence that fermentationarose later inevolution than respiration,and thatmakesperfectsenseinlightofthesethermodynamiclimitations.

Perhaps surprisingly, fermentation is theonlyknownalternative tochemiosmoticcoupling.Allformsof respiration, all formsof photosynthesis, and indeed all formsof autotrophy,where cellsgrow from simple inorganic precursors only, are strictly chemiosmotic. We noted some goodreasons for this in Chapter 2. In particular, chemiosmotic coupling is marvellously versatile. Amassive range of electron sources and sinks can all be plugged into a commonoperating system,

allowing small adaptations tohavean immediatebenefit.Likewise,genescanbepassedaroundbylateral gene transfer, and again canbe installed into a fully compatible system, like a newapp.Sochemiosmoticcouplingenablesmetabolicadaptationtoalmostanyenvironmentinalmostnotimeatall.Nowonderitdominates!

But that’s not all.Chemiosmotic coupling also enables the last drops of energy to be squeezedfrom any environment. Take methanogens, which use H2 and CO2 to drive carbon and energymetabolism.Wehavenotedthatit’snoteasytogetH2andCO2toreacttogether:aninputofenergyisneeded toovercome thebarrier to their reaction;methanogensuse that clever trickcalledelectronbifurcationtocoercethemintoreacting.Intermsoftheoverallenergetics,thinkoftheHindenburgairship,theGermandirigiblefilledwithhydrogengas,whicheruptedlikeafirebombaftercrossingtheAtlantic,givinghydrogenabadnameeversince.H2andO2arestableandunreactivesolongasenergyisnotaddedintheformofaspark.Evenasmallsparkimmediatelyreleasesavastamountofenergy. In thecaseofH2andCO2, theproblem is reversed– the ‘spark’has tobe relatively large,whereastheamountofenergyreleasedisrathersmall.

Cellsfaceaninterestinglimitationiftheamountofusableenergyreleasedfromanyonereactionislessthandoubletheenergyinputrequired.Youmayrecallhavingtobalancechemicalequationsatschool.Awholemoleculemustreactwithanothermolecule–it’simpossibleforhalfamoleculetoreactwiththree-quartersofanotherone.Foracell,1ATPmustbespenttogainfewerthan2ATPs.Thereisnosuchthingas1.5ATPs–therecanbeeitheroneortwo.So1ATPhastobespenttogain1ATP.Thereisnonetgain,andthatprecludesgrowthfromH2andCO2bynormalchemistry.ThisistruenotonlyofH2andCO2butalsoofmanyotherredoxcouples(apairingofelectrondonorandacceptor), such asmethane and sulphate.Despite this basic limitationof chemistry, cells still growfromtheseredoxcouplesperfectlyhappily.Theydosobecauseprotongradientsacrossmembranesarebydefinitiongradations.Thebeautyofchemiosmoticcouplingisthatittranscendschemistry.Itallowscellstosaveup‘loosechange’.Ifittakes10protonstomake1ATP,andaparticularchemicalreactiononlyreleasesenoughenergytopump4protons,thenthereactioncansimplyberepeated3timestopump12protons,10ofwhicharethenusedtomake1ATP.Whilethisisstrictlynecessaryfor some forms of respiration, it is beneficial for all of us, as it allows cells to conserve smallamounts of energy that would otherwise bewasted as heat. And that, almost always, gives protongradientsanedgeoverplainchemistry–thepowerofnuance.

The energetic benefits of chemiosmotic coupling suffice to explainwhy it has persisted for 4billion years; but proton gradients also have other facets that have become incorporated into thefunctionofcells.Themoredeeply rootedamechanism, themore it canbecome thebasisofquiteunrelatedtraits.Soprotongradientsarewidelyusedtodrivetheuptakeofnutrientsandexcretionofwaste;theyareusedtoturnthescrewthatisthebacterialflagellum,arotatingpropellerthatmotorscells about; and they are deliberately dissipated to produce heat, as in brown fat cells. Mostintriguingly, their collapse ushers in the abrupt programmed death of bacterial populations. Inessence,whenabacterialcellbecomes infectedwithavirus, it ismost likelydoomed.If itcankillitself quickly, before thevirus copies itself, then its kin (nearbycells sharing relatedgenes)mightsurvive. The genes that orchestrate cell death will spread through the population. But these deathgeneshavetoactquickly,andfewmechanismsarefasterthanperforatingthecellmembrane.Manycellsdoexactly this–wheninfected, theyformpores in themembrane.Thesecollapse theproton-motive force, which in turn trips the latent death machinery. Proton gradients have become theultimatesensorsofcellularhealth,thearbitersoflifeanddeath,arolethatwillloomlargelaterinthischapter.

All in all, the universality of chemiosmotic couplingdoesnot look like a fluke. Its originwas

arguablylinkedtotheoriginoflifeandtheemergenceofcellsinalkalinehydrothermalvents(byfarthemostprobableincubatorsoflife),whileitspersistenceinalmostallcellsmakesverygoodsense.Whatonceseemedtobeapeculiarmechanismnowlookstobeonlysuperficiallycounterintuitive–ouranalysissuggeststhatchemiosmoticcouplingoughttobeliterallyauniversalpropertyoflifeinthecosmos.Andthatmeansthatlifeelsewhereshouldfaceexactlythesameproblemthatbacteriaandarchaeafacehere,rootedinthefactthatprokaryotespumpprotonsacrosstheircellmembrane.Thatdoesn’tconstrainrealprokaryotesinanyway–quitethecontrary–butitdoessetlimitsonwhatispossible.Whatisnotpossible,Iwillargue,ispreciselywhatwedonotsee:largemorphologicallycomplexprokaryoteswithbiggenomes.

The issue isenergyavailabilitypergene. Ihadbeenstumblingblindly towards thisconceptforsomeyears,butitwasthecutandthrustofdialoguewithBillMartinthatreallybroughtmatterstoahead.Afterweeksoftalking,tradingideasandperspectives,itsuddenlydawnedonusthatthekeytotheevolutionofeukaryotesliesinthesimpleideaof‘energypergene’.WithoverflowingexcitementIspentaweekscribblingcalculationsonthebackofanenvelope, in theendlotsofenvelopes,andfinally cameupwith an answer that shocked us both, an answer that extrapolated fromdata in theliterature to put a number on the energy gap that separates prokaryotes from eukaryotes. By ourcalculations, eukaryotes have up to 200,000 times more energy per gene than prokaryotes. Twohundred thousand timesmore energy!At lastwe had a gulf between the two groups, a chasm thatexplainswithvisceralforcewhythebacteriaandarchaeaneverevolvedintocomplexeukaryotes,andbythesametoken,whyweareunlikelyever tomeetanaliencomposedofbacterialcells. Imaginebeingtrappedinanenergylandscape,wherethepeaksarehighenergy,andthetroughslowenergy.Bacteriasitatthebottomofthedeepesttrough,inanenergychasmsoprofoundthatthewallsabovestretchhigh into thesky,utterlyunscalable.Nowonderprokaryotes remained there foraneternity.Letmeexplain.

EnergypergeneByandlarge,scientistscomparelikewithlike.Whenitcomestoenergythefairestcomparisonispergram.Wecancomparethemetabolicrateof1gramofbacteria(measuredasoxygenconsumption)with1gramofeukaryoticcells.Idoubtthatitwillsurpriseyoutolearnthatbacteriausuallyrespirefaster than single-celled eukaryotes, on average three times faster. That unsurprising fact iswheremostresearcherstendtoleaveit;togoonriskscomparingappleswithpears.Wewenton.Whatifwecompared the metabolic rate per cell? What an unfair comparison! In our sampling of about 50bacterial species and 20 single-celled eukaryotic species, the eukaryoteswere (on average) 15,000timeslargerthanthebacteriaintheircellvolume.3Giventhattheyrespireatathirdofthebacterialrate, theaverageeukaryote consumesabout5,000 timesmoreoxygenper second than the averagebacterium. That simply reflects the fact that the eukaryote is much bigger, with far more DNA.Nonetheless,asingleeukaryoticcellstillhas5,000timesmoreenergy.Whatisitspendingiton?

NotmuchofthisextraenergyisspentonDNAitself;onlyabout2%oftheoverallenergybudgetof a single-celled organism goes on replicating DNA. In contrast, according to Frank Harold,distinguishedelderstatesmanofmicrobialbioenergetics(andaheroofmine,eventhoughwedon’talwaysagree),cellsspendasmuchas80%oftheir totalenergybudgetonproteinsynthesis.That’sbecause cells are mostly made of proteins; about half the dry weight of a bacterium is protein.Proteinsarealsoverycostlytomake–theyarestringsofaminoacids,usuallyafewhundredofthemlinkedtogetherinalongchainby‘peptide’bonds.Eachpeptidebondrequiresatleast5ATPstoseal,five times as much as is needed to polymerise nucleotides into DNA. And then each protein isproduced in thousandsofcopies,whicharecontinuously turnedover to repairwearand tear.Toa

first approximation, then, the energy costs of cells equate closely to the costs ofmaking proteins.Each different protein is encoded by a single gene. Assuming that all genes are translated intoproteins(whichisgenerally thecase,despitedifferences ingeneexpression), themoregenes thereareinagenome,thehigherthecostsofproteinsynthesis.Thisisborneoutbythesimpleexpedientofcountingribosomes(theprotein-buildingfactoriesincells),asthereisastraightcorrelationbetweenribosome number and the burden of protein synthesis. There are about 13,000 ribosomes in anaveragebacteriumsuchasE.coli;andatleast13millioninasinglelivercell,about1,000to10,000timesasmany.

On average, bacteria have around 5,000 genes, eukaryotes have about 20,000, ranging up to40,000inthecaseoflargeprotozoa,likethefamiliarpond-dwellingparamecium(whichhastwiceasmany genes as we do). The average eukaryote has 1,200 times as much energy per gene as theaverage prokaryote. Ifwe correct for the number of genes by scaling up the bacterial genome of5,000 genes to a eukaryote-sized genome of 20,000 genes, the bacterial energy-per-gene falls tonearly5,000timeslessthantheaverageeukaryote.Inotherwords,eukaryotescansupportagenome5,000 times larger than bacteria, or alternatively, they could spend 5,000 times more ATP onexpressingeachgene,forexamplebyproducingmanymorecopiesofeachprotein;oramixtureofthetwo,whichisinfactthecase.

Bigdeal,Ihearyousay,theeukaryoteis15,000timeslarger.Ithastofillupthislargervolumewith something, and that something is mostly protein. These comparisons onlymake sense if wecorrect forcellvolume too.Let’sexpandourbacteriumup to theaveragesize foreukaryotes,andcalculatehowmuchenergyitwouldhavetospendpergenethen.YoumightthinkalargerbacteriumwouldhavemoreATP,andindeeditdoes;butitalsohasagreaterdemandforproteinsynthesis,andthatconsumesmoreATP.Theoverallbalancedependsonhowthosefactorsinterrelate.Wecalculatedthatbacteriaactuallypayaheftypenaltyforbeingbigger:sizedoesmatter,andforbacteria,biggerisnot better. On the contrary, giant bacteria should have 200,000 times less energy per gene than aeukaryoteofthesamesize.Here’swhy.

Scaling up a bacterium over orders of magnitude immediately runs into a problem with thesurface-area-to-volumeratio.Oureukaryotehasameanvolumethat is15,000times larger thananaverage bacterium.Let’s keep things simple, and assume that cells are just spheres. To inflate ourbacteriumuptoeukaryoticsize,theradiuswouldneedtoincrease25-fold,andthesurfacearea625-fold.4Thismatters,asATPsynthesistakesplaceacrossthecellmembrane.Toafirstapproximation,then,ATPsynthesiswouldincrease625-fold,inlinewiththeexpandedmembranesurfacearea.

But of course ATP synthesis requires proteins: respiratory chains that actively pump protonsacross themembrane, and theATPsynthase, themolecular turbines thatuse the flowofprotons topowerATPsynthesis.Ifthesurfaceareaofthemembraneisincreased625-fold,ATPsynthesiscouldonly expand 625-fold if the total number of respiratory chains and ATP synthase enzymes wereincreased commensurately, such that their concentration remained the same per unit area. That issurelytrue,butthereasoningispernicious.Alloftheseextraproteinsneedtobephysicallymadeandinserted into themembrane, and that requires ribosomes and all kinds of assembly factors. Thesehavetobesynthesisedtoo.Aminoacidsmustbedeliveredtotheribosomes,alongwithRNAs,allofwhich have to be made as well, necessitating in turn the genes and proteins needed to do so. Tosupportthisextraactivity,morenutrientsmustbeshippedacrosstheexpandedmembranearea,andthis requires specific transport proteins. Indeed we need to synthesise the new membrane too,demanding the enzymes for lipid synthesis. And so on. This great tide of activity could not besupported by a single genome. Picture it, one diminutive genome, sitting there all by itself,responsible for producing 625 times as many ribosomes, proteins, RNAs and lipids, somehowshippingthemacrossthevastlyexpandedcellsurface,forwhat?MerelytosustainATPsynthesisat

thesamerate,perunitsurfacearea,asbefore.Plainlythatisnotpossible.Imagineincreasingthesizeofacity625-fold,withnewschools,hospitals,shops,playgrounds,recyclingcentres,andsoon;thelocalgovernmentresponsibleforalltheseamenitiescanhardlyberunonthesameshoestring.

Giventhespeedofbacterialgrowth,andthebenefitsthataccruefromstreamliningtheirgenomes,thelikelihoodisthatproteinsynthesisfromeachgenomeisalreadystretchedquiteclosetoitslimit.Increasingoverallproteinsynthesisby625-foldwouldmostreasonablyrequire625copiesofthefullbacterialgenometocope,witheachgenomeoperatinginexactlythesameway.

Onthefaceofit,thatmightsoundcrazy.Infact,it’snot;we’llreturntothispointinamoment.Fornow,though,let’sjustconsidertheenergycosts.Wehave625timesasmuchATP,but625timesasmanygenomes,eachoneofwhichhasequivalentrunningcosts.Intheabsenceofasophisticatedintracellulartransportsystem,whichwouldtakemanygenerationsandbucketsofenergytoevolve,eachofthesegenomesisresponsibleforanequivalent‘bacterial’volumeofcytoplasm,membrane,andsoon.Probablythebestwaytoseethisscaled-upbacteriumisnotasasinglecellatall,butasaconsortiumof625identicalcells,mergedintoawhole.Plainlythe‘energypergene’remainsexactlythesameforeachofthesemergedunits.Scalingupthesurfaceareaofabacteriumthereforehasnoenergybenefitatall.Scaled-upbacteria remainatamajordisadvantagecomparedwitheukaryotes.Rememberthateukaryoteshave5,000timesmoreenergypergenethan‘normal’bacteria.Ifscalingupthebacterialsurfaceareaby625-foldhasnoeffectontheenergyavailabilitypergene,thenthatremains5,000timeslowerthanineukaryotes.

Itgetsworse.Wehavescaledupthesurfaceareaofourcell625-fold,bymultiplyingtheenergeticcostsandbenefitsofbacteria625times.Butwhatabouttheinternalvolume?Thatisincreasedbyawhopping15,000-fold.Ourscalingsofarhasproducedagiantbubbleofacell,withaninteriorthatisundefinedinmetabolicterms;wehaveleftitwithzeroenergyrequirements.Thatwouldbetrueiftheinsidewasfilledwithagiantvacuole,metabolicallyinert.Butifthatwerethecase,ourscaled-upbacteriumwouldnotcomparewithaeukaryote,whichisnotjust15,000timeslarger,butisstuffedwithcomplicatedbiochemicalmachinery.That’smostlymadeofproteinstoo,withsimilarenergeticcosts.Thesameargumentsapplyifwetakealltheseproteinsintoaccount.Itisinconceivablethatcellvolumecouldbeincreased15,000-foldwithoutraisingthetotalnumberofgenomesbyroughlythesame amount. But ATP synthesis cannot be increased commensurately – it depends on the cellmembranearea,andwe’vealreadytakenthatintoconsideration.SoscalingupabacteriumtothesizeofanaverageeukaryoteincreasesATPsynthesisby625-fold,butincreasestheenergycostsbyupto15,000-fold.Theenergyavailablepersinglecopyofeachgenemustfall25-fold.Multiplythatbythe5,000-fold difference in energy per gene (after correcting for genome size), and we see thatequalisingforbothgenomesizeandcellvolumemeans thatgiantbacteriahave125,000 times lessenergy per gene than eukaryotes. That’s an average eukaryote. Large eukaryotes such as amoebaehavemorethan200,000timestheenergypergenethanagiantscaled-upbacterium.That’swhereournumbercamefrom.

Youmight thinkthis is justplayingtrivialgameswithnumbers, that itholdsnorealmeaning.Imustconfessthatworriedmetoo–thesenumbersarequiteliterallyincredible–butthistheorisingdoes at leastmake a clear prediction.Giant bacteria should have thousands of copies of their fullgenome.Well, thatpredictioniseasilytestable.Therearesomegiantbacteriaoutthere; they’renotcommon,buttheydoexist.Twospecieshavebeenstudiedindetail.Epulopisciumisknownonlyfromtheanaerobichindgutofsurgeonfish.Itisabattleshipofacell–longandstreamlined,abouthalfamillimetre in length, justvisible to thenakedeye.That’s substantially larger thanmosteukaryotes,includingparamecium(Figure23).WhyEpulopiscium issobig isunknown.Thiomargarita isevenlarger. These cells are spheres, nearly a millimetre in diameter and composed mostly of a hugevacuole.Asinglecell canbeasbigas theheadofa fruit fly!Thiomargarita lives in oceanwaters

periodicallyenrichedinnitratesbyupwellingcurrents.Thecellstrapthenitratesintheirvacuolesforuse as electron acceptors in respiration, enabling them to keep respiring during days orweeks ofnitratedeprivation.But that isnot thepoint.Thepoint is thatbothEpulopiscium andThiomargaritaexhibit‘extremepolyploidy’.Thatmeanstheyhavethousandsofcopiesoftheirfullgenome–upto200,000copiesinthecaseofEpulopisciumand18,000copies in thecaseofThiomargarita (despitemostofthecellbeingahugevacuole).

Suddenly,loosetalkof15,000genomesdoesn’tsoundverycrazyafterall.Notonlythenumberbutthedistributionofthesegenomescorrespondstotheory.Inbothcases,theyarepositionedclosetothecellmembrane,aroundtheperipheryofthecell(Figure23).Thecentreismetabolicallyinert,justvacuoleinthecaseofThiomargarita,andanearlyemptyspawninggroundfornewdaughtercellsinthecaseofEpulopiscium.Thefactthattheinteriorismetabolicallyalmostinertmeanstheysaveonthecostsofproteinsynthesis,andsodon’thoardmoregenomesinsidetheir insides.Intheory, thatmeanstheyshouldberoughlycomparablewithnormalbacteriaintheirenergypergene–theextragenomesareeachassociatedwithmorebioenergeticmembrane,capableofgeneratingall theextraATPneededtosupporttheadditionalcopiesofeachgene.

Figure23Giantbacteriawith‘extremepolyploidy’AshowsthegiantbacteriumEpulopiscium.Thearrowpointstothe‘typical’bacteriumE.coli,forcomparison’ssake.ThecellatthebottomcentreistheeukaryoticprotistParamecium,dwarfedbythisbattleshipofabacterium.BshowsEpulopisciumstainedbyDAPIstainingforDNA.Thewhitedotsclosetothecellmembranearecopiesofthecompletegenome–asmanyas200,000copiesinlargercells,astateknownas‘extremepolyploidy’.Cisanevenlargerbacterium,Thiomargarita,whichisabout0.6mmindiameter.DshowsThiomargaritastainedbyDAPIstainingforDNA.Mostofthecellistakenupwithagiantvacuole,theblackareaatthetopofthemicrograph.Surroundingthevacuoleisathinfilmofcytoplasmcontainingasmanyas20,000copiesofthecompletegenome(markedbywhitearrows).

Figure24EnergypergeneinbacteriaandeukaryotesAshowstheaveragemetabolicratepergeneinbacteria(a,greybar)comparedwithsingle-celledeukaryotes(b,blackbar),whenequalisedforgenomesize.Bshowsmuchthesamething,butthistimeequalisedforcellvolume(15,000-foldlargerineukaryotes)aswellasgenomesize.NoticethattheYaxisonallthesegraphsislogarithmic,soeachunitisa10-foldincrease.Asingleeukaryoticcellthereforehas100,000-foldmoreenergypergenethanbacteria,despiterespiringaboutthreetimesslowerpergramofcells(asshowninC).Thesenumbersarebasedonmeasuredmetabolicrates,butcorrectionsforgenomesizeandcellvolumearetheoretical.Dshowsthatthetheorymatchesrealitynicely.Thevaluesshownarethemetabolicrateforeachsinglegenome,takingintoconsiderationgenomesize,copynumber(polyploidy)andcellvolume.InthiscaseaisE.coli,bisThiomargarita,cisEpulopiscium,disEuglena,andeisthelargeAmoebaproteus.

Andso,itseems,theyare.Themetabolicratesofthesebacteriahavebeenskilfullymeasuredandweknowthetotalcopynumberofthegenome,sowecancalculatetheenergypergenedirectly.Andlo! It is close (within the same order ofmagnitude) to that of the bog-standard bacteriumE. coli.Whateverthecostsandbenefitsofgreatersizeingiantbacteriamaybe,thereisnoenergyadvantage.Exactlyaspredicted,thesebacteriahaveabout5,000timeslessenergypersinglecopyofeachgenethan eukaryotes (Figure24).Note that this figure is not 200,000 times less, as these giant bacteriaonly have multiple genomes around their periphery, and not inside – their inner volume ismetabolicallynearlyinert,givingthegiantsaproblemwithcelldivision,whichhelpstoexplainwhythey’renotabundant.

Bacteria and archaea are happy as they are. Small bacteria with small genomes are notenergeticallylimited.Theproblemonlyemergeswhenwetrytoscaleupbacteriatoeukaryoticsizes.Ratherthanswellinguptheirgenomesizeandenergyavailabilityineukaryoticfashion,energypergene actually falls.Thegulf becomes enormous.Bacteria can’t expand their genome size, nor canthey accumulate the thousands of new gene families, encoding all kinds of new functions, thatepitomiseeukaryotes.Ratherthanevolvingasinglegiganticnucleargenome,theyenduphoarding

thousandsofcopiesoftheirstandard-issuesmallbacterialgenome.

HoweukaryotesescapedWhydon’tthesameproblemsofscalepreventeukaryotesfrombecomingcomplex?Thedifferenceliesinthemitochondria.Recallthateukaryotesarguablyoriginatedinagenomicchimerabetweenanarchaealhostcellandabacterialendosymbiont.Thephylogeneticevidence,Isaid,isconsistentwiththis scenario, but that in itself is not sufficient to prove it. Yet the severe energetic constraints onbacteriacomeveryclose toprovingarequirement for a chimericoriginof complex life.Onlyanendosymbiosisbetweenprokaryotes, Iwill argue,couldbreak theenergeticconstraintsonbacteriaandarchaea–andendosymbiosesbetweenprokaryotesareextremelyrareinevolution.

Bacteriaareautonomousself-replicatingentities–cells–whereasgenomesarenot.Theproblemthat faces giant bacteria is that, to be large, theymust replicate their whole genome thousands oftimes.Eachgenome is copiedperfectly,ornearlyperfectly,but then it just sits there,unable todoanythingelse.Proteinsmayset toworkon it, transcribingand translatinggenes; thehost cellmaydivide, powered by the dynamism of its proteins andmetabolism, but the genome itself iswhollyinert,asincapableofreplicatingitselfastheharddiskofacomputer.

Whatdifferencedoesthatmake?Itmeansthatallthegenomesinthecellareessentiallyidenticalcopiesofeachother.Differencesbetweenthemarenotsubjecttonaturalselection,becausetheyarenotself-replicatingentities.Anyvariationsbetweendifferentgenomesinthesamecellwillevenoutovergenerations,assomuchnoise.Butconsiderwhathappenswhenwholebacteriacompeteamongthemselves. If one line of cells happens to replicate twice as fast as another, it will double itsadvantageeachgeneration,growingexponentiallyfaster.Injustafewgenerations,thefast-growinglinewill dominate the population. Such amassive advantage in growth ratemight be unlikely, butbacteriagrowsoquicklythatevensmalldifferencesingrowthratecanhaveamarkedeffectonthecompositionofapopulationovermanygenerations.Forbacteria,onedaycouldseethepassageof70generations,thedawnofthatdayasremoteasthebirthofChristwhenmeasuredinhumanlives.TinydifferencesingrowthratecanbeachievedbysmalldeletionsofDNAfromagenome,suchasalossofonegenethatisnolongerinuse.Nomatterwhetherthisgenemightbeneededagaininthefuture,thecellsthatloseitwillreplicatealittlefaster,andwithinafewdayswillcometodominatethepopulation.Thosethatretaintheuselessgenewillslowlybedisplaced.

Thenconditionschangeagain.Theuselessgeneregainsitsvalue.Cellslackingitcannolongergrow,unlesstheyreacquireitbylateralgenetransfer.Thisendlesslycirculardynamicofgenelossandgaindominatesbacterialpopulations.Over time,genomesizestabilisesat thesmallest feasiblesize,whileindividualcellshaveaccesstoamuchlarger‘metagenome’(thetotalpoolofgeneswithinthewhole population, and indeed neighbouring populations).A singleE.coli cellmay have 4,000genes,butthemetagenomeismorelike18,000genes.Dippingintothismetagenomecarriesrisks–picking up thewrong gene, or amutated version, or a genetic parasite instead; but over time thestrategypaysoff,asnaturalselectioneliminatesthelessfitcellsandtheluckywinnerstakeall.

Butnowthinkaboutapopulationofbacterialendosymbionts.Thesamegeneralprinciplesapply–thisisjustanotherpopulationofbacteria,albeitasmallpopulationinarestrictedspace.Bacteriathatlose unnecessary geneswill replicate slightly faster and tend to dominate, just as before. The keydifference is the stability of the environment. Unlike the great outdoors, where the conditions arealwayschanging,thecytoplasmofcellsisaverystableenvironment.Itmaynotbeeasytogetthere,or to survive there, but once established, a steady and invariable supply of nutrients can be reliedupon.Theendlesslycirculardynamicofgenelossandgaininfree-livingbacteriaisreplacedwithatrajectorytowardsgenelossandgeneticstreamlining.Genesthatarenotneededwillneverbeneeded

again.Theycanbelostforgood.Genomesshrink.Imentionedthatendosymbiosesarerarebetweenprokaryotes,whicharenotcapableofengulfing

othercellsbyphagocytosis.Wedoknowofacoupleofexamplesinbacteria(Figure25),soplainlytheycanoccur,ifonlyveryoccasionallyintheabsenceofphagocytosis.Afewfungiarealsoknowntohaveendosymbionts,despitebeingnomorephagocytic thanbacteria.Butphagocyticeukaryotesfrequentlyhaveendosymbionts;hundredsofexamplesareknown.5Theyshareacommontrajectorytowardsgene loss.The smallest bacterial genomesareusually found in endosymbionts.Rickettsia,forexample,thecauseoftyphusandscourgeofNapoleon’sarmy,hasagenomesizeofjustover1megabase,barelyaquarterthesizeofE.coli.Carsonella,anendosymbiontofjumpingplantlice,hasthe smallest known bacterial genome, which at 200 kilobases is smaller than some plantmitochondrialgenomes.Althoughweknownexttonothingaboutgenelossinendosymbiontswithinprokaryotes,thereisnoreasontosupposetheywouldbehaveanydifferently.Indeed,wecanbesuretheywouldhavelostgenesinmuchthesameway:mitochondria,afterall,wereonceendosymbiontslivinginanarchaealhost.

Figure25BacterialivingwithinotherbacteriaAApopulationofintracellularbacterialivinginsidecyanobacteria.Thewavyinternalmembranesintheright-handcellarethylakoidmembranes,thesiteofphotosynthesisincyanobacteria.Thecellwallisthedarkerlineenclosingthecell,whichissheathedwithinatransluscentgelatinouscoat.Theintracellularbacteriaareenclosedinalighterspacethatcouldbemistakenforaphagocyticvacuole,butisprobablyanartefactofshrinkage,asnocellswithawallcanengulfothercellsbyphagocytosis.Howthesebacteriagotinsideisamystery,butthere’snodoubtthattheyarereallythere,andsonodoubtthatitispossible,ifveryrare,tohaveintracellularbacteriainsidefree-livingbacteria.BPopulationsofgamma-proteobacteriainsidebeta-proteobacterialhostcells,inturnlivingwithintheeukaryoticcellsofamulticellularmealybug.Ontheleft,thecentralcell(withthenucleusabouttodividebymitosis)hassixbacterialendosymbionts,eachofthemcontaininganumberofrod-shapedbacteria,shownmagnifiedontheright.Thiscaseislesscompellingthanthecyanobacterialexample,astheircohabitationwithinaeukaryoticcellisnotequivalenttoafree-livinghostcell;nonetheless,bothcasesshowthatphagocytosisisnotneededforanendosymbiosisbetweenbacteria.

Genelossmakesahugedifference.Losinggenesisbeneficialtotheendosymbiont,asitspeedsup their replication; but losing genes also saves ATP. Consider this simple thought experiment.Imaginethatahostcellhas100endosymbionts.Eachendosymbiontstartsoutasanormalbacterium,anditlosesgenes.Let’ssayitstartsoutwithafairlystandardbacterialgenomeof4,000genes,anditloses 200 of them (5%), perhaps initially the genes for cell-wall synthesis, which are no longerneededwhenlivinginahostcell.Eachofthese200genesencodesaprotein,whichhasanenergeticcost tosynthesise.Whataretheenergysavingsofnotmaking thoseproteins?Anaveragebacterialprotein has 250 amino acids,with an average of 2,000 copies of each protein. Each peptide bond(whichjoinsaminoacidstogether)costsabout5ATPs.SothetotalATPcostof2,000copiesof200proteinsin100endosymbiontsis50billionATPs.Ifthisenergycostisincurredduringthelifecycleofacell,andthecelldividesevery24hours,thenthecostforsynthesisingtheseproteinswouldbe580,000ATPspersecond!Conversely,thatistheATPsavingifthoseproteinsarenotmade.

There is no necessary reason for theseATPs to be spent on anything else of course (althoughthere are some possible reasons, to which we will return), but let’s just consider what kind ofdifferenceitcouldmaketoacelliftheywerespent.Onerelativelysimplefactorthatsetseukaryotesapart frombacteria is adynamic internal cytoskeleton, capableof remodelling itself andchangingshape in the course of either cellmovement or the transport ofmaterialswithin the cell.Amajorcomponentof theeukaryoticcytoskeletonisaproteincalledactin.Howmuchactincouldwemakefor580,000ATPspersecond?Actinisafilamentcomposedofmonomersjoinedtogetherinachain;andtwosuchchainsarewoundaroundeachothertoformthefilament.Eachmonomerhas374aminoacids,andthereare2×29monomerspermicrometreofactinfilament.WiththesameATPcostperpeptidebond,thetotalATPrequirementpermicrometreofactinis131,000.Soinprinciplewecouldmakeabout4.5micrometresofactinpersecond.Ifthatdoesn’tsoundmuchtoyou,bearinmindthatbacteria are typically a couple of micrometres in length.6 So the energy savings accruing fromendosymbiotic gene loss (just 5%of their genes) could easily support the evolution of a dynamiccytoskeleton, as indeed happened.Bear inmind, aswell, that 100 endosymbionts is a conservativeestimate.Somelargeamoebaehaveasmanyas300,000mitochondria.

Andgene losswentmuch further thanamere5%.Mitochondria lostnearlyall theirgenes.Wehave retained just 13 protein-coding genes, along with all other animals. Assuming that themitochondriaderivedfromancestorsthatwerenotdissimilartomodernα-proteobacteria,theymusthavestartedoutwitharound4,000genes.Overevolutionarytime,theylostmorethan99%oftheirgenome.Byourcalculationabove,if100endosymbiontslost99%oftheirgenes,theenergysavingswouldbecloseto1trillionATPsovera24-hourlifecycle,orastaggering12millionpersecond!Butmitochondriadon’tsaveenergy.TheymakeATP.MitochondriaarejustasgoodatmakingATPas their free-living ancestors, but they reduced the costly bacterial overheadsmassively. In effect,eukaryoticcellshavemultibacteriapower,butsaveonthecostsofproteinsynthesis.Orrather,they

divertthecostsofproteinsynthesis.Mitochondrialostmostoftheirgenes,butsomeofthemweretransferredtothenucleus(moreon

thatinthenextchapter).Someofthesegenescontinuedtoencodethesameproteins,carryingoutthesameoldjob,sotherewerenoenergysavingsthere.Butsomeofthemwerenolongerneeded,eitherby the host cell or the endosymbiont. They arrived in the nucleus as genetic free-booters, free tochangetheirfunction,unconstrainedasyetbyselection.ThesesuperfluousstretchesofDNAarethegeneticrawmaterial foreukaryoticevolution.Someof themspawnedwholefamiliesofgenes thatcouldspecialise fornewdisparate tasks.Weknow that theearliesteukaryoteshadabout3,000newgenefamiliescomparedwithbacteria.Genelossfrommitochondriaenabledtheaccumulationofnewgenesinthenucleusatnoenergeticcost.Inprinciple,ifacellthathad100endosymbiontstransferred200genesfromeachendosymbionttothenucleus(just5%oftheirgenes)thehostcellwouldhave20,000newgenes in thenucleus– awholehumangenome’sworth!–whichcouldbeused for allkinds of novel purposes, all at no net energetic cost. The advantage of mitochondria is simplybreathtaking.

Twoquestionsremain,andtheyaretightlylinked.First,thisentireargumentisbasedontheissueof surface-area-to-volume ratio in prokaryotes. But some bacteria, such as cyanobacteria, areperfectlycapableofinternalisingtheirbioenergeticmembranes,twistingtheirinnermembraneintobaroque convolutions, expanding their surface area considerably. Why can’t bacteria escape theconstraintsofchemiosmoticcouplingbyinternalisingtheirrespirationinthisway?Andsecond,why,ifgenelossissoimportant,didthemitochondrianeverlosetheirfullgenome,takingtheprocesstocompletionandmaximisingtheenergeticbenefitsofgeneloss?Theanswerstothesequestionsmakeitclearwhybacteriaremainedstuckintheirrutfor4billionyears.

Mitochondria–keytocomplexityIt’s not obviouswhymitochondria always retain a handful of genes.Hundreds of genes encodingmitochondrialproteinswere transferred to thenucleusearly ineukaryoticevolution.Theirproteinproductsarenowmadeexternallyinthecytosol,beforebeingimportedintothemitochondria.Yetasmallgroupofgenes,encodingrespiratoryproteins,invariablyremainedinthemitochondria.Why?ThestandardtextbookMolecularBiologyoftheCellstates:‘Wecannotthinkofcompellingreasonswhytheproteinsmadeinthemitochondriaandchloroplastsshouldbemadethere,ratherthaninthecytosol.’ That same sentence appears in the 2008, 2002, 1992 and 1983 editions; one is entitled towonderhowmuchtheauthorsdidactuallythinkaboutthequestion.

Fromthestandpointofeukaryoticorigins,itseemstometherearetwopossibletypesofanswer–trivial,ornecessary.WhenIsay‘trivial’Idon’tmeanthatinatrivialsense–Imeanthatthereisnounmodifiablebiophysicalreasonforthemitochondrialgenestoremainwheretheyare.Thefactthattheyhavenotmovedisnotbecausetheycan’tmove,butbecauseforhistoricalreasonstheysimplyhavenot.Trivialanswersexplainwhygenesstayedinthemitochondria:theycouldhavemovedtothenucleus,butthebalanceofchanceandselectiveforcesmeantthatsomeofthemremainedwheretheyhadalwaysbeen.Possiblereasonsincludethesizeandhydrophobicityofmitochondrialproteins,orminoralterations in thegeneticcode. Inprinciple, the‘trivial’hypothesisargues,all the remainingmitochondrialgenescouldbetransferredtothenucleus,albeitrequiringalittlegeneticengineeringto modify their sequence as necessary, and the cell would work perfectly well. There are someresearchers activelyworking on transferringmitochondrial genes to the nucleus, on the basis thatsuch a transfer could prevent ageing (more on that in Chapter 7). This is a problem beset withchallenges,notatrivialundertakinginthecolloquialuseoftheterm;butitistrivialinthesensethatthese researchersbelieve there isnoneedforgenes to remain in themitochondria.They think that

therearerealbenefitstotransferringthemtothenucleus.Goodlucktothem.Idisagreewiththeirreasoning.The‘necessary’hypothesisarguesthatmitochondriahaveretained

genes because they need genes – without them, mitochondria could not exist at all. The cause isunmodifiable:itisnotpossibletotransferthesegenestothenucleuseveninprinciple.Whynot?Theanswer,inmyview,comesfromJohnAllen,abiochemistandlong-standingcolleague.Ibelievehisanswernotbecauseheisafriend;quitethereverse.WebecamefriendsinpartbecauseIbelievehisanswer.Allenhasafertilemindandhasputforwardanumberoforiginalhypotheses,whichhehasspentdecadestestingandsomeofwhichwehavebeenarguingaboutforyears.Inthisparticularcase,he has good evidence supporting the argument that mitochondria (and chloroplasts, for similarreasons)haveretainedgenesbecausetheyareneededtocontrolchemiosmoticcoupling.Transfertheremainingmitochondrialgenes to thenucleus, theargumentgoes, and thecellwilldie in time,nomatterhowcarefullycraftedthegenesmaybetotheirnewhome.Themitochondrialgenesmustberight there on site, next to the bioenergetic membranes they serve. I’m told the political term is‘bronzecontrol’.7Inawar,goldcontrolisthecentralgovernment,whichshapeslong-termstrategy;silvercontrolisthearmycommand,whoplanthedistributionofmanpowerorweaponryused;butawariswonorlostontheground,underthecommandofbronzecontrol, thebravemenorwomenwhoactuallyengagetheenemy,whotakethetacticaldecisions,whoinspiretheirtroops,andwhoareremembered inhistoryasgreat soldiers.Mitochondrialgenesarebronzecontrol,decision-makersontheground.

Why are such decisions necessary? In Chapter 2 we discussed the sheer power of the proton-motive force. The mitochondrial inner membrane has an electrical potential of about 150–200millivolts. As the membrane is just 5 nanometres thick, we noted that this translates into a fieldstrengthof30millionvoltspermetre,equaltoaboltoflightning.Woebetideyouifyoulosecontroloversuchanelectricalcharge!ThepenaltyisnotsimplyalossofATPsynthesis,althoughthatalonemaywellbeserious.Failuretotransferelectronsproperlydowntherespiratorychainstooxygen(orotherelectronacceptors)canresultinakindofelectricalshort-circuiting,inwhichelectronsescapetoreactdirectlywithoxygenornitrogen,toformreactive‘freeradicals’.ThecombinationoffallingATPlevels,depolarisationofthebioenergeticmembranesandreleaseoffreeradicalsistheclassictrigger for ‘programmed cell death’, which we noted earlier is widespread, even in single-celledbacteria.Inessence,mitochondrialgenescanrespondtolocalchangesinconditions,modulatingthemembranepotentialwithinmodestboundsbeforechangesbecomecatastrophic.If thesegenesweremoved to the nucleus, the hypothesis is simply that themitochondriawould lose control over themembranepotentialwithinminutesofanyseriouschangesinoxygentensionorsubstrateavailability,orfree-radicalleak,andthecellwoulddie.

We need to breathe continuously to stay alive and to exert fine control over muscles in thediaphragm,chest and throat.Downat the levelofmitochondria, themitochondrialgenesmodulaterespiration inmuch thesameway,makingsure thatoutput isalways finely tailored todemand.Nootherreasonisbigenoughtoexplaintheuniversalretentionofmitochondrialgenes.

This ismore than a ‘necessary’ reason for genes to remain inmitochondria. It is a necessaryreasonforgenestobestationednexttobioenergeticmembraneswherevertheymaybe.It’sstrikingthatmitochondriahaveinvariablyretainedthesamesmallsubsetofgenesinalleukaryotescapableofrespiration.Onthefewoccasionsthatcellslostgenesfromthemitochondriaaltogether,theyalsolostthe ability to respire. Hydrogenosomes and mitosomes (the specialised organelles derived frommitochondriafoundinthearchezoa)havegenerallylostall theirgenes,andhavelost thepowerofchemiosmotic coupling into thebargain.Conversely thegiantbacteriawediscussedearlier alwayshavegenes(orratherwholegenomes)stationedrightnexttotheirbioenergeticmembranes.Formethecaseisclinchedbycyanobacteria,withtheirconvolutedinnermembrane.Ifgenesarenecessaryto

controlrespiration,thencyanobacteriashouldhavemultiplecopiesoftheirfullgenome,inmuchthesame way as the giant bacteria, even though they are substantially smaller. They do. The morecomplexcyanobacteriaoftenhaveseveralhundredcopiesof theircompletegenome.Aswithgiantbacteria,thatconstrainstheirenergyavailabilitypergene–theycannotincreasethesizeofanyonegenome up to a eukaryotic-sized nuclear genome, because they are obliged instead to accumulatemultiplesmallbacterialgenomes.

Here, then, is the reason that bacteria cannot inflate up to eukaryotic size. Simply internalisingtheirbioenergeticmembranesandexpandinginsizedoesnotwork.Theyneedtopositiongenesnexttotheirmembranes,andthereality,intheabsenceofendosymbiosis,isthatthosegenescomeintheformoffullgenomes.Thereisnobenefitintermsofenergypergenefrombecominglarger,exceptwhenlargesizeisattainedbyendosymbiosis.Onlythenisgenelosspossible,andonlythencantheshrinkingofmitochondrialgenomesfueltheexpansionofthenucleargenomeoverseveralordersofmagnitude,uptoeukaryoticsizes.

Youmight have thought of another possibility: the use of bacterial plasmids, semi-independentringsofDNAthatcancarryscoresofgenesonoccasions.Whycouldn’tthegenesforrespirationbeplaced on one large plasmid, and then multiple copies of this plasmid be stationed next to themembranes? There are potentially intractable logistical difficulties with this, but could it work inprinciple?Ithinknot.Amongprokaryotesthereisnoadvantagetobeingbiggerforitsownsake,andno advantage to havingmoreATP than necessary. Small bacteria are not short ofATP: they haveplenty.BeingalittlelargerandhavingalittlemoreATPcarriesnobenefit; it isbetter tobealittlesmaller and have just enough ATP – and replicate faster. A second disadvantage to expanding involumeforitsownsakeisthatsupplylinesareneededtoserveremoteregionsofthecell.Alargecellneedstoshipcargotoallquarters,andeukaryotesdoexactlythat.Butsuchtransportsystemsdonotevolveovernight.Thattakesmanygenerations,duringwhichtimetherewouldneedtobesomeotheradvantagetobeingbigger.Soplasmidswon’twork–theyputthecartbeforethehorses.Byfarthesimplestsolutiontotheproblemofdistributionisjusttoside-stepitaltogether,tohavemultiplecopiesofafullgenome,eachcontrollinga‘bacterial’volumeofcytoplasm,asinthegiantbacteria.

Sohowdideukaryotesbreakoutofthesizeloop,andevolvecomplextransportsystems?Whatissodifferentabouta largecellwithmultiplemitochondria,eachoneofwhichhas itsownplasmid-sizedgenome, andagiantbacteriumwithmultipleplasmids, dispersed to control respiration?TheansweristhatthedealattheoriginofeukaryoteshadnothingtodowithATP,aspointedoutbyBillMartin andMiklosMüller in their hypothesis for the first eukaryote.Martin andMüller propose ametabolic syntrophy between the host cell and its endosymbionts, meaning that they trade in thesubstratesofgrowth,not just energy.Thehydrogenhypothesis argues that the first endosymbiontsprovided their methanogen host cells with hydrogen needed for growth.We don’t need to worryabout thedetailshere.Thepoint is thatwithout theirsubstrate(hydrogenin thiscase) thehostcellscannot grow at all. The endosymbionts provide all the substrate needed for growth. The moreendosymbionts, the more substrate, the faster the host cells can grow; and the better theendosymbionts do too. In the case of endosymbioses, then, larger cells do benefit because theycontainmore endosymbionts, and so gainmore fuel for growth. Theywill do even better as theydeveloptransportnetworkstotheirownendosymbionts.Thisalmostliterallyputsthehorses(powersupply)beforethecart(transport).

As the endosymbionts lose genes, their own ATP demands fall. There’s an irony here. CellrespirationproducesATP fromADP,andas theATP isbrokenbackdown toADP itpowersworkaround the cell. If ATP is not consumed, then the entire pool of ADP is converted into ATP, andrespiration grinds to a halt. Under these conditions the respiratory chain accumulates electrons,becominghighly‘reduced’(moreonthisinChapter7).Itisthenreactivewithoxygen,leakingfree

radicalsthatcandamagethesurroundingproteinsandDNA,oreventriggercelldeath.Theevolutionofonekeyprotein, theADP–ATPtransporter,enabledthehostcell tobleedofftheendosymbionts’ATPforitsownpurposes,buttellingly,alsosolvedthisproblemfortheendosymbionts.BybleedingoffexcessATPandresupplyingtheendosymbiontswithADP,thehostcellrestrictedfree-radicalleakwithintheendosymbiont,andsoloweredtheriskofdamageandcelldeath.Thishelpsexplainwhyitwas in the interestsofboth thehostcell andendosymbionts to ‘burn’ATPonextravagantbuildingprojectssuchasadynamiccytoskeleton.8Butthekeypointisthattherewereadvantagesateverystageoftheendosymbioticrelationship,unlikeplasmids,whichoffernobenefittobeinglargerorhavingmoreATPforitsownsake.

Theoriginof theeukaryoticcellwasasingularevent.Hereonearth ithappenedjustonce in4billion years of evolution. When considered in terms of genomes and information, this peculiartrajectory is nearly impossible to understand. But when considered in terms of energy and thephysicalstructureofcellsitmakesagreatdealofsense.Wehaveseenhowchemiosmoticcouplingmay have arisen in alkaline hydrothermal vents, and why it remained universal in bacteria andarchaea for all eternity. We have seen that chemiosmotic coupling made possible the wonderfuladaptabilityandversatilityofprokaryotes.Such factorsare likely toplayoutonotherplanets too,rightbacktothebeginningsoflifefromlittlemorethanrock,waterandCO2.Nowwesee,too,whynaturalselection,operatingon infinitepopulationsofbacteriaover infiniteperiodsof time,shouldnot give rise to large complex cells, what we know as eukaryotes, except by way of a rare andstochasticendosymbiosis.

Thereisnoinnateoruniversaltrajectorytowardscomplexlife.Theuniverseisnotpregnantwiththe ideaofourselves.Complex lifemightariseelsewhere,but it isunlikely tobecommon, for thesame reasons it did not arise repeatedly here. The first part of the explanation is simple –endosymbiosesbetweenprokaryotesarenotcommon(althoughwedoknowofacoupleofexamples,soweknowthattheycanhappen).Thesecondpartislessobvious,andsmacksofSartre’svisionofhell as other people. The intimacy of endosymbiosis might have broken the endless deadlock ofbacteria,butinthenextchapterweshallseethatthetormentedbirthofthisnewentity,theeukaryoticcell,goessomeway towardsexplainingwhysucheventshappenvery rarely,andwhyallcomplexlifesharessomanypeculiartraits,fromsextodeath.

Footnotes1Actuallytechnicallyitcan,asasinglegenecanbesplicedtogetherfromtwoseparatepieceswithdifferenthistories;butingeneralthisdoesnothappen,andintryingtotracehistoryfromsinglegenes,phylogeneticistsdonotusuallysetouttoreconstructconflictingstories.2Muchthefastestandmostreliablewayofremovingtheendproductsoffermentationisburningthem,viarespiration.Theendproduct,CO2,islostsimplybydiffusionintotheair,orprecipitationascarbonaterocks.Fermentationthereforedependslargelyonrespiration.3Forthesecomparisons,weneedtoknowthemetabolicrateofeachofthesecells,aswellascellvolumeandgenomesize.Ifyouthinkthat50bacteriaand20eukaryotesisnotmanyforacomparisonofthiskind,justthinkofthedifficultiesinvolvedinprocuringallthisinformationforeachcelltype.Thereareplentyofcaseswherethemetabolicratehasbeenmeasured,butnotthegenomesizeorcellvolume,orviceversa.Evenso,thevaluesthatweextractedfromtheliteratureseemtobereasonablyrobust.Ifyouareinterestedinthedetailedcalculations,seeLaneandMartin(2010).4Thevolumeofaspherevarieswiththecubeofitsradius,whereasthesurfaceareavarieswiththesquareoftheradius.Increasingtheradiusofthespherethereforeincreasesthevolumefasterthanthesurfacearea,givingcellsaprobleminthattheirsurfaceareabecomesproportionatelysmallerinrelationtotheirvolume.Changingshapehelps:forexample,manybacteriaarerod-shaped,givingthemalargersurfaceareainrelationtotheirvolume;butwhenexpandedinsizeoverseveralordersofmagnitude,suchshapechangesonlymitigatetheproblemtoadegree.5Thefactthatprokaryotescannotengulfothercellsbyphagocytosisissometimescitedasareasonwhythehostcell‘had’tobesomekindof‘primitive’phagocyte,notaprokaryote.Therearetwoproblemswiththisreasoning.Thefirstisthatitisjustnottrue–weknowofrareexamplesofendosymbiontslivingwithinprokaryotes.Thesecondproblemispreciselythatendosymbiontsarecommonineukaryotes,andyetdonotroutinelygiverisetoorganelleslikemitochondria.Indeed,theonlyexamplesknownaremitochondriaandchloroplasts,despite(nodoubt)thousandsormillionsofopportunities.Theoriginoftheeukaryoticcellwasasingularevent.Asnotedin

Chapter1,aproperexplanationmustelucidatewhyithappenedonlyonce:itmustbepersuasiveenoughtobebelievable,butnotsopersuasivethatweareleftwonderingwhyitdidnothappenonmultipleoccasions.Endosymbiosisbetweenprokaryotesisrare,butnotsorarethatitcanaccountforthesingularityofeukaryoticoriginsbyitself.However,theenormousenergeticrewardsofendosymbiosisbetweenprokaryotes,whencombinedwithgravedifficultiesofreconcilinglifecycles(whichwewilldiscussinthenextchapter),dotogetherexplainthisevolutionarysingularity.6Toputsomeperspectiveonthosenumbers,animalcellsgenerallyproduceactinfilamentsatarateofabout1–15micrometresperminute,butsomeforaminiferacanreachspeedsof12micrometrespersecond.Thisistherateofassemblyfrompreformedactinmonomers,however,notdenovosynthesisofactin.7Iwasintroducedtothistermbyaformerdefencesecretary,JohnReid,whoinvitedmetoteaintheHouseofLordsafterreadingLifeAscending.Myattemptstoexplainthedecentralisedregulationofmitochondriatomyintellectuallyvoracioushostturnedouttomakeperfectsenseinmilitaryterms.8ThereisaninstructivebacterialprecedentforburningATP,knownasATPorenergy‘spilling’.Thetermisaccurate:somebacteriacansplashawayuptotwo-thirdsoftheiroverallATPbudgetonfutilecyclingofionsacrossthecellmembraneandotherequallypointlessfeats.Why?OnepossibleansweristhatitkeepsahealthybalanceofATPtoADP,whichkeepsthemembranepotentialandfree-radicalleakundercontrol.Again,itgoestoshowthatbacteriahaveplentyofATPtospare–theyarenotinanywayenergeticallychallenged;onlyscalinguptoeukaryoticsizesrevealstheenergy-per-geneproblem.

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6

SEXANDTHEORIGINSOFDEATH

ature abhors a vacuum, saidAristotle. The ideawas echoed, twomillennia later, byNewton.Bothworriedaboutwhatfillsspace;Newtonbelieved itwasamysterioussubstanceknownas

theæther.Inphysics,theideafellintodisreputeinthetwentiethcentury,butthehorrorvacui retainsallitsstrengthinecology.Thefillingupofeveryecologicalspaceisnicelycapturedinanoldrhyme:‘Big fleas have little fleas upon their backs to bite ’em; little fleas have smaller fleas, and so adinfinitum.’ Every conceivable niche is occupied, with each species exquisitely adapted to its ownspace.Everyplant,everyanimal,everybacterium,isahabitatinitself,ajungleofopportunitiesforall kinds of jumping genes, viruses and parasites, to say nothing of big predators. Anything andeverythinggoes.

Exceptthatitdoesn’t.Itonlylooksthatway.Theinfinitetapestryoflifeisbutasemblance,withablackholeatitsheart.It istimetoaddressthegreatestparadoxinbiology:whyit isthatall lifeonearthisdividedintoprokaryotes,whichlackmorphologicalcomplexity,andeukaryotes,whichshareamassivenumberofdetailedproperties,noneofwhicharefoundinprokaryotes.Thereisagulf,avoid,avacuum,betweenthetwo,whichnaturereallyoughttoabhor.Alleukaryotessharemoreorlesseverything;allprokaryoteshave,fromamorphologicalpointofview,nexttonothing.Thereisnobetterillustrationoftheinequitablebiblicaltenet‘tohimthathathshallbegiven’.

Inthepreviouschapter,wesawthatanendosymbiosisbetweentwoprokaryotesbroketheendlessloopofsimplicity.Itisnoteasyforonebacteriumtogetinsideanotheroneandtosurvivethereforendlessgenerations,butweknowofafewexamples,soweknowthatitdoeshappen,ifveryrarely.Butacellwithinacellwasjustthebeginning,apregnantmomentinthehistoryoflife,yetnomorethanthat.Itisjustacellwithinacell.Somehowwehavetochartacoursefromtheretothebirthoftruecomplexity–toacellthathasaccumulatedeverythingcommontoalleukaryotes.Westartwithbacteria,lackingalmostallcomplextraits,andendwithcompleteeukaryotes,cellswithanucleus,aplethoraof internalmembranesandcompartments,adynamiccellskeletonandcomplexbehavioursuchassex.Eukaryoticcellsexpandedingenomesizeandinphysicalsizeoverfourorfiveordersofmagnitude. The last common ancestor of eukaryotes had accumulated all these traits; the startingpoint,acellwithinacell,hadnoneofthem.Therearenosurvivingintermediates,nothingmuchtotellushoworwhyanyofthesecomplexeukaryotictraitsevolved.

It’ssometimessaidthattheendosymbiosisthatlaunchedtheeukaryoteswasnotDarwinian:thatitwasnotagradualsuccessionofsmallstepsbutasuddenleapintotheunknown,creatinga‘hopefulmonster ’.Toapointthatistrue.Ihavearguedthatnaturalselection,actingoninfinitepopulationsofprokaryotesoverinfiniteperiodsoftime,willneverproducecomplexeukaryoticcellsexceptbyway

of an endosymbiosis. Such events cannot be represented on a standard bifurcating tree of life.Endosymbiosisisbifurcationbackwards,wherethebranchesdonotbranchbutfusetogether.Butanendosymbiosisisasingularevent,amomentinevolutionthatcan’tproduceanucleusoranyoftheother archetypal eukaryotic traits. What it did do was set in motion a train of events, which areperfectlyDarwinianinthenormalsenseoftheword.

So I am not arguing that the origin of eukaryotes was non-Darwinian but that the selectivelandscape was transformed by a singular endosymbiosis between prokaryotes. After that it wasDarwin all theway.The question is, howdid the acquisition of endosymbionts alter the course ofnatural selection?Did it happen in a predictablemanner,whichmight follow a similar course onother planets, or did the elimination of energetic constraints open the floodgates to unfetteredevolution?Ishallargue thatat leastsomeof theuniversal traitsofeukaryoteswerewrought in theintimate relationship between host cell and endosymbiont, and as such are predictable from firstprinciples.Thesetraitsincludethenucleus,sex,twosexes,andeventheimmortalgermline,begetterofthemortalbody.

Startingoutwithanendosymbiosisimmediatelyplacessomeconstraintsontheorderofevents;thenucleusandmembranesystemsmusthavearisenaftertheendosymbiosis,forexample.Butitalsoplacessomeconstraintsonthespeedatwhichevolutionmusthaveoperated.Darwinianevolutionandgradualismareeasilyconflated,butwhatdoes‘gradual’actuallymean?Itmeanssimplythattherearenogreatleapsintotheunknown,thatalladaptivechangesaresmallanddiscrete.Thatisnottrueifweconsider changes to the genome itself,whichmight take the formof large deletions, duplications,transpositionsorabruptrewiringasaresultofregulatorygenesbeinginappropriatelyswitchedonoroff.Butsuchchangesarenotadaptive;likeendosymbioses,theymerelyalterthestartingpointfromwhichselectionacts.Tosuggestthatthenucleus,forexample,somehowjustpoppedintoexistenceistoconfoundgeneticsaltationwithadaptation.Thenucleusisanexquisitelyadaptedstructure,nomererepositoryforDNA.Itiscomposedofstructuressuchasthenucleolus,wherenewribosomalRNAismanufacturedonacolossalscale; thedoublednuclearmembrane,studdedwithstunninglybeautifulprotein pore complexes (Figure 26), each one containing scores of proteins conserved across alleukaryotes; and the elastic lamina, a flexible proteinmeshwork lining the nuclear membrane thatprotectsDNAagainstshearstress.

Thepointisthatsuchastructureistheproductofnaturalselectionactingoverextendedperiodsoftime,andrequirestherefinementandorchestrationofhundredsofseparateproteins.AllofthisisapurelyDarwinianprocess.Butthatdoesnotmeanithadtohappenslowlyingeologicalterms.Inthefossilrecord,weareusedtoseeinglongperiodsofstasis,punctuatedoccasionallybyperiodsoffastchange.Thischangeisfastingeologicaltime,butnotnecessarilyintermsofgenerations:itissimplynothamperedbythesameconstraintsthatopposechangeundernormalcircumstances.Onlyrarelyisnaturalselectionaforceforchange.Mostcommonly,itopposeschange,purgingvariationsfromthepeaksofanadaptivelandscape.Onlywhenthatlandscapeundergoessomekindofseismicshiftdoesselectionpromotechangerather thanstasis.Andthenitcanoperatestartlinglyswiftly.Theeyeisagood example. Eyes arose in the Cambrian explosion, apparently within the space of a couple ofmillionyears.Whenbluntedtotherhythmofhundredsofmillionsofyearsduringthenear-eternalPrecambrian, 2million years seems indecently hasty.Why stasis for so long, then such rapid-firechange?Perhaps because oxygen levels rose, and then, for the first time, selection favoured largeactive animals, predators andprey,with eyes and shells.1A famousmathematicalmodel calculatedhowlongitmighttakeforaneyetoevolvefromasimplelight-sensitivespotonsomesortofworm.Theanswer,assumingalifecycleofoneyear,andnomorethan1%morphologicalchangeineachgeneration,wasjusthalfamillionyears.

Figure26NuclearporesClassicimagesbythepioneerofelectronmicroscopyDonFawcett.Thedoublemembranesurroundingtheeukaryoticnucleusisclearlyvisible,asaretheregularpores,markedbyarrowsinA.Thedarkerareaswithinthenucleusarerelativelyinactiveregions,wherechromatinis‘condensed’,whereaslighterregionsindicateactivetranscription.Thelighter‘spaces’closetothenuclearporesindicateactivetransportinandoutofthenucleus.Bshowsanarrayofnuclearporecomplexes,eachcomposedofscoresofproteinsassembledtoformthemachineryofimportandexport.Thecoreproteinsintheseporecomplexesareconservedacrossalleukaryotes,hencenuclearporesmusthavebeenpresentinLECA(thelasteukaryoticcommonancestor).

Howlongshould it takeanucleus toevolve?Orsex,orphagocytosis?Whyshould it takeanylonger than the eye? This is a project for the future – to calculate theminimum time to evolve aeukaryotefromaprokaryote.Beforeit’sworthembarkingonsuchaproject,weneedtoknowmoreaboutthesequenceofeventsinvolved.Butthereisnoprimafaciereasontoassumeitshouldtakevasttractsof timemeasured inhundredsofmillionsofyears.Whynot2millionyears?Assumingonecelldivisionperday,that’sclosetoabilliongenerations.Howmanyareneeded?Oncetheenergetic

brakes that blocked the evolution of complexity in prokaryotes were lifted, I see no reason whyeukaryoticcellscouldnothaveevolvedinarelativelyshortperiodoftime.Setagainst3billionyearsof prokaryotic stasis, that masquerades as a sudden leap forward; but the process was strictlyDarwinian.

Justbecauseitisconceivableforevolutiontooperatequicklydoesnotmeanthatitactuallydid.But there are strong grounds to think that the evolution of the eukaryotes probably did happenquickly,basedonnature’sabhorrenceofavacuum.Theproblemispreciselythefactthateukaryotesshare everything, and prokaryotes have none of it. That implies instability. In Chapter 1, weconsideredthearchezoa,thoserelativelysimplesingle-celledeukaryotesthatwereoncemistakenforevolutionaryintermediatesbetweenprokaryotesandeukaryotes.Thisdisparategroupturnedouttobederived from more complex ancestors with a full stock of all eukaryotic traits. But they arenonetheless true ecological intermediates – they occupy the niche of morphological complexitybetweenprokaryotesandeukaryotes.Theyfillthevacuum.Toasuperficialfirstglance,then,thereisno vacuum: there is a continuous spectrum of morphological complexity ranging from parasiticgenetic elements to giant viruses, bacteria to simple eukaryotes, complex cells to multicellularorganisms.Onlyrecently,whenittranspiredthatthearchezoaareasham,didthefullhorrorofthevacuumbecomeevident.

Thefactthatthearchezoawerenotoutcompetedtoextinctionmeansthatsimpleintermediatescanthriveinthisspace.Thereisnoreasonwhythesameecologicalnichecouldnothavebeenoccupiedbygenuineevolutionaryintermediates,cellsthatlackedmitochondria,oranucleus,orperoxisomes,ormembranesystemssuchastheGolgiapparatusorendoplasmicreticulum.Iftheeukaryotesaroseslowly,overtensorhundredsofmillionsofyears,theremusthavebeenmanystableintermediates,cells that lackedvariouseukaryotic traits.Theyshouldhaveoccupied thesame intermediatenichesnowfilledwitharchezoa.Someofthemoughttohavesurviveduntiltoday,asgenuineevolutionaryintermediatesinthevacuum.Butno!Nonearetobefound,despitealong,hardlook.Iftheywerenotoutcompeted to extinction, then why did none of them survive? I would say because they weregeneticallyunstable.Therewerenotmanywaystocrossthevoid,andmostperished.

Thatwouldimplyasmallpopulationsize,whichalsomakessense.Alargepopulationindicatesevolutionary success. If the early eukaryoteswere thriving, they should have spread out, occupiednew ecological spaces, diverged. They should have been genetically stable.At least some of themshouldhave survived.But thatdidn’thappen.At facevalue, then, it seemsmost likely that the firsteukaryotesweregeneticallyunstable,andevolvedquicklyinasmallpopulation.

There’sanotherreasontothinkthatthismustbetrue:thefactthatalleukaryotesshareexactlythesametraits.Thinkabouthowpeculiarthisis!Weallsharethesametraitswithotherhumanbeings,suchasuprightposture,furlessbodies,opposingthumbs,largebrainsandafacilityforlanguage,asweareallrelatedbyancestryandinterbreeding.Sex.Thatisthesimplestdefinitionofaspecies–apopulation of interbreeding individuals.Groups that do not interbreed diverge, and evolve distincttraits– theybecomenewspecies.Yet thisdidn’thappenat theoriginofeukaryotes.Alleukaryotessharethesamesetofbasictraits.Itlooksalotlikeaninterbreedingpopulation.Sex.

Couldanyotherformofreproductionhaveachievedthesameendpoint?Idon’tthinkso.Asexualreproduction – cloning – leads to deep divergence, as differentmutations accumulate in differentpopulations. These mutations are subject to selection in disparate environments, facing differentadvantages and disadvantages.Cloningmay produce identical copies, but ironically this ultimatelydrives divergence between populations as mutations accumulate. In contrast, sex pools traits in apopulation, forevermixing andmatching, opposing divergence.The fact that eukaryotes share thesame traits suggests that theyarose inan interbreeding sexualpopulation.This in turn implies thattheirpopulationwassmallenoughtointerbreed.Anycells thatdidnothavesex, inthispopulation,

didnotsurvive.TheBiblewasright:‘Straitisthegateandnarrowistheway,whichleadethuntolife,andfewtherebethatfindit.’

What about lateral gene transfer, rife as it is in bacteria and archaea? Like sex, lateral genetransfer involves recombination, producing ‘fluid’ chromosomes with shifting combinations ofgenes.Unlikesex,though,lateralgenetransferisnotreciprocal,anddoesnotinvolvecellfusionorrecombinationacrossthefullgenome.Itispiecemealandunidirectional:itdoesnotcombinetraitsinapopulation,but increasesdivergencebetween individuals.JustconsiderE.coli.Asinglecellmaycontainabout4,000genes,butthe‘metagenome’(thetotalnumberofgenesfoundindifferentstrainsofE.coli,asdefinedbyribosomalRNA)ismorelike18,000genes.Theoutcomeoframpantlateralgenetransferisthatdifferentstrainsdifferinuptohalfoftheirgenes–morevariationthaninallthevertebrates put together. In short, neither cloning nor lateral gene transfer, the dominantmodes ofinheritanceinbacteriaandarchaea,canexplaintheenigmaofuniformityineukaryotes.

If I werewriting this a decade ago, the idea that sex arose very early in eukaryotic evolutionwould have had little evidence supporting it; numerous species, including many amoebae andsupposedlydeep-branchingarchezoasuchasGiardia,were taken tobe asexual.Evennow,nobodyhascaughtGiardia inflagrante, in theactofmicrobialsex.Butwhatwelackinnaturalhistory,wemakeupforintechnology.Weknowitsgenomesequence.Itcontainsthegenesneededformeiosis(reductivecelldivisiontoproducegametesforsex)inperfectworkingorder,andthestructureofitsgenomebearswitnesstoregularsexualrecombination.Thesamegoesformoreorlesseveryotherspecies we have looked at. With the exception of secondarily derived asexual eukaryotes, whichusually fall extinct quickly, all known eukaryotes are sexual. We can take it that their commonancestorwastoo.Insum:sexaroseveryearlyineukaryoticevolution,andonlytheevolutionofsexinasmallunstablepopulationcanexplainwhyalleukaryotessharesomanycommontraits.

Thatbringsustothequestionofthischapter.Istheresomethingaboutanendosymbiosisbetweentwoprokaryotesthatmightdrivetheevolutionofsex?Youbet,andmuchelsebesides.

ThesecretinthestructureofourgenesEukaryoteshave ‘genes inpieces’.Fewdiscoveries in twentieth-centurybiologycameas agreatersurprise.Wehadbeenmisledbyearlystudiesonbacterialgenestothinkthatgenesarelikebeadsonastring,alllinedupinasensibleorderonourchromosomes.AsthegeneticistDavidPennyputit:‘Iwouldbequiteproud tohave servedon the committee thatdesigned theE.coli genome.There is,however,nowaythatIwouldadmittoservingonthecommitteethatdesignedthehumangenome.Notevenauniversitycommitteecouldbotchsomethingthatbadly.’

So what went wrong? Eukaryotic genes are a mess. They are composed of relatively shortsequences that code for bits of proteins, broken up by long tracts of non-codingDNA, known asintrons.Thereare typically several intronspergene (which isusuallydefinedasa stretchofDNAencodingasingleprotein).Thesevaryenormouslyinlength,butareoftensubstantiallylongerthantheprotein-codingsequencesthemselves.TheyarealwayscopiedintotheRNAtemplatethatspecifiesthe sequence of amino acids in the protein, but are then spliced out before the RNA reaches theribosomes,thegreatprotein-buildingfactoriesinthecytoplasm.Thisisnoeasytask.Itisachievedbyanotherremarkableproteinnanomachineknownasthespliceosome.We’llreturntothesignificanceof thespliceosomesoon.Fornow, let’s justnote that thewholeprocedure isaweirdly roundaboutwayofgoingabout things.Anyfailure tospliceout these intronsmeans that reamsofnonsensicalRNAcode is fed into the ribosomes,whichgo rightaheadandsynthesise thenonsensicalproteins.TheribosomesareasbeholdentotheirredtapeasaKafkabureaucrat.

Whydoeukaryoteshavegenesinpieces?Thereareafewknownbenefits.Differentproteinscan

be pieced together from the same gene by differential splicing, enabling the recombinatorialvirtuosityoftheimmunesystem,forexample.Differentbitsofproteinarerecombinedinmarvellouswaystoformbillionsofdistinctantibodies,whicharecapableofbindingtopracticallyanybacterialorviralprotein, therebysetting inmotion thekillingmachinesof the immunesystem.But immunesystemsarelateinventionsoflarge,complexanimals.Wasthereanearlieradvantage?Inthe1970s,oneof thedoyensof twentieth-centuryevolutionarybiology,FordDoolittle, suggested that intronsmightdatebacktotheveryoriginsoflifeonearth–anideaknownasthe‘intronsearly’hypothesis.The idea was that early genes, lacking sophisticated modern DNA repair machinery, must haveaccumulatederrorsveryrapidly,makingthemextremelypronetomutationalmeltdown.Givenahighmutation rate, thenumberofmutations that accumulatedependson the lengthofDNA.Only smallgenomescouldpossiblyavoidmeltdown.Intronswereananswer.Howtoencodealargenumberofproteinswith a short stretchofDNA? Just recombine small bits andpieces. It’s a beautiful notion,whichstillretainsafewadherents,ifnotDoolittlehimself.Thehypothesis,likeallgoodhypotheses,makesanumberofpredictions;unfortunately,theseturnoutnottobetrue.

The major prediction is that eukaryotes must have evolved first. Only eukaryotes have trueintrons.Ifintronsweretheancestralstate,theneukaryotesmusthavebeentheearliestcells,precedingthe bacteria and archaea,whichmust have lost their introns later on by selection for streamliningtheir genomes. That makes no phylogenetic sense. The modern era of whole-genome sequencingshowsincontrovertiblythateukaryotesarosefromanarchaealhostcellandabacterialendosymbiont.Thedeepestbranchinthetreeoflifeisbetweenarchaeaandbacteria;eukaryotesarosemorerecently,aviewthatisalsoconsistentwiththefossilrecordandtheenergeticconsiderationsofthelastchapter.

Butifintronsarenotanancestralstate,wheredidtheycomefrom,andwhy?Theanswerseemstobetheendosymbiont.Isaidthat‘trueintrons’arenotfoundinbacteria,but theirprecursorsalmostcertainlyarebacterial,orrather,bacterialgeneticparasites,technicallytermed‘mobilegroupIIself-splicing introns’. Don’t worry about words.Mobile introns are just bits of selfishDNA, jumpinggenes which copy themselves in and out of the genome. But I shouldn’t say ‘just’. They areremarkable and purposefulmachines. They are read off intoRNA in the normal fashion, but thenspring to life (whatotherword is there?), forming themselves intopairsofRNA‘scissors’.ThesespliceouttheparasitesfromthelongerRNAtranscripts,minimisingdamagetothehostcell,toformactivecomplexes thatencodea reverse transcriptase–anenzymecapableofconvertingRNAbackintoDNA.These insert copies of the intron back into the genome. So introns are parasitic genes,whichsplicethemselvesinandoutofbacterialgenomes.

‘Big fleas have little fleas upon their backs to bite ’em…’Who would have thought that thegenomeisasnakepit,seethingwithingeniousparasitesthatcomeandgoattheirpleasure.Butthat’swhatitis.Thesemobileintronsareprobablyancient.Theyarefoundinallthreedomainsoflife,andunlikeviruses theyneverneedto leave thesafetyof theirhostcell.Theyarecopiedfaithfullyeachtimethehostcelldivides.Lifehasjustlearnttolivewiththem.

Andbacteriaarequitecapableofdealingwiththem.Wedon’tquiteknowhow.Itmightsimplybethe strengthof selectionactingon largepopulations.Bacteriawithbadlypositioned introns,whichinterferewith theirgenes insomeway, simply loseout in theselectivebattlewithcells thatdonothave badly positioned introns. Or perhaps the introns themselves are accommodating, and invadeperipheralregionsofDNAthatdon’tupsettheirhostcellsmuch.Unlikeviruses,whichcansurviveontheirown,andsodon’tcaremuchaboutkillingtheirhostcells,mobileintronsperishwiththeirhosts,sogainnothingfromobstructingthem.Thelanguagethatlendsitselfbesttoanalysingthiskindofbiologyisthatofeconomics:themathematicsofcostsandbenefits,theprisoner ’sdilemma,gametheory.Bethatasitmay,thefactisthatmobileintronsdonotrunrifeinbacteriaorarchaea,andarenot found within the genes themselves – they are therefore not technically introns at all – but

accumulate at low density in intergenic regions. A typical bacterial genome is unlikely to containmore thanabout30mobile introns (in4,000genes)comparedwith tensof thousandsof introns ineukaryotes. The low number of introns in bacteria reflects the long-term balance of costs andbenefits,theoutcomeofselectionactingonbothpartiesovermanygenerations.

Thisisthekindofbacteriumthatenteredintoanendosymbiosiswithanarchaealhostcell1.5to2billionyearsago.Theclosestmodernequivalentisanα-proteobacteriumofsomesort,andweknowthatmodernα-proteobacteriacontainlownumbersofmobileintrons.Butwhatconnectstheseancientgeneticparasiteswiththestructureofeukaryoticgenes?Littlemorethanthedetailedmechanismofthe RNA scissors that splice out mobile bacterial introns, and simple logic. I mentioned thespliceosomesafewparagraphsago:thesearetheproteinnanomachinesthatcutouttheintronsfromourownRNAtranscripts.Thespliceosomeisnotonlymadeofproteins:atitsheartisapairofRNAscissors, the very same. These splice out eukaryotic introns by way of a telltale mechanism thatbetraystheirancestryasbacterialself-splicingintrons(Figure27).

That’sit.Thereisnothingaboutthegeneticsequenceoftheintronsthemselvestosuggestthattheyderive frombacteria.Theydonotencodeproteinssuchas reverse transcriptase, theydonotsplicethemselvesinandoutofDNA,theyarenotmobilegeneticparasites,theyaremerelylumpentractsofDNAthatsitthereanddonothing.2Butthesedeadintrons,decayedbymutationsthatpuncturedthembelowthewaterline,arenowcorruptedbeyondallrecognition,andfarmoredangerousthanlivingparasites.Theycannolongercutthemselvesout.Theymustberemovedbythehostcell.Andsotheyare, using scissors that were once requisitioned from their living cousins. The spliceosome is aeukaryoticmachinebasedonabacterialparasite.

Figure27Mobileself-splicingintronsandthespliceosomeEukaryoticgenesarecomposedofexons(sequencesthatencodeproteins)andintrons–long,non-codingsequencesinsertedintogenes,whicharesplicedoutfromtheRNAcode-scriptbeforetheproteinissynthesised.IntronsseemtobederivedfromparasiticDNAelementsfoundinbacterialgenomes(leftpanel),butdecayedbymutationstoinertsequencesineukaryoticgenomes.Thesemustbeactivelyremovedbythespliceosome(rightpanel).Therationaleforthisargumentisthemechanismofsplicingshownhere.Thebacterialparasite(leftpanel)splicesitselfouttoformanexcisedintronsequenceencodingareversetranscriptasethatcanconvertcopiesoftheparasiticgenesintoDNAsequences,andinsertmultiplecopiesintothebacterialgenome.Theeukaryoticspliceosome(rightpanel)isalargeproteincomplex,butitsfunctiondependsonacatalyticRNA(ribozyme)atitsheart,whichsharesexactlythesamemechanismofsplicing.Thissuggeststhatthespliceosome,andbyextensioneukaryoticintrons,derivedfrommobilegroupIIself-splicingintronsreleasedfromthebacterialendosymbiontearlyineukaryoticevolution.

Here is the hypothesis, laid out in an exciting 2006 paper by the Russian-born Americanbioinformatician, Eugene Koonin, and Bill Martin. At the origin of eukaryotes, they said, theendosymbiont unleashed a barrage of genetic parasites upon the unwitting host cell. Theseproliferatedacrossthegenomeinanearlyintroninvasion,whichsculptedeukaryoticgenomesanddrove theevolutionofdeep traits suchas thenucleus. Iwouldaddsex. I admit all this sounds likemake-believe,anevolutionary‘just-so’storybasedontheflimsyevidenceofanincriminatingpairofscissors.Buttheideaissupportedbythedetailedstructureofthegenesthemselves.Thesheernumberof introns – tens of thousands of them – combined with their physical position within eukaryoticgenesbearmutewitnesstotheirancientheritage.Thatheritagegoesbeyondtheintronsthemselves,and speaks to the tortured and intimate relationship between host and endosymbiont. Even if theseideasarenotthewholetruth,Ithinktheyarethekindofanswersweseek.

IntronsandtheoriginofthenucleusThepositionsofmanyintronsareconservedacrosseukaryotes.Thisisanotherunexpectedcuriosity.Takeageneencodingaproteinthatisinvolvedinbasiccellmetabolismfoundinalleukaryotes,forexamplecitratesynthase.We’llfindthesamegeneinourselves,aswellasinseaweeds,mushrooms,treesandamoebae.Despitediverging somewhat in sequenceover the incomprehensiblenumberofgenerations that separate us from our common ancestor with trees, natural selection has acted toconserve its function, and thus its specific gene sequence.This is a beautiful illustration of sharedancestry,andthemolecularbasisofnaturalselection.Whatnobodyexpectedisthatsuchgenesshouldtypicallycontain twoor three introns, frequently inserted inexactly thesamepositions in treesandhumans.Whyshould thatbe?Thereareonly twoplausibleexplanations.Either the introns insertedthemselvesinthesameplacesindependently,becausethoseparticularsiteswerefavouredbyselectionforsomereason,ortheyinsertedthemselvesonceintothecommonancestorofeukaryotesandwerethen passed down to their descendants. Some of these descendants may have lost them again, ofcourse.

If therewereonlyahandfulofcasesknown,wemightfavourtheformerinterpretation,but thefactthatthousandsofintronsareinsertedintoexactlythesamepositionsinhundredsofsharedgenesacrossalleukaryotesmakesthisseemimplausible.Sharedancestry ismuchthemostparsimoniousexplanation.Ifso,thentheremusthavebeenanearlywaveofintroninvasion,soonaftertheoriginofthe eukaryotic cell,whichwas responsible for implanting all these introns in the first place. Thenafterthattheremusthavebeensomekindofmutationalcorruptionofintrons,whichrobbedthemoftheirmobility, preserving their positions in all later eukaryotes like the indelible chalkoutlines ofcorpses.

There is also another more compelling reason to favour an early intron invasion. We candistinguishbetweendifferenttypesofgene,knownasorthologsandparalogs.Orthologsarebasically

thesamegenesdoingthesamejobindifferentspecies,inheritedfromacommonancestor,asintheexamplewe’vejustconsidered.Soalleukaryoteshaveanorthologofthegeneforcitratesynthase,whichweallinheritedfromourcommonancestor.Thesecondgroupofgenes,paralogs,alsoshareacommonancestor, but in this case the ancestralgenewasduplicatedwithin the samecell, often onmultipleoccasions,togiveagenefamily.Suchfamiliescancontainasmanyas20or30genes,eachofwhich usually ends up specialising to a slightly different task. An example is the haemoglobinfamily of about 10 genes, all ofwhich encode very similar proteins,with each serving a slightlydifferentpurpose.Inessence,orthologsareequivalentgenesindifferentspecies,whileparalogsaremembersofagenefamilyinthesameorganism.Butofcourseentirefamiliesofparalogscanalsobe found in different species, inherited from their common ancestor. So all mammals haveparalogoushaemoglobingenefamilies.

Wecanbreakdownthesefamiliesofparalogousgenesintoeitherancientorrecentparalogs.Inaningeniousstudy,EugeneKoonindidexactlythis.Hedefinedancientparalogsasgenefamiliesthatarefoundinalleukaryotes,butwhicharenotduplicatedinanyprokaryotes.Wecanthereforeplacethe round of gene duplications that gave rise to the gene family as an early event in eukaryoticevolution,beforetheevolutionofthelasteukaryoticcommonancestor.Recentparalogs,incontrast,aregenefamiliesfoundonlyincertaineukaryoticgroups,suchasanimalsorplants.Inthiscase,wecan conclude that the duplications occurredmore recently, during the evolution of that particulargroup.

Kooninpredicted that if therewas indeedan intron invasionduringearlyeukaryoticevolution,thenmobileintronsshouldhaverandomlyinsertedthemselvesintodifferentgenes.That’sbecausetheancientparalogswerebeingactivelyduplicatedduringthissameperiod.Iftheearlyintroninvasionhad not yet abated, then mobile introns would still be inserting themselves into new positions indifferentmembersof thegrowingparalogousgenefamily.Incontrast,morerecentduplicationsofparalogsoccurredwellaftertheendofthepostulatedearlyintroninvasion.Withnonewinsertions,theold intronpositions shouldbeconserved innewcopiesof thesegenes. Inotherwords, ancientparalogsshouldhavepoorconservationofintronpositionrelativetorecentparalogs.Thisistruetoaremarkabledegree.Practicallyallintronpositionsareconservedinrecentparalogs,whereasthereisverypoorintronconservationinancientparalogs,exactlyaspredicted.

Allthissuggeststhatearlyeukaryotesreallydidsufferaninvasionofmobileintronsfromtheirownendosymbionts.Butifso,whydidtheseproliferateinearlyeukaryoteswhentheyarenormallykept under tight control in both bacteria and archaea? There are two possible answers, and thechancesarethatbotharetrue.Thefirstreasonisthatearlyeukaryotes–basicallystillprokaryotes,archaea – suffered a bombardment of bacterial introns from uncomfortably close quarters, frominside their own cytoplasm. There is a ratchet operating here. An endosymbiosis is a natural‘experiment’whichmightfail.Ifthehostcelldies,theexperimentisover.Butthat’snottruetheotherway around. If there is more than one endosymbiont, and just one of them dies, the experimentcontinues–thehostcellsurvives,withallitsotherendosymbionts.Butthedeadendosymbiont’sDNAspills intothecytosol,whenceit is likelytoberecombinedintothehostcell’sgenomebystandardlateralgenetransfer.

This is not easy to stop, and continues to this day – our nuclear genomes are riddled withthousandsofbitsandpiecesofmitochondrialDNA,called‘numts’(nuclearmitochondrialsequences,sinceyouask),whicharrivedtherebyexactlysuchtransfers.Newnumtscropupoccasionally,callingattention to themselveswhen they disrupt a gene, causing a genetic disease. Back at the origin ofeukaryotes, before there was a nucleus at all, such transfers must have been more common. Thechaotic transfer of DNA from mitochondria to host cell would have been worse if selectivemechanismsdo exist that directmobile introns toparticular siteswithin agenome,while avoiding

others.Ingeneral,bacterialintronsareadaptedtotheirbacterialhosts,andarchaealintronstotheirarchaealhosts.Intheearlyeukaryotes,however,bacterialintronswereinvadinganarchaealgenome,withverydifferentgenesequences.Therewerenoadaptiveconstraints;andwithoutthem,whatcouldhave stopped introns from proliferating uncontrollably?Nothing! Extinction loomed. The bestwecouldhopeforisasmallpopulationofgeneticallyunstable–sickly–cells.

Thesecondreasonforanearlyintronproliferationisthelowstrengthofselectionactingagainstit.Inpart,thisispreciselybecauseasmallpopulationofsicklycellsislesscompetitivethanaheavingpopulationofhealthycells.Butthefirsteukaryotesshouldalsohavehadanunprecedentedtoleranceforintroninvasion.Afterall,theirsourcewastheendosymbiont,thefuturemitochondria,whichareanenergeticboonaswellasageneticcost.Intronsareacosttobacteriabecausetheyareanenergeticandgeneticburden;smallcellswithlessDNAreplicatefaster thanlargecellswithmoreDNAthantheyneed.Aswesawinthelastchapter,bacteriastreamlinetheirgenomestoaminimumcompatiblewith survival. In contrast, eukaryotes exhibit extremegenomic asymmetry: they are free to expandtheir nuclear genomes precisely because their endosymbiont genomes shrink. Nothing is plannedabouttheexpansionofthehostcellgenome;itissimplythatincreasedgenomesizeisnotpenalisedbyselectioninthesamewayasitisinbacteria.Thislimitedpenaltyenableseukaryotestoaccumulatethousandsmoregenes,throughallkindsofduplicationsandrecombinations,butalsototolerateafarheavier load of genetic parasites. The two must inevitably go hand in hand. Eukaryotic genomesbecameoverrunwithintronsbecause,fromanenergeticpointofview,theycouldbe.

Soitseemslikelythatthefirsteukaryotessufferedabombardmentofgeneticparasitesfromtheirown endosymbionts. Ironically, these parasites didn’t posemuch of a problem: the problem reallybeganwhen the parasites decayed anddied, leaving their corpses – introns – littering the genome.Now thehost cellhad tophysicallycut the intronsout,or theywouldbe readoff intononsensicalproteins.Aswe’venoted, this isdonebythespliceosome,whichderivesfromtheRNAscissorsofmobile introns.But the spliceosome, impressivenanomachine though itmightbe, is only apartialsolution. The trouble is that spliceosomes are slow. Even today, after nearly 2 billion years ofevolutionaryrefinement, theytakeseveralminutestocutoutasingleintron.Incontrast,ribosomesworkat a furiouspace–up to10aminoacidsper second. It takesbarelyhalf aminute tomakeastandardbacterialprotein,about250aminoacidsinlength.EvenifthespliceosomecouldgainaccesstoRNA (which is not easy asRNA is often encrusted inmultiple ribosomes) it could not stop theformationofalargenumberofuselessproteins,withtheirintronsincorporatedintact.

Howcouldanerrorcatastrophebeaverted?Simplybyinsertingabarrierintheway,accordingtoMartin andKoonin.Thenuclearmembrane is abarrier separating transcription from translation–inside the nucleus, genes are transcribed intoRNA codescripts; outside the nucleus, theRNAs aretranslated intoproteinson theribosomes.Crucially, theslowprocessofsplicing takesplace insidethe nucleus, before the ribosomes can get anywhere near theRNA.That is thewhole point of thenucleus:tokeepribosomesatbay.Thisexplainswhyeukaryotesneedanucleusbutprokaryotesdon’t–prokaryotesdon’thaveanintronproblem.

Buthangonaminute,Ihearyoucry!Wecan’tjustpulloutaperfectlyformednuclearmembranefromnowhere!Itmusthavetakenmanygenerationstoevolve,sowhydidn’ttheearlyeukaryotesdieout inthemeantime?Well,nodoubtmanydid,but theproblemmightnotbeasdifficultasall that.Thekeyliesinanothercuriosityrelatingtomembranes.Eventhoughitisclearfromthegenesthatthe host cell was a bona fide archaeon, which must have had characteristic archaeal lipids in itsmembranes,eukaryoteshavebacterial lipids in theirmembranes.That’sa fact toconjurewith.Forsomereason,thearchaealmembranesmusthavebeenreplacedwithbacterialmembranesearlyonineukaryoticevolution.Why?

Therearetwofacetstothisquestion.Thefirstisamatterofpracticality:coulditactuallybedone?

Theanswerisyes.Rathersurprisingly,mosaicmembranes,composedofvariousdifferentmixturesofarchaealandbacteriallipids,areinfactstable;weknowthisfromlabexperiments.Itisthereforepossibletotransitgraduallyfromanarchaealtoabacterialmembrane.Sothere’snoreasonwhyitshouldn’t happen, but the fact is that such transitions are rare indeed.That brings us to the secondfacet–whatrareevolutionaryforcemightdrivesuchachange?Theansweristheendosymbiont.

Thechaotic transferofDNAfromendosymbionts to thehostcellmusthave included thegenesforbacteriallipidsynthesis.Wecantakeitthattheenzymesencodedweresynthesisedandactive:theywentrightaheadandmadebacterial lipids,but initiallythissynthesiswaslikelytobeuncontrolled.Whathappenswhenlipidsaresynthesisedinarandomfashion?Whenformedinwater,theysimplyprecipitateaslipidvesicles.JeffErringtoninNewcastlehasshownthatrealcellsbehaveinthesameway:mutationsthatincreaselipidsynthesisinbacteriaresultinprecipitationofinternalmembranes.Thesetendtoprecipitateclosetowheretheyareformed,surroundingthegenomewithpilesoflipid‘bags’.Justasatrampmightinsulatehimselffromthecoldwithplasticbags,ifinadequately,sopilesoflipidbagswouldeasetheintronproblembyprovidinganimperfectbarrierbetweentheDNAandribosomes.Thatbarrierneededtobeimperfect.AsealedmembranewouldpreventexportofRNAouttotheribosomes.Abrokenbarrierwouldmerelyslowitdown,givingthespliceosomesalittlemoretime to cutout the intronsbefore the ribosomescouldget towork. Inotherwords, a random (butpredictable)startingpointgaveselection thebeginningsofasolution.Thebeginningwasapileoflipid bags surrounding a genome; the end point was the nuclear membrane, replete with itssophisticatedpores.

Themorphology of the nuclearmembrane is consistentwith this view.Lipid bags, like plasticbags, can be flattened. In cross section, a flattened bag has two closely aligned parallel sides – adouble membrane. That is precisely the structure of the nuclear membrane: a series of flattenedvesicles, fused together, with the nuclear pore complexes nestling in the interstices. During celldivisionthemembranedisintegratesbackintoseparatesmallvesicles;afterwards,theygrowandfuseagaintoreconstitutethenuclearmembranesofthetwodaughtercells.

Thepatternofgenesencodingnuclearstructuresalsomakessenseinthislight.Ifthenucleushadevolvedbefore theacquisitionofmitochondria, then the structureof itsvariousparts– thenuclearpores,nuclearlaminaandnucleolus–shouldbeencodedbyhost-cellgenes.That’snotthecase.Allofthemarecomposedofachimericmixtureofproteins,someencodedbybacterialgenes,afewbyarchaealgenes,andtherestbygenesfoundonlyineukaryotes.It’spracticallyimpossibletoexplainthispatternunless thenucleusevolvedafter theacquisitionofmitochondria, in thewakeofunrulygene transfers. It’s often said that in the evolution of the eukaryotic cell, the endosymbionts weretransformed,almost(butnotquite)beyondrecognition, intomitochondria. It’s lessappreciated thatthe host cell underwent an evenmore dramaticmakeover. It started out as a simple archaeon, andacquired endosymbionts. These bombarded their unwitting hostwithDNA and introns, driving theevolutionofthenucleus.Andnotjustthenucleus:sexwenthandinhand.

TheoriginofsexWe’venotedthatsexaroseveryearlyineukaryoticevolution.Iimplied,too,thattheoriginsofsexmighthavehadsomethingtodowithintronbombardment.Howso?Let’sfirstquicklyrecapwhatwearetryingtoexplain.

Truesex,aspractisedbyeukaryotes, involvesthefusionof twogametes(forus, thespermandegg),eachgametehavinghalfthenormalquotaofchromosomes.YouandIarediploid,alongwithmostothermulticellulareukaryotes.Thatmeanswehavetwocopiesofeachofourgenes,onefromeach parent. More specifically, we have two copies of each chromosome, known as sister

chromosomes. Iconic images of chromosomes might make them look like unchanging physicalstructures, but that’s far from the case. During the formation of gametes, the chromosomes arerecombined, fusingbitsofonewithpiecesofanother,givingnewcombinationsofgenes thathavemost likely never been seen before (Figure 28). Work your way down a newly recombinedchromosome,genebygene,andyou’llfindsomegenesfromyourmother,somefromyourfather.Thechromosomesarenowseparatedintheprocessofmeiosis(literally,‘reductivecelldivision’)toform haploid gametes, each with a single copy of every chromosome. Two gametes, each withrecombined chromosomes, finally fuse to form the fertilised egg, a new individualwith a uniquecombinationofgenes–yourchild.

The problem with the origin of sex is not that a lot of new machinery had to evolve.Recombination works by lining up the two sister chromosomes side by side. Sections of onechromosome are then physically transferred on to its sister, and vice versa, byway of cross-overpoints.Thisphysicalliningupofchromosomesandrecombinationofgenesalsooccursinbacteriaandarchaeaduring lateralgene transfer,but isnotusually reciprocal: it isused to repairdamagedchromosomes or to reload genes that had been deleted from the chromosome. The molecularmachinery is basically the same; what differs in sex is the scope and the reciprocality. Sex is areciprocal recombination across the entire genome.That entails the fusion ofwhole cells, and thephysicaltransferofentiregenomes,whichisrarelyifeverseeninprokaryotes.

Figure28SexandrecombinationineukaryotesAsimplifieddepictionofthesexualcycle:thefusionoftwogametesfollowedbyatwo-stepmeiosiswithrecombinationtogeneratenew,geneticallydistinctgametes.InAtwogameteswithasinglecopyofanequivalent(butgeneticallydistinct)chromosomefusetogethertoformazygotewithtwocopiesofthechromosomeB.Noticetheblackbars,whichcouldsignifyeitheraharmfulmutation,orabeneficialvariantofspecificgenes.InthefirststepofmeiosisCthechromosomesarealignedandthenduplicated,togivefourequivalentcopies.TwoormoreofthesechromosomesarethenrecombinedD.SectionsofDNAarereciprocallycrossedoverfromonechromosometoanother,tofashionnewchromosomescontainingbitsoftheoriginalpaternalandbitsoftheoriginalmaternalchromosomeE.TworoundsofreductivecelldivisionseparatethesechromosomestogiveFandfinallyanewselectiongametesG.Noticethattwoofthesegametesareidenticaltotheoriginalgametes,buttwonowdiffer.Iftheblackbarsignifiesaharmfulmutation,sexhasheregeneratedonegametewithnomutations,andonegametewithtwo;thelattercanbeeliminatedbyselection.Conversely,iftheblackbarsignifiesabeneficialvariant,thensexhasunitedbothoftheminasinglegamete,allowingselectiontofavourbothsimultaneously.Inshort,sexincreasesthevariance(thedifference)betweengametes,makingthemmorevisibletoselection,soeliminatingmutationsandfavouringbeneficialvariantsovertime.

Sexwasconsideredthe‘queen’ofbiologicalproblemsinthetwentiethcentury,butwenowhaveagood appreciationofwhy it helps, at least in relation to strict asexual reproduction (cloning). Sex

breaksuprigidcombinationsofgenes,allowingnaturalselectionto‘see’individualgenes,toparseallourqualitiesonebyone.Thathelps in fendingoffdebilitatingparasites, aswell as adapting tochanging environments, and maintaining necessary variation in a population. Just as medievalstonemasonsoncecarvedthebackofsculpturesthatarehiddenintherecessesofcathedrals,becausetheywere still visible toGod, so sex allows the all-seeing eye of natural selection to inspect herworks, gene by gene. Sex gives us ‘fluid’ chromosomes, ever-changing combinations of genes(technically alleles3), which allows natural selection to discriminate between organisms withunprecedentedfinesse.

Imagine 100 genes lined up on a chromosome that never recombines. Selection can only everdiscriminatethefitnessofthewholechromosome.Let’ssaythereareafewreallycriticalgenesonthischromosome–anymutationsinthemwouldalmostalwaysresult indeath.Critically,however,mutationsonlesscriticalgenesbecomenearlyinvisibletoselection.Slightlyharmfulmutationscanaccumulate in thesegenes,as theirnegativeeffectsareoffsetby thebigbenefitsof thefewcriticalgenes.Asaresult,thefitnessofthechromosome,andtheindividual,isgraduallyundermined.Thisisroughlywhat happens to theY chromosome inmen – the lack of recombinationmeans thatmostgenesareinastateofslowdegeneration;onlythecriticalgenescanbepreservedbyselection.Intheend,theentirechromosomecanbelost,asindeedhashappenedinthemolevoleEllobiuslutescens.

Butit’sevenworseifselectionactspositively.Considerwhathappensifararepositivemutationinacriticalgeneissobeneficialthatitsweepsthroughthepopulation.Organismsthatinheritthenewmutationdominate,andthegeneultimatelyspreadsto‘fixation’:allorganismsinthepopulationendupwithacopyofthegene.Butnaturalselectioncanonly‘see’thewholechromosome.Thismeansthattheother99genesonthechromosomealsobecomefixedinthepopulation–theygoalongforthe ride and are said to ‘hitch-hike’ to fixation. This is a disaster. Imagine there are two or threeversions(alleles)ofeachgeneinthepopulation.Thatgivesbetween10,000and1milliondifferentpossible combinations of alleles. After fixation, all this variation is wiped out, leaving the entirepopulationwithasinglecombinationofthe100genes–thosethathappenedtosharethechromosomewiththerecentlyfixedgene–acatastrophiclossofvariation.And,ofcourse,amere100genesisagrossoversimplification:asexualorganismshavemanythousandsofgenes,allofwhicharepurgedofvariationinasingleselectivesweep.The‘effective’populationsizeishugelydiminished,makingasexual populations far more vulnerable to extinction.4 That’s exactly what does happen to mostasexuals–almostallclonalplantsandanimalsfallextinctwithinafewmillionyears.

These two processes – accumulation of mildly damaging mutations, and loss of variation inselectivesweeps–aretogetherknownasselectiveinterference.Withoutrecombination,selectiononcertain genes interferes with selection on others. By generating chromosomes with differentcombinationsofalleles–‘fluidchromosomes’–sexallowsselectiontoactonallgenesindividually.Selection,likeGod,cannowseeallourvicesandvirtues,genebygene.That’sthegreatadvantageofsex.

But there are also serious disadvantages to sex, hence its long standing as the queen ofevolutionaryproblems.Sexbreaksupcombinationsofallelesprovedtobesuccessfulinaparticularenvironment,randomisingtheverygenesthathelpedourparentstothrive.Thegenepackisshuffledagain every generation, with never a chance to clone an exact copy of a genius, anotherMozart.Worse,thereisthe‘twofoldcostofsex’.Whenaclonalcelldivides,itproducestwodaughtercells,eachofwhichgoesontoproduceanothertwodaughters,andsoon.Thegrowthofapopulationisexponential. Ifasexualcellproduces twodaughtercells, thesemustfusewitheachother toformanewindividual thatcanproduce twomoredaughtercells.Soanasexualpopulationdoubles insizeeachgeneration,whereasasexualpopulationremainsthesamesize.Andcomparedwithjustcloninga nice copyof yourself, sex introduces the problemof finding amate,with all its emotional (and

financial) costs. And there’s the cost of males. Clone yourself and there’s no need for all thoseaggressive,prancingmales,lockinghorns,fanningtailsordominatingboardrooms.Andwe’dberidof horrible sexually transmitted diseases like AIDS or syphilis, and the opportunity for geneticfreeloaders–virusesand‘jumpinggenes’–toriddleourgenomeswithjunk.

Thepuzzleisthatsexisubiquitousamongeukaryotes.Onemightthinkthattheadvantageswouldoffset thecostsundercertaincircumstancesbutnotothers.Toapoint this is true, in thatmicrobesmaydivideasexuallyfor30generationsorso,beforeindulginginoccasionalsex,typicallywheninastateofstress.Butsexisfarmorewidespreadthanseemsreasonable.Thisisprobablybecausethelastcommonancestorofeukaryoteswasalreadysexual,andhenceallherdescendantsweresexualtoo. While many microorganisms no longer have regular sex, very few ever lost sex altogetherwithoutfallingextinct.Thecostsofneverhavingsexarethereforehigh.Asimilarargumentshouldapplytotheearliesteukaryotes.Thosethatneverhadsex–arguablyallthosethathadnot‘invented’sex–werelikelytofallextinct.

Butherewerunagainintotheproblemoflateralgenetransfer,whichissimilartosexinthatitrecombines genes, producing ‘fluid chromosomes’. Until recently, bacteria were perceived as thegrandmastersofcloning.Theygrowat exponential rates. If totallyunconstrained, a singleE. colibacterium,doublingevery30minutes,wouldproduceacolonywith themassof theearth in threedaysflat.Asithappens,though,E.colicandomuchmorethanthat.Theycanalsoswaptheirgenesaround,incorporatingnewgenesontotheirchromosomesbylateralgenetransferwhilelosingotherunwantedgenes.Thebacteria thatgiveyougastric flumaydiffer in30%of theirgenescomparedwith the same ‘species’ up your nose. So bacteria enjoy the benefits of sex (fluid chromosomes)alongwith the speed and simplicity of cloning.But they don’t fusewhole cells together, and theydon’thavetwosexes,andsotheyavoidmanyofthedisadvantagesofsex.Theywouldseemtohavethebestofbothworlds.Sowhydidsexarisefromlateralgenetransferintheearliesteukaryotes?

Work from the mathematical population geneticists Sally Otto and Nick Barton points to anunholytrinityoffactors thatconspicuouslyrelates tocircumstancesat theoriginofeukaryotes: thebenefitsofsexaregreatestwhenthemutationrateishigh,selectionpressureisstrong,andthereisalotofvariationinapopulation.

Takethemutationratefirst.Withasexualreproduction,ahighmutationrateincreasestherateofaccumulationofmildlydamagingmutations,andalsothelossofvariationfromselectivesweeps:itincreases the severityof selective interference.Givenanearly intron invasion, the first eukaryotesmusthavehadahighmutationrate.Howhighexactlyishardtoconstrain,butitmightbepossibletodosobymodelling.I’mworkingonthisquestionwithAndrewPomiankowskiandJezOwen,aPhDstudentwithabackgroundinphysicsandaninterestinthesebigquestionsinbiology.Jezisrightnowdevelopingacomputationalmodeltofigureoutwheresexscoresoverlateralgenetransfer.There’sasecondfactortoconsiderheretoo–genomesize.Evenif themutationrateremainsthesame(let’ssay one lethal mutation in every 10 billion DNA letters), it is not possible to expand a genomeindefinitelywithoutsomesortofamutationalmeltdown.Inthiscase,cellswithagenomeoflessthan10 billion letterswould be fine, but cellswith a genomemuch larger than thatwould die, as theywouldallsufferalethalmutation.Theacquisitionofmitochondriaattheoriginofeukaryotesmusthaveexacerbatedbothproblems–theyalmostcertainlyincreasedthemutationrate,andtheyenabledamassiveexpansionofgenomesize,overseveralordersofmagnitude.

Itmightwellbethatsexwastheonlysolutiontothisproblem.Whilelateralgenetransfercouldinprincipleavoidselectiveinterferencethroughrecombination,Jez’sworksuggeststhatthiscanonlygosofar.Thelargerthegenome,theharderitbecomestopickupthe‘correct’genebylateralgenetransfer;that’sreallyjustanumbersgame.Theonlywaytoensurethatagenomehasallthegenesitneeds, in fullworking order, is to retain all of them, and to recombine them regularly across the

entiregenome.Thatcan’tbeachievedby lateralgene transfer– itneeds sex, ‘total sex’, involvingrecombinationacrossthewholegenome.

Whataboutthestrengthofselection?Again,intronsmaybeimportant.Inmodernorganisms,theclassic selection pressures favouring sex are parasitic infections and variable environments. Eventhen, selection has to be strong for sex to be better than cloning – for example, parasitesmust becommonanddebilitatingtofavoursex.Nodoubtthesesamefactorsappliedtoearlyeukaryotestoo,but theyalsohad tocontendwithadebilitatingearly intron invasion–parasiticgenes.Whywouldmobileintronsdrivetheevolutionofsex?Becausegenome-widerecombinationincreasesvariance,formingsomecellswith introns indamagingplaces,andothercellswith introns in lesshazardousplaces.Selectionthenactstoweedouttheworstcells.Lateralgenetransferispiecemeal,andcannotproduce systematic variation, in which some cells have their genes cleaned up, while othersaccumulatemore than their shareofmutations. InhisbrilliantbookMendel’sDemon,MarkRidleycompared sex with the New Testament view of sin – just as Christ died for the collected sins ofhumanity, so too sex can bring together the accumulated mutations of a population into a singlescapegoat,andthencrucifyit.

The amount of variation between cells could also relate to introns. Both archaea and bacteriausuallyhaveasinglecircularchromosome,whereaseukaryoteshavemultiplestraightchromosomes.Why?A simple answer is that introns can cause errors as they splice themselves in andout of thegenome.Iftheyfailtorejointhetwoendsofachromosomeaftercuttingthemselvesout,thatleavesabreak in thechromosome.Asinglebreak inacircularchromosomegivesa straightchromosome;several breaks give several straight chromosomes.So recombinatorial errors producedbymobileintronscouldhaveproducedmultiplestraightchromosomesintheearlyeukaryotes.

That must have given early eukaryotes terrible problems with their cell cycle. Different cellswould have had different numbers of chromosomes, each accumulating different mutations ordeletions. They would also have been picking up new genes and DNA from their mitochondria.Copyingerrorswouldnodoubtduplicate chromosomes. It’shard to seewhat lateralgene transfercould contribute in this context. But standard bacterial recombination – lining up chromosomes,loadingupmissinggenes–wouldensurethatcells tendedtoaccumulategenesandtraits.Onlysexcould accumulate genes thatworked, andbe rid of those that didn’t.This tendency to pickupnewgenes and DNA by sex and recombination easily accounts for the swelling of early eukaryoticgenomes. Accumulating genes in this way must have solved some of the problems of geneticinstability,while theenergeticadvantagesofhavingmitochondriameant that,unlikebacteria, therewasnoenergeticpenalty.Allthisisspeculative,tobesure,butthepossibilitiescanbeconstrainedbymathematicalmodelling.

How did cells physically segregate their chromosomes? The answermay lie in themachineryusedbybacteria to separate largeplasmids–mobile ‘cassettes’ofgenes that encode traits suchasantibiotic resistance.Largeplasmids are typically segregated inbacterial divisionon a scaffoldofmicrotubulesthatresemblesthespindleusedbyeukaryotes.It’splausiblethattheplasmidsegregationmachinerywasrequisitionedbyearlyeukaryotestoseparatetheirvariedchromosomes.Itisnotonlyplasmidsthataresegregatedinthisway–somebacterialspeciesseemtoseparatetheirchromosomesonrelativelydynamicspindles,ratherthanusingthecellmembraneasnormal.Itmaybethatbettersamplingoftheprokaryoticworldwillgiveusmorecluesaboutthephysicaloriginsofeukaryoticchromosomalsegregationinmitosisandmeioisis.

It’salmostunknownamongbacteriawithcellwalls,thoughsomearchaeaareknowntofuse.Thelossof thecellwallwouldcertainlyhavemadefusionfarmorelikely;andL-formbacteria,whichhavelost theircellwall,doindeedfusewitheachotherquitereadily.Thenumberofcontrolsovercellfusioninmoderneukaryotesalsoimpliesitmighthavebeenhardtostoptheirancestorsfusing

together.Earlyfusionscouldevenhavebeenpromotedbymitochondria,asarguedbytheingeniousevolutionarybiologistNeilBlackstone.Thinkabouttheirpredicament.Asendosymbionts,theycouldnotleavetheirhostcellsandsimplyinfectanotherone,sotheirownevolutionarysuccesswastiedtothe growth of their hosts. If their hosts were crippled by mutations and unable to grow, themitochondriawouldbestucktoo,unabletoproliferatethemselves.Butwhatiftheycouldsomehowinducefusionwithanothercell?Thisisawin-winsituation.Thehostcellacquiresacomplementarygenome,therebyenablingrecombination,orperhapssimplymaskingmutationsonparticulargeneswith potentially clean copies of the same genes – the benefits of outbreeding. Because cell fusionpermittedrenewedgrowthofthehostcell,themitochondriacouldreverttocopyingthemselvestoo.Soearlymitochondriacouldhavebeenagitating for sex!5Thatmighthave solved their immediateproblem,butironically,itonlyopenedthedoortoanother,evenmorepervasive,issue:competitionbetween mitochondria. The solution might just have been that other puzzling aspect of sex – theevolutionoftwosexes.

Twosexes‘No practical biologist interested in sexual reproduction would be led to work out the detailedconsequencesexperiencedbyorganismshavingthreeormoresexes;yetwhatelseshouldhedoifhewishes tounderstandwhythesexesare, infact,always two?’SosaidSirRonaldFisher,oneof thefoundingfathersofevolutionarygenetics.Theproblemhasyettobeconclusivelysolved.

Onpaper, twosexes seem tobe theworstofallpossibleworlds. Imagine ifeveryonewere thesamesex–wecouldallmatewitheachother.Wewoulddoubleourchoiceofpartners inonego.Surelythatwouldmakeeverythingeasier!If,forsomereason,weareobligedtohavemorethanonesex,thenthreeorfoursexesoughttobebetterthantwo.Evenifrestrictedtomatingwithothersexes,wecould thencouplewith two-thirdsor three-quartersof thepopulation rather thanamerehalf. Itwouldstilltaketwopartners,ofcourse,butthere’snoobviousreasonwhythesepartnerscouldn’tbethe same sex, or multiple sexes, or for that matter hermaphrodites. The practical difficulties withhermaphroditesgivesawaypartof theproblem:neitherpartnerwants tobear thecostofbeing the‘female’.Hermaphroditicspeciessuchasflatwormsgotobizarrelengthstoavoidbeinginseminated,fightingpitchedbattleswiththeirpenises,theirsemenburninggapingholesinthevanquished.Thisislivelynaturalhistory,but it iscircularasanargument,as it takesforgranted that therearegreaterbiologicalcoststobeingfemale.Whyshouldtherebe?Whatactuallyisthedifferencebetweenmaleandfemale?ThesplitrunsdeepandhasnothingtodowithXandYchromosomes,orevenwitheggcellsandsperm.Twosexes,oratleastmatingtypes,arealsofoundinsingle-celledeukaryotes,suchassomealgaeandfungi.Theirgametesaremicroscopicandthetwosexeslookindistinguishablebutthey’restillasdiscriminatingasyouandme.

Oneofthedeepestdistinctionsbetweenthetwosexesrelatestotheinheritanceofmitochondria–onesexpassesonitsmitochondria,whiletheothersexdoesnot.Thisdistinctionappliesequallytohumans(allourmitochondriacomefromourmother,100,000ofthempackedintotheegg)andtoalgae such asChlamydomonas. Even though such algae produce identical gametes (or isogametes)onlyonesexpasseson itsmitochondria; theother suffers the indignityofhaving itsmitochondriadigestedfromwithin.Infactit’sspecificallythemitochondrialDNAthatgetsdigested;theproblemseems tobe themitochondrial genes, not themorphological structure.Sowehave a verypeculiarsituation,inwhichmitochondriaapparentlyagitateforsex,aswe’vejustseen,buttheoutcomeisnotthattheyspreadfromcelltocellbutthathalfofthemgetdigested.What’sgoingonhere?

Themostgraphicpossibilityisselfishconflict.Thereisnorealcompetitionbetweencellsthatareallgeneticallythesame.That’showourowncellsaretamed,sothattheycooperatetogethertoform

ourbodies.Allourcellsaregeneticallyidentical;wearegiantclones.Butgeneticallydifferentcellsdocompete,with somemutants (cellswithgeneticchanges)producingcancer; andmuch the samehappensifgeneticallydifferentmitochondriamixinthesamecell.Thosecellsormitochondriathatreplicatethefastestwilltendtoprevail,evenifthatisdetrimentaltothehostorganism,producingakindofmitochondrial cancer.That’s because cells are autonomous self-replicating entities in theirownright,andtheyarealwayspoisedtogrowanddivideiftheycan.FrenchNobellaureateFrançoisJacoboncesaidthatthedreamofeverycellistobecometwocells.Thesurpriseisnotthattheyoftendo,butthattheycanberestrainedforlongenoughtomakeahumanbeing.Forthesereasons,mixingtwopopulationsofmitochondriainthesamecellisjustaskingfortrouble.

Thisideagoesbackseveraldecades,andcomeswiththesealofsomeofthegreatestevolutionarybiologists,includingBillHamilton.Buttheideaisnotbeyondchallenge.Forastart,thereareknownexceptions,inwhichmitochondriamixfreely,anditdoesnotalwaysendindisaster.Thenthereisapracticalproblem.Imagineamitochondrialmutation thatgivesa replicativeadvantage.Themutantmitochondriaoutgrowtherest.Eitherthisislethal,inwhichcasethemutantswilldieoutalongwiththehostcells,orit isnot,andthemutantsspreadthroughthepopulation.Anygeneticconstraintontheirspread(forexample,somechangeinanucleargenethatpreventsmitochondrialmixing)hastoarisequickly,tocatchthemutantintheactofspreading.Ifjusttherightgenedoesnotariseintime,it’stoolate.Nothingisgainedifthemutanthasalreadyspreadtofixation.Evolutionisblindandhasnoforesight.Itcan’tanticipatethenextmitochondrialmutant.Andthere’sathirdpointthatmakesmesuspectthatfast-replicatingmitochondriaarenotasbadasallthat–thefactthatmitochondriahaveretainedso fewgenes.Theremaybemanyreasons for this,butselectiononmitochondria for fastreplication is surely among them. That implies there have been numerous mutations that sped upmitochondrialreplicationovertime.Theywerenoteliminatedbytheevolutionoftwosexes.

Forthesereasons,Isuggestedanewideainanearlierbook:perhapstheproblemrelatesrathertotherequirementformitochondrialgenestoadapttogenesinthenucleus.I’llsaymoreaboutthisinthenextchapter.Fornow,let’sjustnotethekeypoint:forrespirationtoworkproperly,genesinthemitochondriaandthenucleusneedtocooperatewitheachother,andmutationsineithergenomecanunderminephysicalfitness.Iproposedthatuniparentalinheritance,inwhichonlyonesexpassesonthemitochondria,might improve thecoadaptationof the twogenomes.The ideamakes reasonablesensetome,butthereitwouldhaverestedhadnotZenaHadjivasiliou,anablemathematicianwithabuddinginterestinbiology,embarkedonaPhDwithmeandAndrewPomiankowski.

Zenadidindeedshowthatuniparentalinheritanceimprovesthecoadaptationofthemitochondrialandnucleargenomes.Thereasonissimpleenoughandrelatestotheeffectsofsampling,athemethatwillreturnwithintriguingvariations.Imagineacellwith100geneticallydifferentmitochondria.Youremoveoneof them, place it all by itself inside another cell, and then copy it, until youhave100mitochondriaagain.Barringa fewnewmutations, thesemitochondriawillallbe thesame.Clones.Nowdothesamewiththenextmitochondrionandkeepgoinguntilyouhavecopiedall100.Eachofyour100newcellswillhavedifferentpopulationsofmitochondria,someofthemgood,somebad.Youhaveincreasedthevariancebetweenthesecells.Ifyouhadjustcopiedthewholecell100times,eachdaughtercellwouldhavehadroughlythesamemixofmitochondriaastheparentcell.Naturalselectionwouldnotbeable todistinguishbetweenthem–theyareall toosimilar.Butbysamplingandcloningthesample,youproducedarangeofcells,someofthemfitterthantheoriginal,otherslessfit.

Thisisanextremeexample,butillustratesthepointofuniparentalinheritance.Bysamplingafewmitochondria from only one of the two parents, uniparental inheritance increases the variance ofmitochondriabetween fertilisedeggcells.Thisgreatervariety ismorevisible tonatural selection,which can eliminate the worst cells, leaving the better cells behind. The fitness of the population

improvesovergenerations. Intriguingly, this ispractically thesameadvantageassex itself,butsexincreases the variance of nuclear genes,whereas two sexes increase the variance ofmitochondriabetweencells.It’sassimpleasthat.Orsowethought.

Ourstudywasastraightcomparisonoffitnesswithandwithoutuniparentalinheritance,butatthispointwehadnotconsideredwhatwouldhappenifageneimposinguniparental inheritanceweretoarise inapopulationofbiparentalcells, inwhichbothgametespasson themitochondria.Would itspreadtofixation?Ifso,wewouldhaveevolvedtwosexes:onesexwouldpassonitsmitochondriaandtheothersexwouldhaveitsmitochondriakilled.Wedevelopedourmodeltotestthispossibility.Forgoodmeasurewecomparedourcoadaptationhypothesiswiththeoutcomesarisingfromselfishconflict,asdiscussedabove,andasimpleaccumulationofmutations.6Theresultscameasasurprise,andatleastinitiallyweredisappointing.Thegenewouldnotspread,certainlynottofixation.

Theproblemwasthat thefitnesscostsdependonthenumberofmutantmitochondria: themoremutants, thehigher thecosts.Conversely, thebenefitsofuniparental inheritancealsodependon themutationload,butthistimetheotherwayround:thesmallerthemutantload,thelowerthebenefit.Inother words the costs and benefits of uniparental inheritance are not fixed, but change with thenumber ofmutants in the population; and that can be lowered by just a few rounds of uniparentalinheritance(Figure29).We found that uniparental inheritance did indeed improve the fitness of apopulationinallthreemodels,butasthegeneforuniparentalinheritancebeginstospreadthroughapopulation, its benefits dwindle until they are offset by the disadvantages – themain disadvantagebeing that uniparental cells mate with a smaller fraction of the population. The trade-off reachesequilibriumwhenbarely20%ofthepopulationisuniparental.Highmutationratescouldforceitupto 50% of the population; but the other half of the population could continue to mate amongthemselves,giving,ifanything,threesexes.Thebottomlineisthatmitochondrialinheritancewillnotdrive the evolution of two mating types. Uniparental inheritance increases the variance betweengametes, improving fitness, but this benefit isn’t strong enough by itself to drive the evolution ofmatingtypes.

Figure29The‘leakage’offitnessbenefitsinmitochondrialinheritanceAandaaregameteswithdifferentversions(alleles)ofaparticulargeneinthenucleus,denotedAanda.Gameteswithapassontheirmitochondriawhentheyfusewithanotheragamete.GameteswithAare‘uniparentalmutants’:ifanAgametefuseswithanagamete,onlytheAgametepassesonitsmitochondria.ThefirstmatinghereshowsafusionofAandagametes,toproduceazygotewithbothnuclearalleles(Aa),butallmitochondriaderivingfromA.Ifacontainssomedefectivemitochondria(paleshade)theseareeliminatedbyuniparentalinheritance.Thezygotenowproducestwogametes,onewiththeAallele,andonewiththeaallele.Eachofthesefuseswithanagametecontainingdefectivemitochondria(paleshade).Intheuppercross,AandagametesgenerateanAazygote,withallmitochondriaderivingfromtheAgamete,therebyeliminatingthedefective(pale)mitochondria.Inthelowercross,twoagametesfuse,andthedefectivemitochondriaarepassedontotheaazygote.Eachofthesezygotes(Aaandaa)nowformsgametes.Theamitochondriahavenowbeen‘cleanedup’byacoupleofroundsofuniparentalinheritance.Thatimprovesthefitnessofbiparentalgametes,sothefitnessbenefit‘leaks’throughthepopulation,ultimatelyarrestingitsownspread.

Well,thiswasadirectdisproofofmyownidea,soIdidn’tlikeitmuch.Wetriedeverythingwecould think of to make it work, but eventually I had to concede that there are no realisticcircumstances inwhichauniparentalmutantcoulddrive theevolutionof twomating types.Matingtypesmusthaveevolvedforsomeotherreason.7Evenso,uniparentalinheritanceexists.Ourmodelwouldsimplybewrongifwecouldnotexplainthat.Infact,weshowedthatiftwomatingtypesdidalready exist, for some other reason, then certain conditions could fix uniparental inheritance:specifically,alargenumberofmitochondria,andahighmitochondrialmutationrate.Ourconclusionseemed incontrovertible; and our explanation sitsmore comfortablywith the known exceptions touniparentalinheritanceinthenaturalworld.Italsomadesenseofthefactthatuniparentalinheritanceis practically universal among multicellular organisms, animals such as ourselves, which do ingeneralhavelargenumbersofmitochondriaandhighmutationrates.

Thisisafineexampleofwhymathematicalpopulationgeneticsisimportant:hypothesesneedtobetestedformally,bywhatevermethodsarepossible;inthiscase,aformalmodelshowedclearlythatuniparental inheritance cannot fix in a populationunless twomating types already exist.This is asclosetorigorousproofaswecanget.Butallwasnotyetlost.Thedifferencebetweenmatingtypesand ‘true’ sexes (inwhichmales and females are obviously different) is opaque.Many plants andalgaehavebothmating typesandsexes.Perhapsourdefinitionof sexeswaswrong,andwe reallyshouldbeconsideringtheevolutionoftruesexes,ratherthantwoostensiblyidenticalmatingtypes.Coulduniparentalinheritanceaccountforthedistinctionbetweentruesexesinanimalsandplants?Ifso,matingtypesmighthavearisenforotherreasons,buttheevolutionoftruesexescouldstillhavebeendrivenbymitochondrial inheritance.Frankly, thatseemedaweak idea,butwortha look.Thatreasoningdidnotbegintoprepareusfortherevelatoryanswerthatweactuallyfound,ananswerthatemerged precisely because we did not start out with the normal assumption that uniparentalinheritanceisuniversal,butwiththedisappointingconclusionsofourownpreviousstudy.

Immortalgermline,mortalbodyAnimals have large numbers of mitochondria, and we use them ceaselessly to power oursupercharged lifestyles,givingushighmitochondrialmutationrates, right?Moreor lessright.Wehavehundredsorthousandsofmitochondriaineachcell.Wedon’tknowtheirmutationrateforsure(it’s difficult tomeasure directly) butwe do know that overmany generations, ourmitochondrialgenes evolve some 10–50 times faster than genes in the nucleus. This implies that uniparentalinheritance ought to fix readily in animals. In our model, we did indeed find that uniparentalinheritancewillfixmoreeasilyinmulticellularthansingle-celledorganisms.Nosurprisesthere.

Butweareeasilymisledbythinkingaboutourselves.Thefirstanimalswerenotlikeus:theyweremorelikespongesorcorals,sessilefilterfeedersthatdon’tmovearound,atleastnotintheiradult

forms.Notsurprisingly,theydon’thavemanymitochondria,andthemitochondrialmutationrateislow– lower, if anything, than in thenucleargenes.Thiswas the startingpoint for thePhDstudentArunas Radzvilavicius, yet another gifted physicist attracted to the big problems of biology. Onebeginstowonderifallthemostinterestingproblemsinphysicsarenowinbiology.

WhatArunasrealisedisthatsimplecelldivisioninmulticellularorganismshasarathersimilareffect to uniparental inheritance: it increases the variance between cells.Why? Each round of celldivisionapportionsthemitochondrialpopulationrandomlybetweenthedaughtercells.Ifthereareafewmutants, thechancesof thembeingdistributedexactlyequally is low– it’sfarmore likely thatonedaughtercellwillreceiveafewmoremutantsthantheother.Ifthisisrepeatedovermanyroundsofcelldivision,theoutcomeisgreatervariance;somegreat-great-great-granddaughtercellswillendup inheriting a greatermutant load thanothers.Whether this is a goodor a bad thingdepends onwhichcellsreceivethebadmitochondria,andhowmanythereare.

Imagineanorganismlikeasponge,inwhichallthecellsarequitesimilar.It’snotdifferentiatedintolotsofspecialisedtissues,likebrainandintestine.Cutupalivingspongeintosmallpieces(don’tdothisathome)anditcanregenerateitselffromthosebitsandpieces.Itcandosobecausestemcells,lurkingmoreorlessanywhere,cangiverisetonewgermcellsaswellasnewsomatic(body)cells.Inthisregard,spongesaresimilartoplants–neitherofthemsequestersaspecialisedgermlineearlyindevelopment,butinsteadtheygenerategametesfromstemcellsinmanytissues.Thisdifferenceiscritical.Wehaveadedicatedgermline,whichishiddenawayearlyoninembryonicdevelopment.Amammalwouldnormallyneverproducegermcellsfromstemcellsintheliver.Sponges,coralsandplants,however,cangrownewsexualorgansproducinggametesfrommanydifferentplaces.Thereare explanations for these differences, rooted in competition between cells, but they are not reallycompelling.8WhatArunasfoundisthatalltheseorganismshaveonethingincommon:theyhaveasmallnumberofmitochondriaandalowmitochondrialmutationrate.Andthefewmutationsthatdooccurcanbeeliminatedbysegregation.Itworkslikethis.

Recall thatmultiple rounds of cell division increase the variance between cells. That goes forgerm cells too. If the germ cells are sequestered early on in development, there can’t be muchdifferencebetweenthem–thefewroundsofcelldivisiondon’tgeneratemuchvariance.Butifgermcellsareselectedatrandomfromadulttissues,thentherewillbemuchgreaterdifferencesbetweenthem (Figure 30). Multiple rounds of cell division mean that some germ cells accumulate moremutations thanothers.Somewillbenearlyperfect,othersadreadfulmess– there ishighvariancebetweenthem.Thatiswhatnaturalselectionneeds:itcanweedoutallthebadcells,soonlythegoodonessurvive.Overgenerations, thequalityofgermcells increases; selecting themrandomly fromadulttissuesworksbetterthanhidingthemaway,puttingthem‘onice’earlyindevelopment.

Sogreatervarianceisgoodforthegermline,butitcanbedevastatingforthehealthofanadult.Badgermcellsareeliminatedbyselection, leaving thebetterones toseed thenextgeneration;butwhat about bad stem cells, which give rise to new adult tissues? These will tend to producedysfunctional tissues thatmaybeunable to support theorganism.The fitnessof theorganismasawholedependsonthefitnessofitsworstorgan.IfIhaveaheartattack,thefunctionofmykidneysisimmaterial:myhealthyorganswilldiealongwiththerestofme.Sotherearebothadvantagesanddisadvantages to increasing mitochondrial variance in an organism, and the advantage to thegermlinemaywellbeoffsetby thedisadvantage to thebodyasawhole.Thedegree towhich it isoffsetdependsonthenumberoftissuesandthemutationrate.

Themoretissuesthereareinanadult,thegreaterthelikelihoodthatavitaltissuewillaccumulatealltheworstmitochondria.Conversely,withonlyonetissuetype,thisisnotaproblem,asthereisnointerdependence–noorganswhosefailurecanunderminethefunctionofthewholeindividual.Inthecaseofasimpleorganismwithasingle tissue, then, increasedvariance isunequivocallygood: it’s

beneficialfor thegermlineandnotparticularlydetrimental tothebody.Wethereforepredictedthatthe first animals,with (presumably) lowmitochondrialmutation rates andvery few tissues, shouldhavehadbiparental inheritanceand lackedasequesteredgermline.Butwhenearlyanimalsbecameslightlymore complex,withmore than a couple of different tissues, increasedvariancewithin thebodyitselfbecomesdisastrousforadultfitness,asitinevitablyproducesbothgoodandbadtissues–theheartattackscenario.Toimproveadultfitness,mitochondrialvariancemustbedecreasedsothatnascenttissuesallreceivesimilar,mostlygood,mitochondria.

Figure30RandomsegregationincreasesvariancebetweencellsIfacellstartswithamixtureofdifferenttypesofmitochondria,whicharedoubledandthendividedroughlyequallybetweentwodaughtercells,theproportionswillvaryslightlywitheachcelldivision.Overtimethesedifferencesareamplified,aseachcellpartitionsanincreasinglydistinctpopulationofmitochondria.Ifthefinaldaughtercellsattherightbecomegametes,thenrepeatedcelldivisionhastheeffectofincreasingvariancebetweengametes.Someofthesegametesareverygood,andothersverybad,increasingthevisibilitytonaturalselection:exactlythesameeffectasuniparentalinheritance,andaGoodThing.Conversely,ifthecellsattherightareprogenitorcellsthatgiverisetoanewtissueororgan,thenthisincreasedvarianceisadisaster.Nowsometissueswillfunctionwellbutotherswillfail,underminingthefitnessoftheorganismasawhole.Onewaytolowerthevariancebetweentissueprogenitorcellsistoincreasethenumberofmitochondriainthezygote,suchthatthenumberofmitochondriapartitionedinitiallyismuchgreater.Thiscanbeachievedbyincreasingthesizeoftheeggcell,givingriseto‘anisogamy’(largeegg,smallsperm).

Thesimplestwaytodecreasevarianceinadulttissuesistostartoutwithmoremitochondriaintheeggcell.Asastatisticalrule,varianceislowerifalargefounderpopulationispartitionedbetweennumerousrecipientsthanifasmallpopulationisrepeatedlydoubledandthenpartitionedtothesamenumberofrecipients.Theupshotisthatincreasingthesizeofeggcells,packingthemwithmoreandmore mitochondria, is beneficial. By our calculations, a gene specifying larger eggs will spreadthroughapopulationof simplemulticellularorganisms,because itdecreases the variance betweenadulttissues, ironingoutanypotentiallydevastatingdifferencesinfunction.Ontheotherhand,lessvarianceisnotgoodforgametes,whichbecomemoresimilartoeachother,andsoless‘visible’tonaturalselection.Howcanthesetwoopposingtendenciesbereconciled?Simple!Ifonlyoneofthetwogametes,theeggcell,increasesinsize,whereastheothershrinks,becomingsperm,thatsolvesbothproblems.The large egg cell decreases the variance between tissues, improving adult fitness,

whereastheexclusionofmitochondriafromspermultimatelyresultsinuniparentalinheritance,withonly one parent passing on its mitochondria. We’ve already noted that uniparental inheritance ofmitochondria increases the variance between gametes, so improving their fitness. In other words,fromthesimplestofstartingpoints,bothanisogamy(distinctgametes,spermandegg)followedbyuniparentalinheritancewilltendtoevolveinorganismswithmorethanonetissue.

Ihave to stress that all this assumesa lowmitochondrialmutation rate.That’sknown tobe thecase in sponges, corals and plants, but it is not the case in ‘higher ’ animals.What happens if themutationraterises?Thebenefitofdelayinggerm-cellproductionisnowlost.Ourmodelshowsthatmutationsaccumulatequickly,leavinglategermcellsriddledwithmutations.AsthegeneticistJamesCrow put it, the greatestmutational health hazard in the population is fertile oldmen. Thankfully,uniparental inheritance means that men don’t pass on their mitochondria at all. Given a fastermutation rate, we find that a gene which induces early sequestration of the germline will spreadthrough a population: hiving off an early germline, putting female gametes on ice, limits theaccumulationofmitochondrialmutations.Adaptationsthatspecificallylowerthegermlinemutationrateshouldalsobefavoured.Infact,mitochondriainthefemalegermlineseemtobeswitchedoff,hidden in the primordial egg cells that are sequestered early in the embryonic development ofovaries,asshownbymycolleagueJohnAllen.Hehaslongarguedthatthemitochondriaineggcellsaregenetic‘templates’,which,beinginactive,havealowermutationrate.Ourmodelsupportstheseideasformodernfast-livinganimalswithnumerousmitochondriaandrapidmutationrates,butnotfortheirslower-livingancestors,orforwidergroupssuchasplants,algaeandprotists.

Whatdoesallthismean?Itmeans,astonishingly,thatmitochondrialvariationalonecanexplainthe evolution of multicellular organisms that have anisogamy (sperm and eggs), uniparentalinheritance,andagermline,inwhichfemalegermcellsaresequesteredearlyindevelopment–whichtogether form the basis for all sexual differences betweenmales and females. In otherwords, theinheritanceofmitochondria can account formost of the real physical differencesbetween the twosexes.Selfish conflictbetweencellsmayplaya role too,but isnotnecessary: the evolutionof thegermline–soma distinction can be explained without reference to selfish conflict. Critically, ourmodelspecifiesanorderofeventsthatisnotwhatIwouldhaveguessedattheoutset.Ihadimaginedthat uniparental inheritance was the ancestral state, that the germline evolved next, and that theevolutionofspermandeggcellswasconnectedwiththedivergenceoftruesexes.Instead,ourmodelimplies that the ancestral state was biparental; anisogamy (sperm and egg) arose next, thenuniparental inheritance, and finally the germline. Is this revised order correct? There’s littleinformationeitherway.Butitisanexplicitpredictionthatcanbetested,andwehopetodoso.Theplacestolookfirstarespongesandcorals.Bothgroupshavespermandeggs,butlackasequesteredgermline.Wouldtheydeveloponeifweselectedforahighermitochondrialmutationrate?

Let’sdrawtoaclosewithafewimplications.Whywouldthemitochondrialmutationraterise?Anincreasedturnoverofcellsandproteinswoulddoit,reflectingphysicalactivity.TheoxygenationoftheoceanssoonbeforetheCambrianexplosionfavouredtheevolutionofactivebilateralanimals.Theirgreateractivitywouldhave raised theirmitochondrialmutation rate (which ismeasurable inphylogeneticcomparisons),andthatshouldhaveforcedthesequestrationofadedicatedgermlineintheseanimals.Thiswastheoriginoftheimmortalgermlineandthemortalbody–theoriginofdeathasaplannedandpredeterminedendpoint.Thegermlineisimmortalinthesensethatgermcellscancontinue dividing forever. They never age or die. Each generation sequesters a germline early indevelopment,whichproducesthecellsthatseedthenextgeneration.Individualgametesmaybecomedamaged,butthefactthatbabiesarebornyoungmeansthatgermcellsaloneretainthepotentialforimmortalityseeninorganismslikespongesthatregeneratethemselvesfrombitsandpieces.Assoonasthisspecialisedgermlineishiddenaway,therestofthebodycanspecialiseforspecificpurposes,

nolongerrestrainedbytheneedtoretainimmortalstemcellsintheirmidst.Weseeforthefirsttimetissues that can no longer regenerate themselves, such as the brain. The disposable soma. Thesetissueshavea limited lifespan,whichdependsonhow long theorganism takes to reproduce itself.That dependsonhowquickly the animal reaches reproductivematurity, thedevelopmental rate, itsanticipatedlifespan.Weseefor thefirst timea tradeoffbetweensexanddeath, therootsofageing.We’lllookintothatinthenextchapter.

Thischapterhasexploredtheeffectsofmitochondriaontheeukaryoticcell,someofwhichweredramatic.Recallthecentralquestion:whydidalleukaryotesevolveawholeseriesofsharedtraitsthatare never found in bacteria or archaea? In the previous chapter, we saw that prokaryotes areconstrainedbytheircellstructure,andspecificallytherequirementforgenestocontrolrespiration.Theacquisitionofmitochondria transformed the selective landscape for eukaryotes, enabling theirexpansionincellvolumeandgenomesizeoverfourorfiveordersofmagnitude.Thattriggerwasarare endosymbiosis between two prokaryotes, not far from a freak accident, but the consequenceswerebothsevereandpredictable.Severe,becauseacell lackinganucleusishighlyvulnerabletoabarrageofDNAandgeneticparasites(introns)fromitsownendosymbionts.Predictable,becausetheresponseofthehostcellateachstage–theevolutionofanucleus,sex,twosexesandagermline–canbeunderstoodintermsofclassicalevolutionarygenetics,albeitfromanunconventionalstartingpoint. Some of the ideas in this chapter may turn out to be wrong, as did my hypothesis on theevolutionoftwosexes;butinthatcaseafullerunderstandingturnedouttobefarricherthanIhadimagined, accounting instead for the germline–soma distinction, the origins of sex and death.Theunderlyinglogic,excavatedthroughrigorousmodelling,isatoncebeautifulandpredictable.Lifeislikelytofollowasimilarpathtocomplexityelsewhere.

Thisviewoflife’shistory,a4-billion-yearstory,placesthemitochondriarightatthecentreoftheevolutionoftheeukaryoticcell.Inrecentyears,medicalresearchhascometoarathersimilarview:wenowappreciate thatmitochondria are instrumental in controlling cell death (apoptosis), cancer,degenerativedisease, fertility,andmore.Butmyarguments thatmitochondria reallyare thehubofphysiologyareprone tomakesomemedical researcherscross; thecharge is that I lackaproperlybalanced perspective. Look at any human cell down a microscope, and you will see a wonderfulassemblyofworkingparts,ofwhich themitochondriaare justone,admittedly important, cog.Butthatisnottheviewfromevolution.Theviewfromevolutionseesmitochondriaasequalpartnersintheoriginofcomplexlife.Alleukaryotictraits–allcellphysiology–evolvedintheensuingtugofwarbetweenthesetwopartners.Thattugofwarcontinuestothisday.Inthefinalpartofthisbook,weshallseehowthisinterplayunderpinsourownhealth,fertilityandlongevity.

Footnotes1I’mnotclaimingthatariseinoxygenconcentrationdrovetheevolutionofanimals(asdiscussedinChapter1),butthatitenabledmoreactivebehaviourinlargeanimals.Thereleasefromenergeticconstraintspromotedapolyphyleticradiationofmanydifferentgroupsofanimals,butanimalshadalreadyevolvedbeforetheCambrianexplosion,beforethemajorriseinoxygentowardstheendofthePrecambrian.2OK,mostlynothing.Someintronshaveacquiredfunctions,suchasbindingtranscriptionfactors,andsometimestheyareactiveasRNAthemselves,interferingwithproteinsynthesisandthetranscriptionofothergenes.Weareinthemidstofanera-definingargumentaboutthefunctionofnon-codingDNA.Someofitiscertainlyfunctional,butIalignmyselfwiththedoubters,whoarguethatmostofthe(human)genomeisnotactivelyconstrainedinitssequences,andthereforedoesnotserveapurposethatisdefinedbyitssequence.Toallintentsandpurposes,thatmeansitdoesn’thaveafunction.Ifforcedtohazardaguess,Iwouldsaythatperhaps20%ofthehumangenomeisfunctional,andtherestisbasicallyjunk.Butthatdoesn’tmeanitisnotusefulforsomeotherpurpose,suchasfillingspace.Natureabhorsavacuum,afterall.3Variantsofthesamegenearetermed‘alleles’.Specificgenesremaininthesamepositiononachromosome,the‘locus’,buttheactualsequenceofaspecificgenecanvarybetweenindividuals.Ifparticularvariantsarecommoninapopulation,theyareknownasalleles.Allelesarepolymorphicvariantsofthesamegene,atthesamelocus.Theydifferfrommutantsinfrequency.Newmutationsarepresent

atalowfrequencyinapopulation.Iftheyofferanadvantage,theymayspreadthroughthepopulationuntilthisadvantageiscounterbalancedbysomedisadvantage.Theyhavebecomealleles.4Theeffectivepopulationsizereflectstheamountofgeneticvariationinapopulation.Intermsofaparasiticinfection,aclonalpopulationmightaswellbeasingleindividual,asanyparasiticadaptationthatallowsittotargetaparticulargenecombinationcouldtearthroughtheentirepopulation.Conversely,largesexualpopulationstendtohavealotofgeneticvariationinalleles(whileallsharingthesamegenes).Thatvariationmeansthatsomeorganismsarelikelytoberesistanttothisparticularparasiticinfection.Theeffectivepopulationsizeisgreater,evenifthenumberofindividualsisthesame.5Blackstonehasevensuggestedapossiblemechanismthatderivesfromthebiophysicsofmitochondria.HostcellswhosegrowthiscrippledbymutationswouldhavelowATPdemands,sotheywouldbreaklittleATPbackdownintoADP.BecauseelectronflowinrespirationdependsonADPconcentration,therespiratorychainwouldtendtofillupwithelectronsandbecomemorereactive,formingoxygenfreeradicals(moreonthisinthenextchapter).Insomealgaetoday,free-radicalleakagefrommitochondriainducestheformationofgametesandsex;andthisresponsecanbeblockedbygivingthemantioxidants.Couldfreeradicalshavetriggeredmembranefusiondirectly?It’spossible.Radiationdamageisknowntocausemembranefusionthroughafree-radicalmechanism.Ifso,anaturalbiophysicalprocesscouldhaveservedasthebasisforsubsequentnaturalselection.6Fromamathematicalpointofview,allthreetheoriesturnedouttobevariantsofeachother:eachonedependsonthemutationrate.Inasimplemutationmodel,therateofaccumulationofmutantsobviouslydependsonthemutationrate.Likewise,whenaselfishmutantarises,itreplicatesalittlefasterthanthewildtype,meaningthatthenewmutantspreadsthroughthepopulation.Mathematicallythatequatestoafastermutationrate,whichistosay,therearemoremutantsinagiventime.Thecoadaptationmodeldoestheopposite.Theeffectivemutationrateisloweredbecausenucleargenescanadapttomitochondrialmutants,meaningtheyarenolongerdetrimental,hencebyourdefinitionthey’renotmutants.7Thereareplentyofotherpossibilities,rangingfromensuringoutbreedingtosignallingandpheromones.Giventhattwocellsfuseinsex,theyfirsthavetofindeachother,andmakesuretheyfusewiththerightcell–anothercellofthesamespecies.Cellstypicallyfindeachotherby‘chemotaxis’,whichistosaytheyproduceapheromone,ineffecta‘smell’,andtheymovetowardsthesourceofthesmell,upaconcentrationgradient.Ifbothgametesproducethesamepheromone,theycanconfusethemselves.They’relikelytoswimaroundinsmallcircles,smellingtheirownpheromone.Itisgenerallybetterforonlyonegametetoproduceapheromoneandtheothertoswimtowardsit,sothedistinctionbetweenmatingtypescouldrelatetotheproblemoffindingamate.8ThedevelopmentalbiologistLeoBusshasargued,forexample,thatanimalcells,beingmobile,aremorelikelytoinvadethegermline,inaselfishattempttoperpetratethemselves,thanplantcells,whosecumbersomecellwallrendersthemvirtuallyimmobile.Butisthattrueofcoralsandspongestoo,whicharecomposedofperfectlymobileanimalcells?Idoubtit.Yettheyhavenomoreofagermlinethandoplants.

PARTIV

PREDICTIONS

C

7

THEPOWERANDTHEGLORY

hrist Pantocrator: Ruler of the World. Even beyond Orthodox iconography, there can be nogreaterartisticchallengethantheportraitofChristinhis‘twonatures’,atonceGodandman,the

sternbut lovingjudgeofallhumanity.Inhis lefthand,hemaycarrytheGospelofJohn:‘Iamthelightof theworld,whofollowsmewillnotwander in thedarknessbutwillhave the lightof life.’Unsurprisingly, given this sober task, the Pantocrator tends to look rather melancholic. From theartist’spointofview,capturingthespiritofGodinthefaceofmanisnotenough:itmustbedoneinmosaic,insideadome,highoverthealtarofafinecathedral.Ican’timaginetheskillsrequiredtogettheperspectivejustright, tocatchthelightandshadeofalivingface, toinvest tinypiecesofstonewithmeaning,eachpieceobliviousof itsplacein thegranddesign,yeteachonecrucial to thefullconception. I do know that marginal errors can destroy the whole effect, giving the Creator adisturbingly comical expression; but when done supremely well, as in Cefalù Cathedral in Sicily,even the least religious will recognise the face of God, an eternal monument to the genius offorgottenhumancraftsmen.1

Iamnotabouttodepartinsomeunanticipateddirection.I’mstruckbytheappealofmosaicstothehumanmind, and by the strikingly parallel importance of mosaics in biology – could there be asubconsciousconnectionbetween themodularityofproteinsandcells,andoursenseofaesthetics?Oureyesarecomposedofmillionsofphotoreceptorcells,rodsandcones;eachreceptorisswitchedonoroffwitharayoflight,forminganimageasamosaic.Thisisreconstructedinourmind’seyeas a neuronal mosaic, conjured up from splintered features of the image – brightness, colour,contrast,edge,movement.Mosaicsstirouremotionsinpartbecausetheysplinterrealityinasimilarwaytoourminds.Cellscandothisbecausetheyaremodularunits,livingtiles,eachonewithitsownvitalplace,itsownjob,40trillionpiecesmakingupthewonderfulthree-dimensionalmosaicthatisahumanbeing.

Mosaicsrunevendeeperinbiochemistry.Considermitochondria.Thegreatrespiratoryproteins,which transfer electrons from food to oxygen while pumping protons across the mitochondrialmembrane, aremosaicsofnumerous subunits.The largest, complex I, is composedof45 separateproteins, each one made up of hundreds of amino acids linked together in a long chain. Thesecomplexes are often grouped into larger ensembles, ‘supercomplexes’, which funnel electrons tooxygen.Thousandsofsuper-complexes,eachoneanindividualmosaic,adornthemajesticcathedralofthemitochondrion.Thequalityofthesemosaicsisvitallyimportant.AcomicalPantocratormaybenolaughingmatter,buttinyerrorsinthepositionofindividualpiecesintherespiratoryproteinscancarryaburdenas terribleasanybiblicalpunishment.If justoneaminoacidisoutofplace–a

singlestoneinthefullmosaic–theconsequencesmaybeacripplingdegenerationofthemusclesandbrain, and an early death: a mitochondrial disease. These genetic conditions are horriblyunpredictableintheirseverityand theirageofonset,dependingonexactlywhichpiece isaffected,andhowoften;butallofthemreflectthecentralityofmitochondriatotheveryboneofourexistence.

Somitochondria aremosaics, and their qualitymatters in terms of life and death; but there ismore. Like the Pantocrator, the respiratory proteins are unique in having ‘two natures’, themitochondrial and the nuclear, and these had better be a match made in heaven. The peculiararrangementoftherespiratorychain–theassemblyofproteinsthatconveyselectronsfromfoodtooxygen– is shown inFigure31.Most of the core proteins in themitochondrial innermembrane,shownindarkershade,areencodedbygeneslocatedinthemitochondriathemselves.Theremainingproteins(lightershading)areencodedbygenesinthenucleus.Wehaveknownaboutthisstrangestateofaffairssincetheearly1970s,whenitfirstbecameclearthatthemitochondrialgenomeissosmallthat it cannot possibly encode most of the proteins found in the mitochondria. The old idea thatmitochondria are still independent of their host cells is therefore nonsense. Their ostensibleautonomy–theygiveaneerieimpressionofreplicatingthemselveswhenevertheyfeellikeit–isamirage. The fact is that their function depends on two distinct genomes. They can only grow orfunctioniftheyarewhollyprovisionedwithproteinsencodedbybothofthesegenomes.

Letme ram home just how odd this is. Cell respiration – without which wewould die withinminutes–dependsonmosaicrespiratorychainsthatarecomposedofproteinsencodedbytwoverydifferentgenomes.Toreachoxygen,electronsmusthopdownarespiratorychainfromone‘redoxcentre’ to thenext.Redoxcentres typicallyacceptordonateelectronsoneat a time– theseare thestepping stones that we discussed in Chapter 2. The redox centres are embedded deep inside therespiratoryproteins,theirprecisepositionsdependingonthestructureoftheproteins,henceonthesequence of the genes that encode the proteins, and hence on both the mitochondrial and nucleargenomes. As noted, electrons hop by a process known as quantum tunnelling. They appear anddisappearfromeachcentrewithaprobabilitythatdependsonseveralfactors–thetuggingpowerofoxygen (more specifically, the reduction potential of the next redox centre), the distance betweenadjacent redox centres, and the occupancy (whether the next centre is already occupied by anelectron).Theprecisedistancebetween redoxcentres iscritical.Quantum tunnellingwillonly takeplaceoververyshortdistances,lessthanabout14Å(recallthatanångström(Å)isaboutthediameterofanatom).Redoxcentresspacedfurtherapartmightaswellbeinfinitelydistant,asthelikelihoodofelectronshoppingbetweenthemfallstozero.Withinthiscriticalrange,therateofhoppingdependsonthedistancebetweencentres.Andthatdependsonhowthetwogenomesinteractwitheachother.

Figure31ThemosaicrespiratorychainProteinstructuresforcomplexI(left),complexIII(centreleft),complexIV(centreright)andtheATPsynthase(right),allembeddedintheinnermitochondrialmembrane.Thedarkersubunits,mostlyburiedwithinthemembrane,areencodedbygenesthatarephysicallylocatedinthemitochondria,whereasthepalersubunits,mostlyperipheraloroutsidethemembrane,areencodedbygenesthatresideinthenucleus.Thesetwogenomesevolveindramaticallydifferentways–themitochondrialgenesarepassedonasexuallyfrommothertodaughter,whereasnucleargenesarerecombinedbysexeverygeneration;andmitochondrialgenes(inanimals)alsoaccumulatemutationsatuptofiftytimestherateofnucleargenes.Despitethispropensitytodiverge,naturalselectioncangenerallyeliminatedysfunctionalmitochondria,maintainingnearlyperfectfunctionoverbillionsofyears.

Foreachångströmincreaseindistancebetweenredoxcentres,thespeedofelectrontransferfallsabout10-fold.Letmesaythatagain.Thereisa10-folddecreaseintherateofelectrontransferforevery 1 Å increase in distance between redox centres! That’s roughly the scale of electricalinteractions between adjacent atoms, for example the ‘hydrogen bonds’ between negatively andpositively charged amino acids in proteins. If amutation alters the identity of an amino acid in aprotein,hydrogenbondsmaybebroken,ornewonesformed.Wholewebsofhydrogenbondsmayshiftalittle,includingthosethatpinionaredoxcentreintoitscorrectposition.Itmightwellmovebyan ångströmor so. The consequences of such tiny shifts aremagnified by quantum tunnelling: anångströmthiswayorthatcouldslowdownelectrontransferbyanorderofmagnitude,orspeeditbyanequivalentfactor.That’sonereasonwhymitochondrialmutationscanbesocatastrophic.

This precarious arrangement is exacerbated by the fact that the mitochondrial and nucleargenomesaredivergingcontinuously. In thepreviouschapter,wesawthat theevolutionofbothsexandtwosexescouldhavebeenrelatedtotheacquisitionofmitochondria.Sexisneededtomaintainthe functionof individual genes in large genomes,whereas two sexes helpmaintain the quality ofmitochondria. The unforeseen consequencewas that these two genomes evolve in totally differentways. Nuclear genes are recombined by sex every generation, whereas mitochondrial genes passfrommother to daughter in the egg cell, rarely if ever recombining. Even worse, mitochondrialgenesevolve10–50timesfasterthangenesinthenucleus,intermsoftheirrateofsequencechangeovergenerations,at least inanimals.Thismeans thatproteinsencodedbymitochondrialgenesaremorphingfasterandindifferentwayscomparedwithproteinsencodedbygenesinthenucleus;yetstilltheymustinteractwitheachotheroverdistancesofångströmsforelectronstotransferefficientlydowntherespiratorychain.It’shardtoimagineamorepreposterousarrangementforaprocesssocentraltoalllivingthings–respiration,thevitalforce!

Howdidmatterscometosuchapass?Therearefewbetterexamplesoftheshort-sightednessofevolution.Thiscrazysolutionwasprobably inevitable.Recall the startingpoint–bacteria that liveinside other bacteria.Without such an endosymbiosis,we saw that complex life is not possible, asonly autonomous cells are capable of losing superfluous genes, leaving themultimatelywith onlythose genes necessary to control respiration locally. That sounds reasonable enough, but the onlylimitongene loss isnatural selection–andselectionactsonboth thehostcellsandmitochondria.What leads to gene loss? In part, simply replication speed: the bacteriawith the smallest genomesreplicate the fastest, hence tend to dominate over time. But replication speed cannot explain thetransferofgenestothenucleus,onlythelossofgenesfrommitochondria.Inthepreviouschapter,wesawwhymitochondrialgenesarriveinthenucleus–somemitochondriadie,spillingtheirDNAintothehostcell,andthisistakenupintothenucleus.That’shardtostop.SomeofthisDNAinthenucleusnow acquires a targeting sequence, an address code, which targets the protein back to themitochondria.

Thismaysound likea freakevent,but in fact it applies toalmost all the1,500knownproteinstargetedtothemitochondria;plainlyitisnotsohard.Theremustbeatransientsituationwherecopiesofthesamegenearepresentinthesurvivingmitochondriaandinthenucleusatthesametime.Inthe

end one of the two copies is lost. Excepting the 13 protein-coding genes that remain in ourmitochondria (<1% of their initial genome), the nuclear copywas retained and themitochondrialcopylostineverycase.Thatdoesn’tsoundlikechance.Whyisthenuclearcopyfavoured?Therearevariousplausiblereasons,buttheoreticalworkhasnotyetprovedthecaseonewayoranother.Onepossiblereasonismalefitness.Asmitochondriapassdownthefemaleline,frommothertodaughter,it isnotpossibletoselectformitochondrialvariants thatfavourmalefitness,asanygenes inmalemitochondria that happen to improve male fitness are never passed on. Transferring thesemitochondrial genes to the nucleus, where they are passed on in both males and females, couldthereforeimprovemalefitnessaswellasfemalefitness.Genesinthenucleusarealsorecombinedbysex every generation, perhaps improving fitness even more. And then there’s the fact thatmitochondrial genes physically take up space, which could be better filled with the machinery ofrespiration or other processes. Finally, reactive free radicals escape from respiration, which canmutate the neighbouringmitochondrialDNA;wewill return to the effects of free radicals on cellphysiology later. All in all, there are very good reasons why genes are transferred from themitochondriatothenucleus;fromthispointofview,it’smoresurprisingthatanygenesremainthereatall.

Whydothey?Thebalancingforce,whichwediscussedinChapter5,istherequirementforgenestocontrol respiration locally.Recall that theelectricalpotentialacross the thin innermitochondrialmembraneis150–200millivolts,givingafieldstrengthof30millionvoltspermetre,equaltoaboltoflightning.Genesareneededtocontrolthiscolossalmembranepotentialinresponsetochangesinelectronflux,oxygenavailability,ADPandATPratios,numberofrespiratoryproteins,andmore.Ifagenethatisneededforcontrollingrespirationinthiswayistransferredtothenucleus,anditsproteinproduct fails tomake it back to themitochondria in time toprevent a catastrophe, then thenatural‘experiment’endsrightthere.Animals(andplants)whichdidnottransferthatparticulargenetothenucleussurvive,while those that transferred thewronggenedie, theirunfortunatelymisconfiguredgeneswiththem.

Selectionisblindandmerciless.Genesarecontinuouslytransferredfromthemitochondriatothenucleus.Eitherthenewarrangementworksbetter,andthegenestaysinitsnewhome,oritdoesnot,and some penalty is exacted – probably death. In the end, nearly all themitochondrial geneswereeither lost altogether or transferred to the nucleus, leaving a handful of critical genes in themitochondria.Thisis thebasisofourmosaicrespiratorychains–blindselection.Itworks.Idoubtthatanintelligentengineerwouldhavedesigneditthatway;butthiswas,Ihazard,theonlywaythatnaturalselectioncouldfashionacomplexcell,giventherequirementforanendosymbiosisbetweenbacteria.Thispreposteroussolutionwasnecessary.Inthischapter,we’llexaminetheconsequencesofmosaicmitochondria:towhatextentdoesthisrequirementpredictthetraitsofcomplexcells?Iwillarguethatselectionformosaicmitochondriacanindeedexplainsomeofthemostpuzzlingcommontraitsofeukaryotes.Allofus.Thepredictedoutcomesofselectionincludeeffectsonourhealth,ourfitness,fertilityandlongevity,evenourhistoryasaspecies.

OntheoriginofspeciesHowandwheredoesselectionact?Weknowthatitdoes.Thesmokinggunofmanygenesequencestestifiestoahistoryofselectionforcoadaptationofmitochondrialandnucleargenes:thetwosetsofgenes change in related ways.We can compare the rates of change ofmitochondrial and nucleargenesovertime–say,themillionsofyearsthatseparatechimpanzeesfromhumansorgorillas.Weimmediatelyseethatthegeneswhichinteractdirectlywitheachother–thosethatencodeproteinsinthe respiratory chain, for example – change at about the same speed, whereas other genes in the

nucleusgenerallychange(evolve)muchmoreslowly.Plainlyachangeinamitochondrialgenetendstoelicitacompensatorychangeinaninteractingnucleargene,orviceversa.Soweknowthatsomeformofselectionhastakenplace;thequestionis,whatprocessesfashionedsuchcoadaptation?

The answer lies in the biophysics of the respiratory chain itself. Picture what happens if thenuclearandmitochondrialgenomesdonotmatchproperly.Electronsenter therespiratorychainasnormal, but the mismatched genomes encode proteins that do not sit comfortably together. Someelectricalinteractionsbetweenaminoacids(hydrogenbonds)aredisrupted,meaningthatoneortworedoxcentresmightnowbeanångströmfurtherapart thannormal.Asa result, theelectrons flowdowntherespiratorychaintooxygenatafractionoftheirnormalspeed.Theybegintoaccumulateinthefirstfewredoxcentres,unabletomoveon,asthedownstreamredoxcentresarealreadyoccupied.Therespiratorychainbecomeshighlyreduced,meaningthattheredoxcentresfillupwithelectrons(Figure32).Thefirstfewredoxcentresareiron-sulphurclusters.TheironisconvertedfromtheFe3+totheFe2+ (orreduced)form,whichcanreactdirectlywithoxygentoformthenegativelychargedsuperoxideradicalO2

•–.Thedotheresymbolisesasingleunpairedelectron,thedefiningsignatureofafreeradical.Andthatputsthecatamongthepigeons.

Figure32MitochondriaincelldeathAshowsnormalelectronflowdowntherespiratorychaintooxygen(wavyarrow),withthecurrentofelectronspoweringtheextrusionofprotonsacrossthemembrane,andprotonfluxthroughtheATPsynthase(right)drivingATPsynthesis.Thepalegreycolourofthethreerespiratoryproteinsinthemembraneindicatesthatthecomplexesarenothighlyreduced,aselectronsdonotaccumulateinthecomplexesbutarepassedonquicklytooxygen.Bshowstheconcertedeffectsofslowingelectronfluxastheresultofanincompatibilitybetweenmitochondrialandnucleargenomes.Slowelectronfluxtranslatesintoloweroxygenconsumption,limitedprotonpumping,fallingmembranepotential(becausefewerprotonsarepumped),andcollapsingATPsynthesis.Theaccumulationofelectronsintherespiratorychainissignifiedbythedarkershadingoftheproteincomplexes.ThehighlyreducedstateofcomplexIincreasesitsreactivitywithoxygen,formingfreeradicalssuchassuperoxide(O2

•-).CIfthissituationisnotresolvedwithinminutes,thenfreeradicalsreactwithmembranelipidsincludingcardiolipin,resultinginthereleaseofcytochromec(thesmallproteinlooselyassociatedwiththemembraneinAandBandnowreleasedinC.Lossofcytochromecprecludeselectronfluxtooxygenaltogether,reducingtherespiratorycomplexesevenmore(nowshowninblack),increasingfree-radicalleak,andcollapsingmembranepotentialandATPsynthesis.Thesefactorstogethertriggerthecelldeathpathway,resultinginapoptosis.

There are various mechanisms, notably the enzyme superoxide dismutase, which quicklyeliminate an accumulation of superoxide radicals.But the abundance of such enzymes is carefullycalibrated.Toomuchwouldriskinactivatingavitallyimportantlocalsignal,whichworksabitlikeafirealarm.Freeradicalsactlikesmoke:eliminatethesmokeandyoudon’tsolvetheproblem.Inthiscase,thetroubleisthatthetwogenomesdonotfunctionwelltogether.Electronflowisimpaired,andthis generates superoxide radicals – a smoke signal.2 Above some threshold, free radicals oxidisenearby membrane lipids, notably cardiolipin, resulting in release of the respiratory proteincytochromec,whichisnormallylooselytetheredtocardiolipin.Thatscupperstheflowofelectronsaltogether, as they need to hop on to cytochrome c to get to oxygen. Remove cytochrome c andelectronscannolongerreachtheendoftherespiratorychain.Withnoelectronflowtherecanbenofurtherprotonpumping,andthatmeanstheelectricalmembranepotentialwillsooncollapse.Thuswehave threealterations toelectronflux in respiration: first,electron transferslowsdown,andso therateofATPsynthesisalsofalls.Second,thehighlyreducediron–sulphurclustersreactwithoxygentoproduce a burst of free radicals, resulting in the releaseof cytochromec from its tethering to themembrane.And third, if nothing is done to compensate for these changes, themembranepotentialcollapses(Figure32).

Ihavejustdescribedacurioussetofcircumstancesfirstdiscoveredinthemid1990sandgreetedat thetimewith‘generalstupefaction’.This is thetriggerforprogrammedcelldeath,orapoptosis.When a cell undergoes apoptosis, it kills itself via a carefully choreographed ballet, the cellularequivalent of thedying swan.Far from simply falling topieces anddecomposing, in apoptosis anarmyofproteinexecutioners,calledcaspaseenzymes,issetloosefromwithin.Thesecutupthegiantmoleculesofthecell–DNA,RNA,carbohydratesandproteins–intobitsandpieces.Thepiecesareboundupinlittlepacketsofmembrane,blebs,andfedtosurroundingcells.Withinafewhours,alltracesofitsformerexistencehavegone,airbrushedfromhistoryaseffectivelyasaKGBcover-upattheBolshoi.

Apoptosis makes perfect sense in the context of a multicellular organism. It is necessary forsculpting tissuesduringembryonicdevelopment,andremovingandreplacingdamagedcells.Whatcameasacomplete surprisewas thecentral involvementofmitochondria,especially thebona fiderespiratory protein cytochrome c. Why on earth would the loss of cytochrome c from themitochondriaactasasignalforcelldeath?Sincethediscoveryofthismechanism,themysteryhasonlydeepened.Itturnsoutthatthissamecombinationofevents–fallingATPlevels,free-radicalleak,lossofcytochromec,andacollapseofmembranepotential–isconservedrightacrosseukaryotes.Plantcellsandyeastkillthemselvesinresponsetoexactlythesamesignal.Nobodyexpectedthat.Yetit emerges fromfirstprinciples, asan inevitableconsequenceof selection for twogenomes– it is

predictablyauniversalpropertyofcomplexlife.Let’s thinkback toourelectronsmaking theirwaydownamismatchedrespiratorychain. If the

mitochondrial andnuclear genesdon’tworkproperly together, the natural biophysical outcome isapoptosis.This is abeautiful exampleofnatural selectionhoningaprocess that cannotbe stoppedfromhappening: anatural tendency is elaboratedby selection, ultimatelybecominga sophisticatedgeneticmechanism,whichretainsatitsheartacluetoitsorigin.Twogenomesareneededforlargecomplexcellstoexistatall.Theymustworkwelltogether,orrespirationwillfail.Iftheydon’tworkproperlytogether,thecelliseliminatedbyapoptosis.Thiscannowbeseenasaformoffunctionalselection against cells with mismatched genomes. Once again, as the Russian-born geneticistTheodosiusDobzhansky famouslyobserved,nothing inbiologymakes senseexcept in the lightofevolution.

Sowehaveamechanismfortheeliminationofcellswithmismatchedgenomes.Conversely,cellswith genomes that doworkwell together will not be eliminated by selection. Over evolution, theoutcomeispreciselywhatwesee:thecoadaptationofmitochondrialandnucleargenomes,suchthatsequencechangesinonegenomearecompensatedforbysequencechangesintheother.Asnotedinthepreviouschapter, theexistenceoftwosexesincreasesthevariancebetweenfemalegermcells–differenteggcellscontainmainlyclonalpopulationsofmitochondria,withdifferenteggsamplifyingdifferent clones ofmitochondria. Some of these cloneswill happen toworkwell against the newnuclearbackgroundofthefertilisedegg,otherslesswell.Thosethatdon’tworksufficientlywellareeliminatedbyapoptosis;thosethatdoworkwelltogethersurvive.

Survive what exactly? In multicellular organisms, the broad answer is development. From afertilised egg cell (zygote), cells divide to form a new individual. The process is exquisitelycontrolled. Cells that die unexpectedly by apoptosis during development jeopardise the entiredevelopmentalprogrammeandmayresultinmiscarriage,afailureofembryonicdevelopment.Thatisn’t necessarily a bad thing.Much better, from the dispassionate viewof natural selection, to haltdevelopment early, before toomany resources have been dedicated to the new individual, than toallow development to run to full term. In the latter case, the offspring would be born withincompatibilitiesbetween thenuclear andmitochondrialgenes,potentiallycausingamitochondrialdisease,breakdownofhealth,andanearlydeath.Ontheotherhand,terminatingdevelopmentearly–sacrificing an embryo if it shows serious incompatibilities between mitochondrial and nucleargenomes–obviouslyreducesfertility.Ifahighproportionofembryosfailtodeveloptofullterm,the outcome is infertility. The costs and benefits here are absolutely central to natural selection:fitness versus fertility. Plainly theremust be refined controls over which incompatibilities triggerapoptosisanddeath,andwhichonesaretolerated.

Allthismayseemalittledryandtheoretical.Doesitactuallymatter?Yes!–atleastinafewcases,and thesecouldbe the tipofan iceberg.Thebestexamplecomes fromRonBurton,at theScrippsMarineResearchInstitute,whohasbeenworkingonmitochondrial–nuclear incompatibilities in themarinecopepodTigriopuscalifornicusformorethanadecade.Copepodsaresmallcrustaceans,1–2mminlength,foundinalmostallwetenvironments;inthiscaseintheintertidalpoolsofSantaCruzIsland in southernCalifornia.Burtonhasbeencrossingbetween twodifferentpopulationsof thesecopepods from opposite sides of the island, which were reproductively isolated for thousands ofyears,despite livingonlya fewmilesapart.Burtonandhiscolleaguescataloguewhat isknownas‘hybridbreakdown’inmatingsbetweenthe twopopulations.Intriguingly, there is littleeffect in thefirst generation, the result of a single cross between the two populations; but if the female hybridoffspringare thenmatedwithamale from theoriginalpaternalpopulation,herownoffspringareterriblysickly,ina‘sorrystate’toborrowfromthetitleofoneofBurton’spapers.Whiletherewasquite a spectrum of outcomes, on average the hybrid fitness was substantially lower – their ATP

synthesiswasreducedbyabout40%,andthiswasreflectedinsimilardecreasesinsurvival,fertilityand developmental time (in this case, time tometamorphosis, which depends on body size, hencegrowthrate).

This entire problem could be ascribed to incompatibilities betweenmitochondrial and nucleargenesby a simple expedient –backcrossingmalehybridoffspringwith females from theoriginalmaternal population.Their offspringwerenow restored to full andnormal fitness. In theoppositeexperiment, however – crossing female hybrid offspring with males from the original paternalpopulation–therewasnopositiveeffectonfitness.Theoffspringremainedsickly;indeedtheywereworsethanever.Theresultsareeasyenoughtounderstand.Themitochondriaalwayscomefromthemother,andtofunctionproperlyneedtointeractwithgenesinthenucleusthataresimilartothoseofthemother.Bycrossingwithmalesfromageneticallydistinctpopulation,themother ’smitochondriaarepairedwithnucleargenesthatdon’tfunctionwellwithhermitochondria.Inthefirstgenerationcross,theproblemisnottoosevere,as50%ofnucleargenesstillcomefromthemother,andtheseworkwellwithhermitochondria.Bythesecondgenerationofhybrids,though,75%ofnucleargenesarenowmismatchedtothemitochondria,andweseeaseriousbreakdowninfitness.Crossinghybridmaleswith females from theoriginalmaternalpopulationmeans that62.5%ofnucleargenesnowderive from the maternal population andmatch the mitochondria. Full health is restored. But thereverse cross has the opposite effect: the maternal mitochondria are nowmismatched with some87.5%ofnucleargenes.Nowondertheywereasicklybunch.

Hybrid breakdown. Most of us are familiar with the idea of hybrid vigour. Outcrossing isbeneficial,becauseunrelatedindividualsarelesslikelytosharethesamemutationsinthesamegenes,sothecopiesinheritedfromthefatherandmotherarelikelytobecomplementary,improvingfitness.Buthybridvigouronlygoessofar.Crossesbetweendistinctspeciesarelikelytoproduceoffspringthat are unviable or sterile.This is hybrid breakdown.The sexual barriers between closely relatedspecies are farmore permeable than the textbookswould have us believe – species that prefer toignoreeachotherinthewild,forbehaviouralreasons,willoftenmatesuccessfullyincaptivity.Thetraditional definition of a species – the failure to produce fertile offspring in crosses betweenpopulations–issimplynottrueformanycloselyrelatedspecies.Nonetheless,aspopulationsdivergeovertime,reproductivebarriersdobuildupbetweenthem,andultimatelysuchcrossesdoindeedfailto produce fertile offspring. These barriers must begin to assert themselves in crosses betweenpopulationsof the samespecies thathavebeen reproductively isolated for longperiods, as inRonBurton’s copepods. In this case, the breakdown is entirely attributable to incompatibilities betweenmitochondrial and nuclear genes. Could similar incompatibilities cause hybrid breakdown in theoriginofspeciesmoregenerally?

I suspect so. This is of course only one mechanism among many, but other examples of‘mitonuclear ’ breakdownhave been reported acrossmany species, from flies andwasps towheat,yeast,andevenmice.Thefactthatthismechanismemergesfromarequirementfortwogenomestoworkproperlytogetherimpliesthatspeciationfollowsinevitablyineukaryotes.Evenso,theeffectsaresometimesmorepronouncedthanothers.Thereasonapparentlyrelatestotherateofchangeofmitochondrialgenes.In thecaseofcopepods, themitochondrialgenesevolveupto50timesfasterthangenes in thenucleus. In thecaseof the fruit flyDrosophila,however, themitochondrialgenesevolvemuchmoreslowly,barelytwicethespeedofgenesinthenucleus.Accordingly,mitonuclearbreakdown ismoreserious incopepods than in fruit flies.The faster rateofchange translates intomore differences in sequence within a given time, hence a greater likelihood of incompatibilitiesbetweengenomesincrossesbetweendifferentpopulations.

Exactly why the mitochondrial genes of animals evolve much faster than nuclear genes isunknown. Doug Wallace, the inspirational pioneer of mitochondrial genetics, argues that

mitochondriaarethefrontlineinadaptation.Rapidchangesinmitochondrialgenesenableanimalstoadapt swiftly to changing diets and climates, the first steps that precede slower morphologicaladaptations.Iliketheidea,althoughthereisasyetlittlegoodevidenceeitherfororagainstit.ButifWallace is right, then adaptation is improved by continually throwing up new variants inmitochondrial sequence on which selection can act. These changes, in being the first to facilitateadaptationstonewenvironments,arealsoamongthefirstharbingersofspeciation.Thatcorrespondstoacuriousoldruleinbiology,firstlaiddownbytheinimitableJ.B.S.Haldane,oneofthefoundingfathers of evolutionary biology. A new interpretation of this rule suggests that mitonuclearcoadaptationmightindeedplayanimportantroleintheoriginofspecies,andinourownhealth.

SexdeterminationandHaldane’sruleHaldane was given tomemorable pronouncements, and in 1922 he came up with this remarkableproclamation:

When in the offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous[heterogametic]sex.

It would have been easier if he had said the ‘male’, but that would actually have been lesssweeping.Themale isheterozygousorheterogametic inmammals,meaning that themalehas twodifferent sex chromosomes – an X and a Y chromosome. Female mammals have two Xchromosomes, and are therefore homozygous for their sex chromosomes (homogametic). It’s theotherwayroundforbirdsandsomeinsects.Herethefemaleisheterogametic,havingaWandaZchromosome,whereasthemaleishomogametic,withtwoZchromosomes.Imagineacrossbetweenamaleandfemalefromtwocloselyrelatedspecies,whichproducesviableoffspring.Butnowlookmorecarefullyattheseoffspring:theyareallmale,orallfemale;orifbothsexesarepresent,thenoneofthetwosexesissterileorotherwisemaimed.Haldane’srulesaysthatthissexwillbethemaleinmammalsandthefemaleinbirds.Thecatalogueofexamplesthathasbeenpiecedtogethersince1922isimpressive:hundredsofcasesconformtotherule,acrossmanyphyla,withsurprisinglyfewexceptionsforasubjectasconfoundedbyexceptionsasbiology.

There have been various plausible explanations for Haldane’s rule, though none of them canaccount for all cases, hence none of them is intellectually entirely satisfying. For example, sexualselectionisstronger inmales,whichmustcompeteamongthemselvesfor theattentionsoffemales(technically there is greater variance in reproductive success betweenmales than females,makingmale sexual traitsmore visible to selection). That in turnmakesmalesmore vulnerable to hybridbreakdown in a cross between different populations. The trouble is that this particular explanationdoesnotexplainwhymalebirdsarelessvulnerabletohybridbreakdownthanfemales.

Another difficulty is thatHaldane’s rule arguably goes beyondmere sex chromosomes,whichlook parochial on a wider view of evolution. Many reptiles and amphibians don’t have sexchromosomesatall,butdefinetheirsexesonthebasisof temperature;eggsincubatedatawarmertemperature develop into males, or occasionally vice versa. In fact, given its apparently basicimportance, themechanismsofsexdeterminationareperplexinglyvariableacrossspecies.Sexcanbe determined by parasites, or by the number of chromosomes, or by hormones, environmentaltriggers,stress,populationdensity,orevenmitochondria.Thefactthatoneofthetwosexestendstobe worse affected in crosses between populations, even when sex is not determined by anychromosomesatall,suggeststheremightbeadeepermechanismafoot.Indeed,theveryfactthatthedetailedmechanisms of sex determination are so variable, yet the development of two sexes is soconsistent, implies that there could be some conserved underlying basis to sex determination (the

processdrivingmaleorfemaledevelopment)whichdifferentgenesmerelyembroider.Onepossibleunderlyingbasis ismetabolicrate.EventheancientGreeksappreciated thatmales

are,literally,hotterthanfemales–the‘hotmale’hypothesis.Inmammalssuchashumansandmice,theearliestdistinctionbetween the twosexes is thegrowthrate:maleembryosgrowslightly fasterthan females, a difference that can be measured within hours of conception, using a ruler (butdefinitelydon’ttrythatathome).OntheYchromosome,thegenethatdictatesmaledevelopmentinhumans,theSRYgene,speedsupthegrowthratebyturningonanumberofgrowthfactors.Thereisnothingsexspecificaboutthesegrowthfactors:theyarenormallyactiveinbothmalesandfemales,it’s just that their activity is set at a higher level inmales than females.Mutations that increase theactivity of these growth factors, speeding growth rate, can induce a sex change, forcing maledevelopmentonfemaleembryoslackingaYchromosome(orSRYgene).Conversely,mutationsthatlower their activity can have the opposite effect, converting males with a perfectly functional Ychromosome into females. All this implies that growth rate is the real force behind sexualdevelopment,at least inmammals.Thegenesare justholding the reins,andcaneasilybe replacedoverevolution–onegenethatsetsthegrowthrateisreplacedbyadifferentgenethatsetsthesamegrowthrate.

Theideathatmaleshaveafastergrowthratecorrespondsintriguinglytothefactthattemperaturedetermineswhichsexdevelopsinamphibiansandreptilessuchasalligators.Thisisrelatedbecausemetabolicratealsodependsinpartontemperature.Withinlimits,raisingthebodytemperatureofareptileby10°C(bybaskinginthesun,forexample)roughlydoublesthemetabolicrate,whichinturnsustains a faster growth rate. While it’s not always the case that males develop at the highertemperature(forvarioussubtlereasons)thelinkbetweensexandgrowthrate,seteitherbygenesortemperature, is more deeply conserved than any particular mechanism. It does look as if variousopportunistic genes have from time to time grabbed the reins of developmental control, setting adevelopmental rate that inducesmaleor femaledevelopment.This isone reason, incidentally,whymenneednot fear thedemiseof theY chromosome– its functionwill probablybe takenover bysomeotherfactor,possiblyageneonadifferentchromosome,whichsets thefastermetabolicrateneededformaledevelopment.Itmightalsoaccountforthestrangevulnerabilityofexternaltesticlesinmammals;gettingthetemperaturerightismuchmoredeeplyembeddedinourbiologythanscrota.

These ideas, I must say, came as a revelation to me. The hypothesis that sex is determinedultimatelybymetabolicratehasbeenadvancedoverseveraldecadesbyUrsulaMittwoch,acolleagueatUCL,whoisstillremarkablyactiveandpublishingimportantpapersattheageof90.Herpapersare not as well known as they should be, perhaps because measurements of ‘unsophisticated’parameters such as growth rate, embryo size, and gonadal DNA and protein content seemed old-fashionedintheeraofmolecularbiologyandgenesequencing.Nowthatweareenteringaneweraofepigenetics(whatfactorscontrolgeneexpression?)herideasresonatebetter,andIhopewilltaketheirproperplaceinthehistoryofbiology.3

But howdoes all this relate toHaldane’s rule? Sterility or inviability corresponds to a loss offunction.Beyondsomethreshold,theorganortheorganismfails.Thelimitsoffunctiondependontwo simple criteria – the metabolic demands required to accomplish the task (making sperm, orwhatever)andthemetabolicpoweravailable.Ifthepoweravailableislowerthanthatrequired,thenthe organ or organismdies. In the subtleworld of gene networks, thesemay seem to be absurdlybluntcriteria,butthey’renonethelessimportantforthat.Putaplasticbagoveryourhead,andyoucutoffyourmetabolicpowerinrelationtoyourneeds.Functionceasesinlittlemorethanaminute,atleastinthebrain.Themetabolicrequirementsofyourbrainandheartarehigh;theywillbethefirstto die. Cells in your skin or intestines might survive much longer; they have much lowerrequirements.Theresidualoxygenwillbesufficienttomeettheirlowmetabolicneedsforhoursor

perhapsevendays.From theviewpointofour constituent cells,death isnot allornothing, it is acontinuum.We are a constellationof cells, and theydonot all die at once.Thosewith the highestdemandsgenerallyfailtomeetthemfirst.

This is precisely the problem in mitochondrial diseases. Most involve neuromusculardegeneration,affectingthebrainandskeletalmuscle,essentiallythetissueswiththehighestmetabolicrate. Sight is especially vulnerable: themetabolic rate of cells in the retina and optic nerve is thehighestinthebody,andmitochondrialdiseasessuchasLeber ’shereditaryopticneuropathyaffecttheoptic nerve, causing blindness. It is difficult to generalise about mitochondrial diseases, as theirseverity depends on many factors – the type of mutation, the number of mutants, and also theirsegregationbetweentissues.Butsettingthataside,thefactremainsthatmitochondrialdiseasesmostlyaffectthetissueswiththehighestmetabolicdemands.

Imaginethatthenumberandtypeofmitochondriaintwocellsarethesame,givingthemmatchingcapacity to generate ATP. If the metabolic demands imposed on these two cells are different, theoutcomewillbedifferent (Figure33).For thefirstcell, let’ssay itsmetabolicdemandsare low: itmeetsthemcomfortably,producingmorethanenoughATP,andspendingitonwhateveritstaskmaybe. Now imagine that the demands on the second cell aremuch greater – actually higher than itsmaximal capacity for producingATP.The cell strains tomatch its demands, itswhole physiologygeared tomeet thishighoutput.Electronspour into therespiratorychains,but theircapacity is toolow: the electrons are entering faster than they can leave again. The redox centres become highlyreduced,and reactwithoxygen toproduce free radicals.Theseoxidise the surroundingmembranelipids, releasingcytochromec.Membranepotential falls.The cell diesby apoptosis.This is still aformoffunctionalselection,eveninatissuesetting,ascellsthatcan’tmeettheirmetabolicdemandsareeliminated,leavingthosethatcan.

Ofcourse,theremovalofcellsthatdon’tworkwellenoughonlyimprovesoveralltissuefunctioniftheyarereplacedwithnewcellsfromthestem-cellpopulation.Amajorproblemwithneuronsandmusclecellsisthattheycannotbereplaced.Howcouldaneuronbereplaced?Ourlife’sexperienceiswritten into synaptic networks, each neuron forming as many as 10,000 different synapses. If theneurondiesbyapoptosis, thosesynapticconnectionsare lostforever,alongwithall theexperienceandpersonalitythatmighthavebeenwrittenintothem.Thatneuronisirreplaceable.Infact,whilelessobviously necessary, all terminally differentiated tissues are irreplaceable – their very existence isimpossiblewithoutthedeepdistinctionbetweengermlineandsomadiscussedinthepreviouschapter.Selectionisallaboutoffspring.Iforganismswithlargeandirreplaceablebrainsleavemoreviableoffspring thanorganismswithsmall replaceablebrains, then theywill thrive.Onlywhen there isadistinctionbetweenthegermlineandsomacanselectionact in thisway;butwhenitdoes, thebodybecomesdisposable.Lifespanbecomesfinite.Andcellsthatcan’tmeettheirmetabolicrequirementswillkillusintheend.

That’swhythemetabolicratematters.Cellswithafastermetabolicratearemorelikelytofailtomeettheirdemands,giventhesamemitochondrialoutput.Notonlymitochondrialdiseases,butalsonormalageingandage-relateddiseases,aremostlikelytoaffectthetissueswiththehighestmetabolicdemands.Andtocomefullcircle–thesexwiththehighestmetabolicdemands.Maleshaveafastermetabolicratethanfemales(inmammalsatleast).Ifthereissomegeneticdefectinthemitochondria,that defect will be unmasked largely in the sex with the faster metabolic rate – the male. Somemitochondrial diseases are indeedmore common inmen than inwomen;Leber ’s hereditary opticneuropathy,forexample,isfivetimesmoreprevalentinmen,whileParkinson’sdisease,whichalsohas a strongmitochondrial component, is twice as common.Males should also bemore seriouslyaffected by mitonuclear incompatibilities. If such incompatibilities are produced by outcrossingbetween reproductively isolated populations, the result should be hybrid breakdown. Thus hybrid

breakdownismostmarkedinthesexwiththehighestmetabolicrate,andinthatsex,withinthetissueswiththehighestmetabolicrate.Again,allofthisisapredictableconsequenceoftherequirementfortwogenomesinallcomplexlife.

Figure33FatedependsoncapacitytomeetdemandTwocellswithequivalentmitochondrialcapacity,facingdifferentdemands.InAthedemandismoderate(signifiedbythearrows);themitochondriacanmeetitcomfortablywithoutbecominghighlyreduced(denotedbythepalegreyshading).InBtheinitialdemandismoderate,butthenincreasestoafarhigherlevel.Electroninputtothemitochondriaincreasescommensurately,buttheircapacityisinsufficientandtherespiratorycomplexesbecomehighlyreduced(darkshading).Unlesscapacitycanbeincreasedswiftly,theoutcomeiscelldeathbyapoptosis(asdepictedinFigure32).

TheseconsiderationsofferabeautifulandsimpleexplanationofHaldane’srule:thesexwiththefastestmetabolicrateismorelikelytobesterileorinviable.Butisittrue,orindeedimportant?Anideacanbetruebuttrivial,andnoneofthisisincompatiblewithothercausesofHaldane’srule.Thereisnothingtosaythatmetabolicrateshouldbethesolecause;butisitasignificantcontributor?Ithinkso.Temperatureiswellknowntoexacerbatehybridbreakdown,forexample.WhentheflourbeetleTriboliumcastaneumiscrossedwithacloselyrelatedspeciesTriboliumfreeman,thehybridoffspringarehealthyattheirnormalrearingtemperatureof29°C–butiftheyareraisedat34°C,thefemales(inthiscase)developdeformitiesintheirlegsandantenna.Thatkindofsensitivitytotemperatureiswidespread,oftencausingsex-specificsterility,andismosteasilyunderstoodintermsofmetabolicrate.Aboveacertainthresholdofdemand,particulartissueswillstarttobreakdown.

Those particular tissues often include the sex organs, especially in males, where spermproduction continues throughout life.A striking example is found inplants, knownas cytoplasmicmalesterility.Mostfloweringplantsarehermaphrodites,butalargeproportionexhibitmalesterility,giving them two ‘sexes’ –hermaphrodites and (male-sterilized) females.Thismishap is causedbymitochondria and has usually been interpreted in terms of selfish conflict.4 But molecular datasuggest that male sterility may simply reflect metabolic rate. The plant scientist Chris Leaver, inOxford,has shown that the causeof cytoplasmicmale sterility in sunflowers is ageneencodingasinglesubunitoftheATPsynthaseenzymeinthemitochondria.Theprobleminthiscaseisanerrorin recombination, which affects a relatively small proportion (importantly, not all) of the ATPsynthaseenzymes.ThatlowersthemaximumrateofATPsynthesis.Inmosttissues,theeffectsofthismutation are unnoticeable – only the male sex organs, the anthers, actually degenerate. Theydegenerate because their constituent cells die by apoptosis, involving the release of cytochrome cfrom theirmitochondria in exactly the sameway as inourselves.The anthers seem tobe theonlytissueinthesunflowerwithametabolicratethatishighenoughtotriggerdegeneration:onlytheredo

thefaultymitochondriafailtomeettheirmetabolicdemands.Theoutcomeismale-specificsterility.SimilarfindingshavebeenreportedinthefruitflyDrosophila.Bytransferringthenucleusfrom

onecell toanother, it’spossible toconstructhybridcells (cybrids) inwhich thenucleargenome ismore or less identical, but the mitochondrial genes differ.5 Doing this with egg cells producesembryonic flies that are genetically identical in their nuclear background, but which havemitochondrialgenes from related species.Theoutcomesare strikinglydifferent,dependingon themitochondrial genes. In thebest cases, there is nothingwrongwith thenewborn flies. In theworstcrosses, themalesaresterile, themalebeing theheterogameticsex inDrosophila.Most interestingare the intermediatecases,where thefliesseemtobefine.Acloser lookat theactivityofgenes invariousorgans shows that they are troubled in their testes, however.More than1,000genes in thetestesandaccessorysexualorgansareup-regulatedinmaleflies.Quitewhatisgoingonisnotwellunderstood,but thesimplestexplanation, inmyeyes, is that theseorganscan’t reallycopewith themetabolicdemandsplaceduponthem.Theirmitochondriaarenotwhollycompatiblewithgenes inthenucleus.Cellsinthetestes,withtheirhighmetabolicdemands,arephysiologicallystressed,andthisstressproducesaresponsethatinvolvesasubstantialpartofthegenome.Asincytoplasmicmalesterilityinplants,onlythemetabolicallychallengedsexorgansareaffected–andonlyinmales.6

Ifall that’s thecase,whyare femalesaffected inbirds?Roughly the same reasoningholds,butwithsomeintriguingdifferences.Inafewbirds,notablybirdsofprey,thefemaleislargerthanthemale,andsopresumablygrowsfaster.Butthatisnotuniversal.UrsulaMittwoch’searlyworkshowsthatinchickenstheovariesoutgrowthetestes,afteraslowstartforthefirstweekorso.Inthesecasesthepredictionwouldbethatfemalebirdsshouldsufferfromsterilityratherthaninviability,asonlytheirsexorgansgrowfaster.Butthatisnottrue.MostcasesofHaldane’sruleinbirdsinfactseemtobeinviabilityratherthansterility.IwasbaffledbythatuntillastyearwhenGeoffHill,aspecialistinsexual selection in birds, sentmehis paper onHaldane’s rule in birds.Hill pointed out that a fewnucleargenesencodingrespiratoryproteinsinbirdsarefoundontheZchromosome(recallthat,inbirds,maleshavetwoZchromosomes,whilefemaleshaveoneZandoneWchromosome,makingthem theheterogametic sex).Whydoes thatmatter? If femalebirdsonly inheritonecopyof theZchromosome, theyonlygetonecopyof severalcriticalmitochondrialgenesand they inherit themfrom their father. If themother did not choose the fatherwith care, then hermitochondrial genesmightnotmatchthesinglecopyofhisnucleargenes.Breakdowncouldbeimmediateandserious.

Hillarguesthatthisarrangementplacestheburdenonthefemaletoselecthermatewithextremecare,orpaythegravepenalty(herfemaleoffspringdie).Thatinturncouldaccountforthevibrantplumageandcolorationofmalebirds.IfHillisright,thedetailedpatternoftheplumagesignalsthemitochondrial type: sharp demarcations in pattern are postulated to reflect sharp demarcations inmitochondrialDNAtype.Thefemalethereforeusespatternasaguidetocompatibility.Butamaleofthe right type can still be a pretty poor specimen.Hill argues that the vibrancy of colour reflectsmitochondrial function, asmostpigmentsare synthesised in themitochondria.Abrightlycolouredmalemust have top-qualitymitochondrial genes. There’s little evidence to back this hypothesis atpresent,butitgivesasenseofhowpervasivetherequirementformitonuclearcoadaptationcouldturnouttobe.It’sasoberingthoughtthattherequirementfortwogenomesincomplexlifecouldexplainevolutionary conundrums as disparate as the origin of species, the development of sexes, and thevividcolorationofmalebirds.

Anditmayrunevendeeper.Therearepenaltiesforgettingmitonuclear incompatibilitywrong,but also costs to getting it right, achieving good compatibility. The balance of costs and benefitsshoulddifferbetweendifferentspecies,dependingontheiraerobicrequirements.Thetrade-off,we’llsee,isbetweenfitnessandfertility.

ThethresholdofdeathImagineyoucanfly.Grampergram,youhavemorethantwicethepowerofacheetahinfullflight,aremarkable combination of strength, aerobic capacity, and lightness.You have no hope of gettingairborneifyourmitochondriaarenotpracticallyperfect.Considerthecompetitionforspaceinyourflightmuscles.Youneedmyofibrils,ofcourse,theslidingfilamentsthatproducemusclecontraction.Themoreoftheseyoucanpackin,thestrongeryouwillbe,asthestrengthofamuscledependsonitscross-sectionalarea,likearope.Unlikearope,however,musclecontractionhastobepoweredbyATP.TosustainexertionformuchmorethanaminuterequiresATPsynthesisonthespot.Thatmeansyou need mitochondria right there in your muscle. They take up space that could otherwise beoccupied by more myofibrils. Mitochondria also need oxygen. That means capillaries, to deliveroxygen and removewaste. The optimal space distribution in aerobicmuscle is around a third formyofibrils,athirdformitochondria,andathirdforcapillaries.That’strueforus,andcheetahs,andhummingbirds,whichhavebyfarthefastestmetabolicratesofallvertebrates.Thebottomlineiswecan’tgetmorepowerjustbyaccumulatingmoremitochondria.

Allthismeansthattheonlywaybirdscangenerateenoughpowertoremainairborneforlongistohave‘supercharged’mitochondria,abletogeneratemoreATPpersecondperunitofsurfaceareathan‘normal’mitochondria.Theelectronfluxfromfoodtooxygenmustbefast.Thattranslatesintofast protonpumping and a fast rate ofATP synthesis, necessary to sustain thehighmetabolic rate.Selectionmust act at every step, speeding up themaximum rate atwhich each respiratory proteinoperates.Wecanmeasuretheserates,andweknowthattheenzymesinthemitochondriaofbirdsdoindeedoperatefasterthanthoseinmammals.But,aswe’veseen,therespiratoryproteinsaremosaics,composedofsubunitsencodedbytwodifferentgenomes.Fastelectronfluxentailsstrongselectionfor the twogenomes toworkwell together, formitonuclear coadaptation.Thegreater the aerobicrequirements, the stronger this selection for coadaptation must be. Cells with genomes that don’twork well together are eliminated by apoptosis. The most reasonable place for such selection tooccur,aswe’veseen,isduringembryonicdevelopment.Fromadispassionatelytheoreticalpointofview, it makes more sense to terminate embryonic development very early on if the embryo hasincompatiblegenomes,whichdon’tworkwellenoughtogethertosustainflight.

Buthowincompatibleisincompatible,howbadisbad?Presumably,theremustbesomesortofathreshold, a point atwhich apoptosis is triggered.Above that threshold, the speed of electron fluxthroughthemosaicrespiratorychainisjustnotgoodenough–it’snotuptothejob.Individualcells,andbyextensionthewholeembryo,diebyapoptosis.Conversely,belowthethreshold,electronfluxisfastenough.Ifso,thenitfollowsthatthetwogenomesmustfunctionwelltogether.Thecells,andbyextensionthewholeembryo,donotkillthemselves.Instead,developmentcontinues,andallbeingwell ahealthychick isborn, itsmitochondria ‘pretested’ and stamped fit forpurpose.7The crucialpoint is that‘fit forpurpose’mustvarywith thepurpose. If thepurpose isflight, thenthegenomeshavetomatchnearlyperfectly.Thecostofhighaerobiccapacityislowfertility.Moreembryosthatcouldhavesurvivedsomelesserpurposemustbesacrificedonthealtarofperfection.Wecanevenseetheconsequencesinmitochondrialgenesequences.Theirrateofchangeinbirdsislowerthaninmostmammals(exceptbats,whichfacethesameproblemasbirds).Flightlessbirds,whichdon’tfacethesameconstraints,haveafasterrateofchange.Thereasonthatmostbirdshavelowratesofchangeis that theyhavealreadyperfected theirmitochondrial sequence for flight.Changes fromthis idealsequence are not readily tolerated, so are typically eliminated by selection. If most change iseliminated,thenwhatremainsisrelativelyunchanging.

Whatifweembracealesserpurpose?Let’ssayIamarat(asmyson’sschoolsonggoes,‘there’sno escaping that’) and I have no interest in flying. It would be stupid to sacrifice most of my

prospectiveoffspringonthealtarofperfection.We’veseenthatthetriggerforapoptosis–functionalselection – is free-radical leak. A sluggish flux of electrons in respiration betokens a poorcompatibilitybetweenthemitochondrialandnucleargenomes.Therespiratorychainsbecomehighlyreducedand leak free radicals.Cytochromec is releasedandmembranepotential falls. If Iwere abird, this combination is the trigger for apoptosis.My offspring would die in embryo again andagain.ButI’maratandIdon’twantthat.Whatif,bysomebiochemicalsleightofhand,I‘ignore’thefree-radicalsignalthatheraldsthedeathofmyoffspring?Iraisethedeaththreshold,meaningIcantoleratemorefree-radicalleakbeforetriggeringapoptosis.Igainanimmeasurablebenefit:mostofmyoffspringsurviveembryonicdevelopment.Ibecomemorefertile.WhatpricewillIpayformyburgeoningfertility?

CertainlyI’mnevergoing tofly.Andmoregenerally,myaerobiccapacitywillbe limited.Thechanceofmyoffspringhavinganoptimalmatchbetweenmitochondrialandnucleargenesisremote.That leads straight to another important pairingof costs andbenefits – adaptabilityversusdisease.Recall Doug Wallace’s hypothesis that the rapid evolution of mitochondrial genes in animalsfacilitatestheiradaptationtodifferentdietsandclimates.Wedon’treallyknowhow,orindeedif,thisreallyworks,butitwouldbesurprisingiftherewerenotruthinitatall.Thefirstlineofadaptationrelatestodietandbodytemperature(wewon’tsurviveforlongifwecan’tgetthebasicsright)andmitochondria are absolutely central to both. The performance of mitochondria depends in largemeasureon theirDNA.DifferentsequencesofDNAsupportdifferent levelsofperformance.Somewillworkbetterincoolerenvironmentsthanhotterones,oringreaterhumidity,orburningupafattydiet,andsoon.

There have been hints from the apparently non-random geographical distribution of differenttypes ofmitochondrialDNA in human populations that selection in particular environmentsmightindeed exist, but littlemore thanhints.Yet there is undoubtedly less variation in themitochondrialDNAofbirds, aswe’ve justnoted.Thevery fact thatmostchanges from theoptimal sequence forflightareeliminatedbyselectionmeansthatlessvariedmitochondrialDNAremains–sothereislessscopeforselectiontochoosesomemitochondrialvariantthatjusthappenstobeparticularlygoodinthecold,orwithafattydiet.It’scuriousinthisregardthatbirdsfrequentlymigrateratherthansufferaseasonalchangeinenvironmentalconditions.Coulditbethattheirmitochondriaarebetterabletosupport theexertionofmigration than function in theharsherenvironment theywould face if theystayedput?Conversely, ratshaveagreatdealmorevariation,andfromfirstprinciples that shouldgivethemtherawmaterialforbetteradaptation.Doesitreally?Frankly,Idon’tknow;thoughratsareprettyadaptablebeasts.There’snoescapingthat.

But,ofcourse,mitochondrialvariationcomesatacost–disease.Toapoint,thatcanbeavoidedbyselectiononthegermline,inwhicheggcellswithmitochondrialmutationsareweededoutbeforetheycanmature.Thereissomeevidenceforsuchselection–severemitochondrialmutationstendtobe eliminated over several generations, though less severemutations persist almost indefinitely inmiceandrats.Butthinkaboutthatremarkagain–severalgenerations!Selectionhereisprettyweak.If you are bornwith a seriousmitochondrial disease it can be little consolation to think that yourgrandchildren,ifyouareluckyenoughtohaveany,maybediseasefree.Evenifselectiondoesactagainst mitochondrial mutations in the germline, this is still no guarantee against mitochondrialdisease.Immatureeggcellsdonothaveanestablishednuclearbackground.Notonlyaretheyheldinlimboformanyyears,pausedhalfwaythroughmeiosis,butatthispointthefather ’sgeneshaveyettobeaddedtothefray.Selectionformitonuclearcoadaptationcanonlyoccurafterthematureeggcellhasbeen fertilisedby the sperm,andanew,geneticallyuniquenucleushasbeen fashioned.Hybridbreakdown is not causedbymitochondrialmutations, but by incompatibilitiesbetweennuclear andmitochondrialgenes,allofwhichareperfectlyfunctionalinsomeothercontext.We’vealreadyseen

that strong selection against mitonuclear incompatibilities necessarily increases the likelihood ofinfertility.Ifwedon’twanttobeinfertile,wehavetoacceptthecost–agreaterriskofdisease.Again,this equation between fertility and disease is a predictable outcome of the requirement for twogenomes.

So there is a hypothetical death threshold (Figure 34). Above the threshold the cell, and byextension the whole organism, dies by apoptosis. Below the threshold the cell and the organismsurvive.Thisthresholdisnecessarilyvariablebetweendifferentspecies.Forbatsandbirdsandothercreatureswithhighaerobicrequirements,thethresholdmustbesetlow–evenamodestrateoffree-radical leak from mildly dysfunctional mitochondria (with slight incompatibilities betweenmitochondrialandnucleargenomes)signalsapoptosisandterminationof theembryo.Forratsandslothsandcouchpotatoeswithlowaerobicrequirements,thethresholdissethigher:amodestrateoffree-radical leak is now tolerated, dysfunctional mitochondria are good enough, the embryodevelops.Therearecostsandbenefitstobothsides.Alowthresholdgivesahighaerobicfitnessandalowriskofdisease,butatthecostofahighrateofinfertilityandpooradaptability.Ahighthresholdgivesalowaerobiccapacityandhigherriskofdiseasebutwiththebenefitsofgreaterfertilityandbetteradaptability.Thesearewords toconjurewith.Fertility.Adaptability.Aerobic fitness.Disease.We can’t cutmuch closer to the grain of natural selection than that. I reiterate: all these trade-offsemergeinexorablyfromtherequirementfortwogenomes.

Figure34ThedeaththresholdThethresholdatwhichfree-radicalleaktriggerscelldeath(apoptosis)shouldvarybetweenspecies,dependingontheaerobiccapacity.Organismswithhighaerobicdemandsneedaverygoodmatchbetweentheirmitochondrialandnucleargenomes.Apoormatchisbetrayedbyahighrateoffree-radicalleakfromthedysfunctionalrespiratorychain(seeFigure32).Ifaverygoodmatchisrequired,cellsshouldbemoresensitivetofree-radicalleak;evenlowleaksignalsthatthematchisnotgoodenough,triggeringcelldeath(alowthreshold).Conversely,ifaerobicdemandsarelowthenthereisnothingtobegainedbykillingthecell.Suchorganismswilltoleratehigherlevelsoffree-radicalleakwithouttriggeringapoptosis(ahighthreshold).Thepredictionsforhighandlowdeaththresholdareshowninthesidepanels.Pigeonsarehypothesisedtohavealowdeaththreshold,ratstheopposite.Bothhavethesamebodysizeandbasalmetabolicrate,butpigeonshaveamuchlowerrateoffree-radicalleak.Whiletheveracityofthesepredictionsisunknown,itisstrikingratsliveforjustthreeorfouryears,pigeonsforupto30.

I just called this a hypothetical death threshold, and so it is. Does it really exist? If so, is itgenuinely important? Just think about ourselves. Apparently 40% of pregnancies end in what is

knownas‘earlyoccultmiscarriage’.‘Early’inthiscontextmeansveryearly–withinthefirstweeksofpregnancy, and typicallybefore the first overt signsofpregnancy.You’dneverknowyouwerepregnant.And‘occult’means‘hidden’–notclinicallyrecognised.Generallywedon’tknowwhyithappened.It’snotcausedbyanyoftheusualsuspects–chromosomesthatfailedtoseparate,givinga‘trisomy’andsuchlike.Could theproblembebioenergetic? It ishard toprove thatonewayor theother,butinthisbravenewworldoffastgenomesequencing,itshouldbepossibletofindout.Theemotionaldistressofinfertilityhassanctionedsomeratherinsalubriousresearchintothefactorsthatpromoteembryogrowth.TheshockinglyclumsyexpedientofinjectingATPintoafalteringembryocanprolongitssurvival.Plainlybioenergeticfactorsmatter.Bythesametoken,perhapsthesefailuresare ‘for thebest’.Perhaps theyhadmitonuclear incompatibilities that triggeredapoptosis. It isbestnottomakeanymoraljudgementsfromevolution.IcanonlysaythatIwillnotforgetmyownyearsofsharedanguish(thankfullynowover),andI,likemostpeople,wanttoknowwhy.Isuspectthatalargenumberofearlyoccultmiscarriagesdoreflectmitonuclearincompatibilities.

But there’sanotherreasonto thinkthedeath threshold isrealandimportant.There isonefinal,indirect cost tohavingahighdeath threshold– a faster rateof ageingandagreaterpropensity tosuffer age-related diseases. That statement will raise hackles in some quarters. A high thresholdmeansahightoleranceforfree-radicalleakbeforetriggeringapoptosis.Thatmeansspecieswithalowaerobiccapacity, likerats,shouldleakmorefreeradicals.Andconversely,specieswithahighaerobiccapacity,suchaspigeons,shouldleakfewerfreeradicals. Ichoosethesespecieswithcare.Theyhavealmostequalbodymassesandbasalmetabolicrates.Onthatbasisalone,mostbiologistswouldpredictthattheyshouldhaveasimilarlifespan.YetaccordingtotheexquisiteworkofGustavoBarja inMadrid,pigeons leak fewer free radicals from theirmitochondria thando rats.8The free-radicaltheoryofageingarguesthatageingiscausedbyfree-radicalleak:thefastertherateoffree-radical leak, the faster we age. The theory has had a bad decade, but in this case makes a clearprediction–pigeonsshould livemuch longer than rats.Theydo.Arat lives threeor fouryears,apigeonfornearlythreedecades.Apigeonisassuredlynotaflyingrat.Soisthefree-radicaltheoryofageingcorrect?Initsoriginalformulation,theansweriseasy:no.ButIstillthinkamoresubtleformistrue.

Thefree-radicaltheoryofageingThefree-radicaltheoryhasitsrootsinradiationbiologyinthe1950s.Ionisingradiationsplitswatertoproducereactive‘fragments’withsingleunpairedelectrons:oxygenfreeradicals.Someofthese,like the notorious hydroxyl radical (OH•), are very reactive indeed; others, like the superoxideradical (O2

•–) are tame in comparison.The pioneers of free-radical biology –RebecaGerschman,Denham Harman and others – realised that the same free radicals can be formed from oxygendirectly,deepdowninthemitochondria,withnoneedforradiationatall.Theysawfreeradicalsasfundamentallydestructive, capableofdamagingproteins andmutatingDNA.All that is true– theycan. Worse than that, they can initiate long chain reactions, in which one molecule after another(typicallymembranelipids)grabsanelectron,wreakinghavocrightacrossthedelicatestructuresofa cell. Free radicals, the theory went, ultimately cause a crescendo of damage. Picture it. Themitochondria leak free radicals, which react with all kinds of neighbouring molecules includingnearby mitochondrial DNA. Mutations accumulate in the mitochondrial DNA, some of whichundermine its function, producing respiratory proteins that leak evenmore radicals.They damagemore proteins and DNA, and before long the rot spreads to the nucleus culminating in an ‘errorcatastrophe’.Lookatademographicchartofdiseaseandmortality,andyou’llseethattheirincidenceincreasesexponentiallyinthedecadesbetween60and100.Theideaofanerrorcatastrophe(damage

feedingonitself)seemstomatchthatgraph.Andtheideathatthewholeprocessofageingisdrivenbyoxygen,theverygasweneedtolive,holdsthehorrifyingfascinationofabeautifulkiller.

Iffreeradicalsarebad,antioxidantsaregood.Antioxidantsinterferewiththenoxiouseffectsoffreeradicals,blockingthechainreactions,andsopreventingthespreadofdamage.Iffreeradicalscause ageing, antioxidants should slow it down, delaying the onset of diseases and perhapsprolonging our lives. Some celebrated scientists, notably Linus Pauling, bought into the myth ofantioxidants,takingseveralspoonfulsofvitaminCeveryday.Hedidlivetotheripeoldageof92,but that’s still squarely in the normal range, including some people who drank and smokedthroughouttheirlives.Plainlyit’snotassimpleasthat.

This black-and-white view of free radicals and antioxidants is still current in many glossymagazines and healthfood stores, even thoughmost researchers in the field realised itwaswronglong ago.A favourite quote ofmine is fromBarryHalliwell and JohnGutteridge, authors of theclassictextbookFreeRadicalsinBiologyandMedicine:‘Bythe1990sitwasclearthatantioxidantsarenotapanaceaforageinganddisease,andonlyfringemedicinestillpeddlesthisnotion.’

Thefree-radicaltheoryofageingisoneofthosebeautifulideaskilledbyuglyfacts.Andboy,arethefactsugly.Notonetenetofthetheory,asitwasoriginallyformulated,haswithstoodthescrutinyof experimental testing. There are no systematicmeasurements of an increase in free-radical leakfrom the mitochondria as we age. There is a small increase in the number of mitochondrialmutations,butwiththeexceptionoflimitedregionsoftissue,theyaretypicallyfoundatsurprisinglylowlevels,wellbelowthoseknowntocausemitochondrialdiseases.Sometissuesshowevidenceofaccumulatingdamage,butnothingthatresemblesanerrorcatastrophe,andthechainofcausalityisquestionable.Antioxidantsmostcertainlydonotprolonglifeorpreventdisease.Quitethecontrary.Theideahasbeensopervasivethathundredsofthousandsofpatientshaveenrolledinclinicaltrialsoverthepastfewdecades.Thefindingsareclear.Takinghigh-doseantioxidantsupplementscarriesamodestbutconsistentrisk.Youaremorelikelytodieearlyifyoutakeantioxidantsupplements.Manylong-livedanimalshavelowlevelsofantioxidantenzymesintheirtissues,whileshort-livedanimalshavemuchhigher levels.Bizarrely,pro-oxidantscanactuallyextend the lifespanofanimals.Takentogether,it’snotsurprisingthatmostofthefieldofgerontologyhasmovedon.Idiscussedallthisatlengthinmyearlierbooks.I’dliketothinkIwasprescientindismissingthenotionthatantioxidantsslowageingaslongagoas2002,inOxygen,butfranklyIwasn’t.Thewritingwasonthewalleventhen. Themyth has been perpetrated by a combination ofwishful thinking, avarice, and a lack ofalternatives.

Sowhy,youmaywellwonder,doIstillthinkthatamoresubtleversionofthefree-radicaltheoryistrue?Forseveralreasons.Twocriticalfactorsweremissingfromtheoriginaltheory:signallingand apoptosis. Free-radical signals are central to cell physiology, including apoptosis, as we’vealready noted. Blocking free-radical signals with antioxidants is hazardous and can suppress ATPsynthesisincellculture,asshownbyAntonioEnriquesandhiscolleaguesinMadrid.Itseemsmostlikely that free-radical signals optimise respiration,within individualmitochondria, by raising thenumber of respiratory complexes, so increasing respiratory capacity.Becausemitochondria spendmuchoftheirtimefusingtogetherandthensplittingapartagain,makingmorecomplexes(andmorecopies of mitochondrial DNA) translates into making more mitochondria – what’s known asmitochondrial biogenesis.9 Free-radical leak can therefore increase the number of mitochondria,whichbetweenthemmakemoreATP!Conversely,blockingfreeradicalswithantioxidantspreventsmitochondrialbiogenesis,henceATPsynthesisfallsasshownbyEnriquez(Figure35).Antioxidantscanundermineenergyavailability.

Butwe’veseenthathigherratesoffree-radicalleak,abovethedeaththreshold,triggerapoptosis.Soarefreeradicalsoptimisingrespirationoreliminatingcellsbyapoptosis?Actually, that’snotas

contradictoryasitsounds.Freeradicalssignaltheproblemthatrespiratorycapacityislow,relativetodemand. If the problem can be fixed by making more respiratory complexes, raising respiratorycapacity,thenalliswellandgood.Ifthatdoesnotfixtheproblem,thecellkillsitself,removingitspresumablydefectiveDNAfromthemix.Ifthedamagedcellisreplacedbyanicenewcell(fromastemcell),thentheproblemhasbeenfixed,orrather,eradicated.

Thiscentralroleoffree-radicalsignallinginoptimisingrespirationexplainswhyantioxidantsdonotprolonglife.Theycansuppressrespirationincellculturebecausethenormalsafeguardsimposedbythebodyarenotpresentincellculture.Inthebody,massivedosesofantioxidantssuchasvitaminCarebarelyabsorbed;theytendtocausediarrhoea.Anyexcessthatdoesgetintothebloodisswiftlyexcretedintheurine.Bloodlevelsarestable.That’snottosayyoushouldavoiddietaryantioxidants,especiallyvegetablesandfruit–youneedthem.Youmightevenbenefitfromtakingsupplementaryantioxidants,ifyouhaveapoordietoravitamindeficiency.Butcrammingantioxidantsupplementsontopofabalanceddiet(whichincludespro-oxidantsaswellasantioxidants)iscounterproductive.Ifthebodyallowedhighlevelsofantioxidantsintocells,theywouldcausehavocandpotentiallykillusthroughanenergydeficiency.So thebodydoesn’t let them in.Their levelsarecarefully regulatedbothinsideandoutsidecells.

Figure35AntioxidantscanbedangerousCartoondepictingtheresultsofanexperimentusinghybridcells,orcybrids.Ineachcasethegenesinthenucleusarenearlyidentical;themaindifferenceliesinthemitochondrialDNA.TherearetwotypesofmitochondrialDNA:onefromthesamestrainofmiceasthenucleargenes(top,‘lowROS’),andtheotherfromarelatedstrainwithanumberofdifferencesinitsmitochondrialDNA(middle,‘highROS’).ROSstandsforreactiveoxygenspeciesandequatestotherateoffree-radicalleakfromthemitochondria.TherateofATPsynthesisisdepictedbythelargearrows,andisequivalentinthelowROSandhighROScybrids.However,thelowROScybridgeneratesthisATPcomfortably,withlowfree-radicalleak(denotedbylittle‘explosions’inthemitochondria)andalowcopynumberofmitochondrialDNA(squiggles).Incontrast,thehigh-ROScybridhasmorethandoubletherateoffree-radicalleak,anddoublethecopynumberofmitochondrialDNA.Free-radicalleakappearstopower-uprespiration.Thatinterpretationissupportedbythebottompanel:antioxidantslowertherateoffree-radicalleak,butalsoreducethecopynumberofmitochondrialDNAand,critically,therateofATPsynthesis.Soantioxidantsdisruptthefree-radicalsignalthatoptimisesrespiration.

And apoptosis, by eradicating damaged cells, eliminates the evidence of damage. Thecombinationof free-radicalsignalsandapoptosis togetherconfoundmostof thepredictionsof theoriginalfree-radicaltheoryofageing,whichwasformulatedlongbeforeeitherprocesswasknown.Wedon’tseeasustainedincreaseinfree-radicalleak,oralargenumberofmitochondrialmutations,oranaccumulationofoxidativedamage,oranybenefittoantioxidants,oranerrorcatastrophe,forthesereasons.Itallmakesperfectlyreasonablesense,explainingwhythepredictionsoftheoriginalfree-radical theoryofageingaremostlywrong.But itdoesn’tgiveanyindicationofwhythefree-radical theorymight still be right.Why, if they are so well regulated and beneficial, should freeradicalshaveanythingtodowithageingatall?

Well,theycanexplainthevarianceinlifespanbetweenspecies.We’veknownsincethe1920sthatlifespantendstovarywithmetabolicrate.TheeccentricbiometricianRaymondPearlentitledanearlyarticle on the subject, ‘Why lazy people live longer ’. They don’t; quite the opposite. But thiswasPearl’s introduction to his famous ‘rate-of-living theory’, which does have some basis of truth.Animalswithalowmetabolicrate(oftenlargespeciessuchaselephants)typicallylivelongerthananimalswithahighmetabolicrate,likemiceandrats.10Theruleusuallyholdswithinmajorgroupssuchasreptiles,mammalsandbirds,butdoesn’tholdatallwellbetweenthesegroups;hencetheideahasbeensomewhatdiscredited,orat least ignored.But in fact there isasimpleexplanation,whichwe’vealreadynoted–free-radicalleak.

As originally conceived, the free-radical theory of ageing envisioned free radicals as anunavoidable by-product of respiration; about 1–5% of oxygen was thought to be convertedunavoidablyintofreeradicals.Butthat’swrongontwocounts.First,alltheclassicalmeasurementsweremadeoncellsortissuesexposedtoatmosphericlevelsofoxygen,muchgreaterthananythingthatcellsareexposedtowithinthebody.Theactualratesofleakmaybeordersofmagnitudelower.Wejustdon’tknowhowbigadifferencethismakesintermsofmeaningfuloutcomes.Andsecond,free-radical leak isnot anunavoidableby-productof respiration– it is adeliberate signal, and theleakratevariesenormouslybetweenspecies,tissues,timesoftheday,hormonalstatus,calorieintake,andexercise.Whenyouexercise,youconsumemoreoxygen,soyourfree-radicalleakrises,right?Wrong.Itremainssimilarorevengoesdown,becausetheproportionofradicalsleakedrelativetooxygen consumed falls off considerably. That happens because electron flux speeds up in therespiratory chains, meaning that the respiratory complexes become less reduced, and so are lesslikelytoreactdirectlywithoxygen(Figure36).Thedetailsdon’tmatterhere.Thepointisthatthereisnosimplerelationshipbetweentherateoflivingandfree-radicalleak.We’venotedthatbirdslivefarlongerthantheyreally‘oughtto’onthebasisoftheirmetabolicrate.Theyhaveafastmetabolicrate,butleakrelativelyfewfreeradicals,andlivealongtime.Theunderlyingcorrelationisbetweenfree-radicalleakandlifespan.Correlationsarenotoriousasaguidetocausalrelationship,butthisisanimpressiveone.Coulditbecausal?

Figure36WhyrestisbadforyouThetraditionalviewofthefree-radicaltheoryofageingisthatasmallproportionofelectrons‘leak’outfromtherespiratorychainduringrespirationtoreactdirectlywithoxygenandformfreeradicalssuchasthesuperoxideradical(O2

•–).Becauseelectronsflowfasterandweconsumemoreoxygeninactiveexercise,theassumptionhasbeenthatfree-radicalleakincreasesduringexercise,eveniftheproportionofelectronsleakingoutremainsconstant.Thatisnotso.Theupperpanelhereindicatestheactualsituationduringexercise:electronflowdowntherespiratorychainisfastbecauseATPisconsumedquickly.ThatallowsprotonstofluxthroughtheATPsynthase,whichlowersmembranepotential,whichallowstherespiratorychaintopumpmoreprotons,whichdrawselectronsfasterdowntherespiratorychaintooxygen,whichpreventstheaccumulationofelectronsinrespiratorycomplexes,loweringtheirreductionstate(representedbythepalegreyshading).Thatmeansfree-radicalleakismodestduringexercise.Theoppositeistrueatrest(lowerpanel),meaningtherecanbehigherratesoffree-radicalleakduringinactivity.LowconsumptionofATPmeansthereisahighmembranepotential,itbecomeshardtopumpprotons,sotherespiratorycomplexesgraduallyfillupwithelectrons(darkergreyshading)andleakmorefreeradicals.Bestgoforarun.

Considertheconsequencesoffree-radicalsignallinginthemitochondria:optimisingrespirationandeliminatingdysfunctionalmitochondria.Themitochondria that leak themost free radicalswillmake themost copies of themselves, precisely because free-radical signals correct the respiratorydeficitbyincreasingcapacity.Butwhatiftherespiratorydeficitdidnotreflectashiftinsupplyanddemand,butratheranincompatibilitywiththenucleus?Somemitochondrialmutationsdooccurwithageing, giving rise to amixture of differentmitochondrial types, someofwhichwork better thanothers with the genes in the nucleus. Think about the problem here. The most incompatiblemitochondriawilltendtoleakthemostfreeradicals,andsomakemorecopiesofthemselves.Thatcan have one of two effects. Either the cell dies by apoptosis, removing its load ofmitochondrialmutations,or itdoesn’t.Let’s considerwhathappens if thecelldies first.Either it’s replaced,or itisn’t.Ifitisreplaced,alliswell.Butifit’snotreplaced,forexampleinbrainorheartmuscle,thenthe

tissuewillslowlylosemass.Fewercellsremaintodothesamejob,sotheyareplacedundergreaterpressure.Theybecomephysiologicallystressed,withtheactivityofthousandsofgeneschanging,asinthetestesofthosefruitflieswithmitonuclearincompatibilities.Atnostageinthisprocessdidfree-radicalleaknecessarilydamageproteinsorcauseanerrorcatastrophe.Everythingisdrivenbysubtlefree-radicalsignalswithinthemitochondria,buttheoutcomeistissueloss,physiologicalstress,andchangesingeneregulation–allchangesassociatedwithageing.

Whathappenswhenthecelldoesn’tdiebyapoptosis?Ifitsenergyneedsarelow,theycanbemetbydeficientmitochondriaorfermentation toproduce lacticacid(what isoften,erroneously,calledanaerobic respiration).Herewemay see the accumulationofmitochondrialmutations in cells thatbecome‘senescent’.Theseno longergrow,butcanbeanangrypresence in tissues,beingstressedthemselves,andoftenprovokechronic inflammationand thedysregulationofgrowth factors.Thatstimulatescellsthatliketogrowanyway,stemcells,vascularcells,andsoon,goadingthemtogrowwhen theyhadbetternot. Ifyou’reunlucky theywilldevelop intocancer,anage-relateddisease inmostcases.

It’s worth emphasising again that this whole process is driven by energetic deficiencies thatultimatelyderivefromfree-radicalsignalswithinthemitochondria.Incompatibilitiesthataccumulatewithageunderminemitochondrialperformance.Thisistotallydifferentfromtheconventionalfree-radical theory, as it does not call upon oxidative damage in the mitochondria or anywhere else(thoughofcourse thatdoesn’t rule itout; it issimplynotnecessary).Aswe’venoted,becausefreeradicalsactassignalsthatincreaseATPsynthesis,thepredictionisthatantioxidantsshouldnotwork:theywill not prolong lifespan, nor protect against disease, because theywould undermine energyavailability if they did ever gain access to mitochondria. This view can also account for theexponentialincreaseindiseaseandmortalitywithage:tissuefunctionmaydeclinegentlyovermanydecades, eventually falling below the threshold required for normal function.Webecome less andless able to cope with exertions, and ultimately not even passive existence. This process isrecapitulated in everyone, over our dying decades, giving rise to the exponential decline onmorticians’graphs.

Sowhatcanwedoaboutageing? I said thatRaymondPearlwaswrong: lazypeopledon’t livelonger–exerciseisbeneficial.So,withinlimits,iscalorierestrictionandlow-carbohydratediet.Allpromoteaphysiologicalstressresponse(asdopro-oxidants)whichtendstoclearoutdefectivecellsand badmitochondria, promoting survival in the short term, but typically at the cost of loweringfertility.11 Again we see the link between aerobic capacity, fertility and longevity. But there isinevitablyalimittowhatcanbeachievedbymodulatingourownphysiology.Wehaveamaximumlifespansetbyourownevolutionaryhistory,whichultimatelydependsonthecomplexityofsynapticconnectionsinourbrains,andthesizeofstemcellpopulationsinothertissues.HenryFordissaidtohave visited a scrap heap to determine which parts of derelict Fords were still operational, theninsisting that, in newmodels, these pointlessly long-lasting parts should be replacedwith cheaperversionstosavemoney.Likewise,inevolution,thereisnopointinmaintainingalargeanddynamicpopulationof stemcells in the stomach lining if they areneverused,becauseourbrainswearoutfirst.Intheend,weareoptimisedbyevolutiontoourexpectedlifespan.Idoubtwewilleverfindawayoflivingmuchbeyond120bymerelyfine-tuningourphysiology.

Butevolutionisadifferentmatter.Thinkbacktothevariablethresholdofdeath.Specieswithhighaerobic requirements, likebats andbirds,havea low threshold: evenmodest free-radical leakwilltriggerapoptosisduringembryonicdevelopment,andonlytheoffspringwithlowleakwilldeveloptofullterm.Thislowrateoffree-radicalleakcorrespondstoalonglifespan,forthereasonswe’vejustdiscussed.Conversely,animalswith lowaerobicrequirements–mice, rats,andsoon–haveahigherdeaththreshold, toleratehigherlevelsoffree-radical leak,andultimatelyhaveshorter lives.

Thereisastraightforwardpredictionhere:selectionforgreateraerobiccapacity,overgenerations,shouldprolonglifespan.Andsoitdoes.Rats,forexample,canbeselectedfortheircapacitytorunonatreadmill.If thehighest-capacityrunnersineachgenerationarematedamongthemselves,andthesame is done with the lowest-capacity runners, lifespan increases in the high-capacity group anddecreases in the low-capacitygroup.Over tengenerations, thehigh-capacity runners increase theiraerobiccapacityby350%relativetothelow-capacityrunners,andlivenearlyayearlonger(alargedifference,giventhatratsnormally liveforabout threeyears). Iwouldarguethatsimilarselectiontookplaceduring theevolutionofbats andbirds, and indeedendotherms (warm-bloodedanimals)moregenerally,ultimatelyincreasingtheirlifespanbyanorderofmagnitude.12

Wemaynotwishtoselectourselvesonsuchabasis;thatsmacksfartoomuchofeugenics.Evenifitactuallyworked,suchsocialengineeringwouldproducemoreproblemsthanitsolves.Butinfactwemightalreadyhavedoneso.Wedohaveahighaerobiccapacityrelativetoothergreatapes.Wedolivea lot longer than them–we livenearly twiceas longaschimpsandgorillas,whichhaveasimilar metabolic rate. Perhaps we owe that to our formative years as a species, chasing gazelleacrosstheAfricansavannah.Youmightnotgainenormouspleasurefromendurancerunning,butitsculpted us as a species. There’s no gain without pain. From a simple consideration of therequirements for two genomes,we can predict that our ancestors increased their aerobic capacity,decreased their free-radical leak, gave themselves a problem with fertility, and increased theirlifespan.Howmuchtruthisthereinallthat?Thisisatestablehypothesisthatmightprovewrong.Butitemergesinexorablyfromtherequirementformosaicmitochondria,apredictionthatinturnrestson the origin of the eukaryotic cell, which on one singular occasion, nearly 2 billion years ago,overcame the energetic constraints that keepbacteriabacterial.Nowonder the sun settingover theplainsofAfricastillholdssuchastrongemotionalresonance.Ittiesusbyawonderful,ifcontorted,trainofcausalityrightbacktotheveryoriginsoflifeonourplanet.

Footnotes1Cefalùcathedralwasbegunin1131,40yearsaftertheNormanscompletedtheirconquestofSicilyin1091(acampaignthathadextendedover30years,beginningin1061,beforetheirmorecelebratedconquestofEngland).ThecathedralwasbuiltinthanksgivingafterKingRogerIIsurvivedshipwreckoffthecoast.ThewonderfulchurchesandpalacesofNormanSicilycombinearchetypalNormanarchitecturewithByzantinemosaicsandArabiccupolas.ThePantocratoratCefalùwasproducedbyByzantinecraftsmen,andsomesayisevenfinerthanthefamousPantocratorintheHagiaSofia,inwhatwasthenConstantinople.Eitherway,it’swellworthavisit.2Mostfree-radicalleakactuallyderivesfromcomplexI.ThespacingbetweentheredoxcentresincomplexIsuggeststhatthisisdeliberate.Recalltheprincipleofquantumtunnelling:electrons‘hop’fromonecentretoanother,withaprobabilitythatdependsonthedistance,theoccupancyandthe‘tug’ofoxygen(thereductionpotential).WithincomplexIthereisanearlybranchinthepathofelectronflux.Inthemainpathway,mostcentresarespacedwithinabout11Åofeachother,henceelectronswillusuallyhoprapidlyfromonetothenext.Thealternativepathwayisacul-de-sac–electronscanenter,butcan’teasilyleaveagain.Atthebranchpoint,electronshavea‘choice’:itisabout8Åtothenextredoxcentreinthemainpath,and12Åtothealternativecentre(Figure8).Undernormalcircumstances,electronswillfluxdownthemainpath.Butifthatpathwayclogsupwithelectrons–ifitishighlyreduced–thealternativecentrenowaccumulateselectrons.Thisalternativecentreisperipheralandeasilyreactswithoxygentoproducesuperoxideradicals.MeasurementsshowthatthisFeSclusteristhemainsourceoffree-radicalleakfromtherespiratorychain.Iviewthisasamechanismtopromotefree-radicalleakasa‘smokesignal’ifelectronfluxistooslowtomeetdemand.3Mittwochpointsoutaparallelproblemrelatingtotruehermaphrodites–peoplewhoarebornwithbothtypesofsexorgan,forexampleatestisontheright-handsideandanovaryontheleft.It’sfarmorelikelytobethatwayround.Barelyathirdofpeoplewithtruehermaphroditismhavethetestisontheleft-handsideandtheovaryontheright.Thedifferencecanhardlybegenetic.Mittwochshowsthatatcriticalperiods,theright-handsidegrowsslightlyfasterthantheleft,andsoismorelikelytodevelopmaleness.Curiously,inmiceitisexactlytheotherwayaround–theleft-handsidegrowsslightlyfasterandismorelikelytodeveloptestes.4Themitochondriapassdownthefemaleline,intheeggcells,notsperm.Hermaphroditesaretheoreticallyparticularlyvulnerabletosexdistortionbymitochondria.Fromtheirpointofview,themaleisageneticdeadend–thelastplacethemitochondria‘want’toendupisintheanthers.Itisthereforeintheirintereststosterilisethemalesexorgans,toensuretheirpassageinafemaleplant.Manybacterialparasitesininsects,notablyBuchneraandWolbachia,playasimilargame–theycancompletelydistortsexratiosininsectsbyselectivelykillingmales.Thecentralimportanceofmitochondriatothehostorganismmeansthattheyhavelessscopethanbacterialparasitestokillmalesthroughsuchselfishconflict,buttheymightnonethelesscausesterilityorselectivedamagetomales.However,

I’minclinedtothinkconflictplaysalesserroleinHaldane’srule,asitcannotexplainwhyfemalesshouldbeworseaffectedinbirds(andflourbeetles).5Suchcybridsarewidelyusedincellcultureexperiments,astheyallowprecisemeasurementstobemadeofcellfunction,notablyrespiration.Mismatchingmitochondrialandnucleargenesbetweenspeciesreducestherateofrespiration,andasnoted,increasesfree-radicalleak.Themagnitudeoffunctionaldeficitdependsongeneticdistance.CybridsconstructedfromchimpmitochondrialDNAandhumannucleargenes(yes,ithasbeendone,butonlyincellculture)showthattherateofATPsynthesisisabouthalfthatofnormalcells.Cybridsbetweenmiceandratsdon’thavefunctionalrespirationatall.6Thisconjecturemightseemalittleodd:dothetestesreallyhaveahighermetabolicratethanothertissuessuchastheheart,brainorflightmuscle?Notnecessarily.Theproblemisthecapacitytomeetdemand.Itmightbethatpeakdemandisindeedgreaterintestes,orthatthenumberofmitochondriathatarecalledupontomeetthatdemandislower,sothatthedemandpermitochondrionisgreater.Thisisasimpletestableprediction,buttomyknowledgeithasnotbeentested.7Isuspectthatthefree-radicalsignalisdeliberatelyamplifiedatsomepointduringembryonicdevelopment.Forexample,thegasnitricoxide(NO)canbindtocytochromeoxidase,thefinalcomplexoftherespiratorychain,increasingfree-radicalleakandthelikelihoodofapoptosis.IfNOwereproducedinlargeramountsatsomepointduringdevelopment,theeffectwouldbetoamplifythesignalaboveathreshold,terminatingembryoswithincompatiblegenomes–acheckpoint.8GustavoBarjahasfoundthattherateoffree-radicalleakisupto10-foldlowerinbirdssuchaspigeonsandbudgerigarsthaninratsandmiceasaproportionofoxygenconsumed.Theactualratesvarybetweentissues.Barjaalsofoundthatthelipidmembranesofbirdsaremoreresistanttooxidativedamagethanthoseofflightlessmammals,andthisresistanceisreflectedinlessoxidativedamagetoDNAandproteins.Altogether,it’shardtointerpretBarja’sworkinanyotherterms.9Icallthis‘reactivebiogenesis’–individualmitochondriareacttoalocalfree-radicalsignal,whichindicatesthatrespiratorycapacityistoolowtomeetdemand.Therespiratorychainbecomeshighlyreduced(cloggedupwithelectrons).Electronscanescapetoreactdirectlywithoxygentoproducesuperoxideradicals.Theseinteractwithproteinsinthemitochondriathatcontrolthatthereplicationandcopyingofmitochondrialgenes,calledtranscriptionfactors.Sometranscriptionfactorsare‘redoxsensitive’,meaningthattheycontainaminoacids(suchascysteine)thatcanloseorgainelectrons,becomingoxidisedorreduced.Agoodexampleismitochondrialtopoisomerase-1,whichcontrolsaccessofproteinstomitochondrialDNA.Theoxidationofacriticalcysteineonthisproteinincreasesmitochondrialbiogenesis.Thusalocalfree-radicalsignal(thatneverleavesthemitochondria)increasesmitochondrialcapacity,raisingATPproductioninrelationtodemand.Thiskindoflocalsignalinresponsetosuddenchangesindemandmayexplainwhymitochondriahaveretainedasmallgenome(seeChapter5).10Thislookslikeacontradiction–largerspeciestypicallyhavealowermetabolicrate,grampergram,yetIhavetalkedaboutmalemammalsbeinglargerandhavingahighermetabolicrate,theopposite.Withinaspeciesthedifferencesinmassaretrivialcomparedwiththemanyordersofmagnitudeplottedoutbetweenspecies;onthatscalethemetabolicratesofadultsinthesamespeciesarepracticallythesame(thoughchildrendohaveahighermetabolicratethanadults).ThesexualdifferencesinmetabolicratethatIwastalkingaboutearlierrelatetodifferencesinabsolutegrowthratesatparticularstagesofdevelopment.IfUrsulaMittwochiscorrect,thesedifferencesaresosubtlethattheycanaccountfordevelopmentaldifferencesontherightversustheleftsideofthebody;seefootnote3.11Andworse.Thebestwaytoclearoutbadmitochondriaistoforcethebodytousethem,increasingtheirturnoverrate.Forexample,ahigh-fatdiettendstoforcetheuseofmitochondria,whereasahigh-carbohydratedietallowsustoprovidemoreenergybyfermentation,withoutusingourmitochondriaasheavily.Butifyouhaveamitochondrialdisease(andwealldevelopfaultymitochondriawithage)thentheswitchcanbetoomuch.Somepatientswithmitochondrialdiseasewhohaveadopteda‘ketogenicdiet’havecollapsedintoacomabecausetheirdamagedmitochondriacan’tprovidetheenergyneededfornormallifewithouthelpfromfermentation.12IdiscusstheinterplaybetweenaerobiccapacityandtheevolutionofendothermyinsomedetailinPower,Sex,SuicideandLifeAscending.Icanonlyshamelesslyrecommendthem,ifyouwanttoknowmoreaboutthat.

M

EPILOGUE:FROMTHEDEEP

ore than 1,200 metres deep in the Pacific Ocean off the coast of Japan lies an underwatervolcanonamedMyojinKnoll.AteamofJapanesebiologistshavebeentrawlingthesewaters

formorethanadecade,searchingforinterestinglifeforms.Bytheirownaccount, theydidn’tfindanythingterriblysurprisinguntilMay2010,whentheycollectedsomepolychaetewormsclingingtoahydrothermalvent.Itwasn’tthewormsthatwereinterestingbutthemicrobesassociatedwiththem.Well,oneofthemicrobes–onecell thatlookedalotlikeaeukaryote,until theylookedatitmoreclosely(Figure37).Thenitbecamethemostteasingenigma.

Eukaryote means ‘true nucleus’, and this cell has a structure that on first glance looks like anormalnucleus.Italsohasotherconvolutedinternalmembranes,andsomeendosymbiontsthatcouldbehydrogenosomes,derivedfrommitochondria.Likeeukaryoticfungiandalgae,ithasacellwall;and not surprisingly, for a specimen from the deep black ocean, it lacks chloroplasts. The cell ismodestlylarge,around10micrometresinlengthand3micrometresindiameter,givingitavolumeabout100timeslargerthanatypicalbacteriumsuchasE.coli.Thenucleusislarge,takingupnearlyhalfof thevolumeof thecell.Onaquickglance, then, thiscell isn’teasy toclassify intoaknowngroup,but it isplainlyeukaryotic.It’sonlyamatterof timeandgenesequencing,youmight think,beforeitissafelyassignedtoitsproperhomeinthetreeoflife.

Figure37auniquemicroorganismfromthedeepseaIsthisaprokaryoteoraeukaryote?Ithasacellwall(CW),plasmamembrane(PM)andanucleus(N)surroundedbyanuclearmembrane(NM).Italsohasseveralendosymbionts(E)whichlookabitlikehydrogenosomes.It’squitebig,about10micrometresinlength,andthenucleusislarge,takingupnearly40%ofthecellvolume.Plainlyaeukaryote,then.Butno!Thenuclearmembraneisasinglelayer,notadoublemembrane.Therearenonuclearporecomplexes,justoccasionalgaps.Thereareribosomesinthenucleus(mottledgreyregions)andoutsidethenucleus.Thenuclearmembraneiscontinuouswithothermembranesandeventheplasmamembrane.TheDNAisintheformofthinfilaments,2nanometresindiameterasinbacteria,noteukaryoticchromosomes.Plainlynotaeukaryote,then.Isuspectthisenigmaisactuallyaprokaryotethatacquiredbacterialendosymbionts,andisnowrecapitulatingeukaryoticevolution,becominglarger,swellingitsgenome,accumulatingtherawmaterialforcomplexity.Butthisistheonlysample,andwithoutagenomesequencewemayneverknow.

Oh, but look again!All eukaryotes have a nucleus, true, but in all known cases that nucleus issimilar in its structure. It has a doubled membrane, continuous with other cellular membranes, anucleolus, where ribosomal RNA is synthesised, elaborate nuclear pore complexes, and an elasticlamina; and the DNA is carefully packaged in proteins, forming chromosomes – relatively thick

chromatinfibres,30nanometresindiameter.AswesawinChapter6,proteinsynthesistakesplaceonribosomesthatarealwaysexcludedfromthenucleus.Thisistheverybasisofthedistinctionbetweenthe nucleus and cytoplasm. So what about the cell from Myojin Knoll? It has a single nuclearmembrane,witha fewgaps.Nonuclearpores.TheDNAiscomposedof fine fibresas inbacteria,about 2 nanometres in diameter, not thick eukaryotic chromosomes. There are ribosomes in thenucleus.Ribosomesinthenucleus!Andribosomesoutsidethenucleustoo.Thenuclearmembraneiscontinuouswiththecellmembraneinseveralplaces.Theendosymbiontscouldbehydrogenosomes,butsomeofthemhaveabacterialcorkscrewmorphologyon3Dreconstruction.Theylookmorelikerelativelyrecentbacterialacquisitions.Whileithasinternalmembranesthereisnothingresemblinganendoplasmicreticulum,ortheGolgiapparatus,oracytoskeleton,allclassiceukaryotic traits.Inother words, this cell is actually nothing like a modern eukaryote. It just bears a superficialresemblance.

Sowhatis it, then?Theauthorsdidn’tknow.TheynamedthebeastParakaryonmyojinensis, thenewterm‘parakaryote’signifyingitsintermediatemorphology.Theirpaper,publishedintheJournalof Electron Microscopy, had one of the most tantalising titles I’ve ever seen: ‘Prokaryote oreukaryote?Auniquemicroorganismfromthedeepsea.’Havingsetup thequestionbeautifully, thepapergoesnowhereatallinansweringit.Agenomesequence,orevenaribosomalRNAsignature,wouldgivesomeinsightintothetrueidentityofthecell,andturnedthislargelyoverlookedscientificfootnoteintoahigh-impactNaturepaper.Buttheyhadsectionedtheironlysample.Alltheycansayfor sure is that in15yearsand10,000electronmicroscopysections, theyhadnever seenanythingremotelysimilarbefore.Theyhaven’tseenanythingsimilarsince,either.Neitherhasanyoneelse.

Sowhatisit,then?Theunusualtraitscouldbeanartefactofpreparation–apossibilitythatisnottobediscounted,giventhetroubledhistoryofelectronmicroscopy.Ontheotherhand,ifthetraitsarejustanartefact,whywasthissampleauniqueoddity?Andwhydothestructureslooksoreasonableinthemselves? I’d hazard it’s not an artefact.That leaves three conceivable alternatives. It could be ahighlyderivedeukaryote,whichchanged itsnormal structuresas it adapted toanunusual lifestyle,clingingto thebackofadeep-seawormonahydrothermalvent.But thatseemsunlikely.Plentyofothercellsliveinsimilarcircumstances,andtheyhavenotfollowedsuit.Ingeneral,highlyderivedeukaryotes losearchetypaleukaryotic traits,but those that remainarestill recognisablyeukaryotic.That’strueofallthearchezoa,forexample,thosepurportedlylivingfossilsthatwereoncethoughttobeprimitive intermediatesbuteventually turnedout tobederivedfromfully fledgedeukaryotes. IfParakaryonmyojinensisreallyisahighlyderivedeukaryote,thenit’sradicallydifferentinitsbasicplantoanythingwe’veseenbefore.Idon’tthinkthat’swhatitis.

Alternatively, it could be a real living fossil, a ‘genuine archezoan’ that somehow clung toexistence, failing to evolve the modern range of eukaryotic accessories in the unchanging deepoceans.Thisexplanationisfavouredbytheauthorsofthepaper,butIdon’tbelievethateither.Itisnotliving in an unchanging environment: it is attached to the back of a polychaeteworm, a complexmulticellular eukaryote that obviously did not exist in the early evolution of eukaryotes. The lowpopulationdensity–justasinglecelldiscoveredaftermanyyearsoftrawling–alsomakesmedoubtthatitcouldhavesurvivedunchangedfornearly2billionyears.Smallpopulationsarehighlyproneto extinction. If the population expands, fine; but if not, it’s only amatter of time before randomstatistical chance pushes it into oblivion. Two billion years is a very long time – about 30 timeslongerthantheperiodcoelacanthsarethoughttohavesurvivedaslivingfossilsinthedeepoceans.Anygenuinesurvivorsfromtheearlydaysoftheeukaryoteswouldhavetobeatleastaspopulousastherealarchezoatosurvivethatlong.

That leaves the finalpossibility.AsSherlockHolmes remarked, ‘Whenyouhaveeliminatedallwhichisimpossible,thenwhateverremains,howeverimprobable,mustbethetruth.’Whiletheother

twooptionsarebynomeans impossible, this third ismuchthemost interesting: it isaprokaryote,whichhasacquiredendosymbionts,andischangingintoacell thatresemblesaeukaryote, insomekind of evolutionary recapitulation. To my mind, that makes much more sense. It immediatelyexplainswhythepopulationdensity is low;aswe’veseen,endosymbiosesbetweenprokaryotesarerareandarebesetbylogisticaldifficulties.1It’snotatalleasytoreconcileselectionactingatthelevelof the host cell and the endosymbiont in a ‘virgin’ endosymbiosis between prokaryotes. Themostlikelyfate for thiscell isextinction.Anendosymbiosisbetweenprokaryotesalsoexplainswhy thiscellhasvarioustraitsthatlookeukaryotic,butoncloserinspectionarenot.Itisrelativelylarge,withagenomethatlookssubstantiallylargerthananyotherprokaryote,housedina‘nucleus’continuouswith internalmembranes,andsoon.Theseareall traits thatwepredictedwouldevolve, fromfirstprinciples,inprokaryoteswithendosymbionts.

Iwouldwagerasmallbetthattheseendosymbiontshavealreadylostalargepartoftheirgenome,asIhavearguedthatonlytheprocessofendosymbioticgenelosscansupport theexpansionofthehost-cell genomeup to eukaryotic levels.That seems to be happening here: an equivalent extremegenomicasymmetryissupportinganindependentoriginofmorphologicalcomplexity.Certainlythehost-cellgenomeislarge,occupyingmorethanathirdofacellthatisalready100timeslargerthanE.coli.Thisgenomeishousedinastructurethatlookssuperficiallymuchlikeanucleus.Oddly,onlysomeoftheribosomesareexcludedfromthisstructure.Doesthatmeanthattheintronhypothesisiswrong?It’shardtosay,asthehostcellherecouldbeabacterium,notanarchaeon,andsocouldbelessvulnerable to the transferofbacterialmobile introns.The fact that anuclearcompartmenthasevolvedindependentlywouldtendtosuggestthatsimilarforcesareoperatinghere,andbythesametokenwould tend to operate in large cellswith endosymbionts.What about other eukaryotic traitssuchassex,andmatingtypes?Wesimplycan’tsay,withoutagenomesequence.AsInoted,thisreallyisthemostteasingenigma.We’lljusthavetowaitandsee;that’spartandparcelofthenever-endinguncertaintyofscience.

Thiswholebookhasbeenanattempttopredictwhylifeisthewayitis.Toafirstapproximation,it looks as ifParakaryonmyojinensismight be recapitulating a parallel pathway towards complexlife, from bacterial ancestors. Whether that same pathway is followed elsewhere in the universehingesonthestartingpoint–theoriginoflifeitself.Ihavearguedthatthisstartingpointmightwellberecapitulatedtoo.

All life on earth is chemiosmotic, depending on proton gradients across membranes to drivecarbon and energy metabolism. We have explored the possible origins and consequences of thispeculiar trait. We’ve seen that living requires a continuous driving force, an unceasing chemicalreaction that produces reactive intermediates, including molecules like ATP, as byproducts. Suchmoleculesdrivetheenergy-demandingreactionsthatmakeupcells.Thisfluxofcarbonandenergymusthavebeenevengreaterattheoriginsoflife,beforetheevolutionofbiologicalcatalysts,whichconstrainedtheflowofmetabolismwithinnarrowchannels.Veryfewnaturalenvironmentsmeettherequirementsforlife–acontinuous,highfluxofcarbonandusableenergyacrossmineralcatalysts,constrained in a naturally microcompartmentalised system, capable of concentrating products andventingwaste.Whiletheremaybeotherenvironmentsthatmeetthesecriteria,alkalinehydrothermalventsmost certainly do, and such vents are likely to be common onwet rocky planets across theuniverse.Theshoppinglistforlifeintheseventsis justrock(olivine),waterandCO2, threeof themostubiquitoussubstancesintheuniverse.Suitableconditionsfortheoriginoflifemightbepresent,rightnow,onsome40billionplanetsintheMilkyWayalone.2

Alkalinehydrothermalventscomewithbothaproblemandasolution:theyarerichinH2,butthisgas does not react readily with CO2. We have seen that natural proton gradients across thin

semiconductingmineralbarrierscouldtheoreticallydrivetheformationoforganics,andultimatelytheemergenceofcells,withintheporesofthevents.Ifso,lifedependedfromtheverybeginningonprotongradients(andiron–sulphurminerals)tobreakdownthekineticbarrierstothereactionofH2

andCO2.Togrowonnaturalprotongradients,theseearlycellsrequiredleakymembranes,capableofretainingthemoleculesneededforlifewithoutcuttingthemselvesofffromtheenergisingfluxofprotons.That,inturn,precludedtheirescapefromthevents,exceptthroughthestraitgatesofastrictsuccessionofevents(requiringanantiporter),whichenabledthecoevolutionofactiveionpumpsandmodernphospholipidmembranes.Onlythencouldcellsleavethevents,andcolonisetheoceansandrocks of the early earth.We saw that this strict successionof events could explain the paradoxicalpropertiesofLUCA, the lastuniversalcommonancestorof life, aswellas thedeepdivergenceofbacteria and archaea. Not least, these strict requirements can explain why all life on earth ischemiosmotic–whythisstrangetraitisasuniversalasthegeneticcodeitself.

Thisscenario–anenvironmentthatiscommonincosmicterms,butwithastrictsetofconstraintsgoverningoutcomes–makesitlikelythatlifeelsewhereintheuniversewillalsobechemiosmotic,andsowill faceparallelopportunitiesandconstraints.Chemiosmoticcouplinggives lifeunlimitedmetabolicversatility,allowingcells to ‘eat’and ‘breathe’practicallyanything. Justasgenescanbepassedaroundby lateralgene transfer,because thegeneticcode isuniversal, so too the toolkit formetabolicadaptationtoverydiverseenvironmentscanbepassedaround,asallcellsuseacommonoperatingsystem.Iwouldbeamazedifwedidnotfindbacteriarightacrosstheuniverse,includingourownsolarsystem,allworkinginmuchthesameway,poweredbyredoxchemistryandprotongradientsacrossmembranes.It’spredictablefromfirstprinciples.

Butifthat’strue,thencomplexlifeelsewhereintheuniversewillfaceexactlythesameconstraintsaseukaryotesonearth–aliensshouldhavemitochondriatoo.We’veseenthatalleukaryotesshareacommon ancestor which arose just once, through a rare endosymbiosis between prokaryotes.Weknow of two such endosymbioses between bacteria (Figure25) – three, ifwe includeParakaryonmyojinensis–soweknowthatitispossibleforbacteriatogetinsidebacteriawithoutphagocytosis.Presumably there must have been thousands, perhaps millions, of cases over 4 billion years ofevolution. It’s abottleneck,butnot a stringentone. In eachcase,wewouldexpect to seegene lossfrom theendosymbionts, anda tendency togreater sizeandgenomiccomplexity in thehost cell–exactlywhatwedoseeinParakaryonmyojinensis.Butwe’dalsoexpectintimateconflictbetweenthehostandtheendosymbiont–thisisthesecondpartofthebottleneck,adoublewhammythatmakestheevolutionof complex life genuinelydifficult.We saw that the first eukaryotesmost likely evolvedquicklyinsmallpopulations; theveryfact that thecommonancestorofeukaryotessharessomanytraits,noneofwhicharefoundinbacteria,impliesasmall,unstable,sexualpopulation.IfParakaryonmyojinensisisrecapitulatingeukaryoticevolution,asIsuspect,itsextremelylowpopulationdensity(justonespecimenin15yearsofhunting)ispredictable.Itsmostlikelyfateisextinction.Perhapsitwilldiebecauseithasnotsuccessfullyexcludedallitsribosomesfromitsnuclearcompartment,orbecause it has not yet ‘invented’ sex.Or perhaps, chance in amillion, it will succeed, and seed asecondcomingofeukaryotesonearth.

I thinkwe can reasonably conclude that complex lifewill be rare in theuniverse – there is noinnatetendencyinnaturalselectiontogiverisetohumansoranyotherformofcomplexlife.Itisfarmorelikelytogetstuckatthebacteriallevelofcomplexity.Ican’tputastatisticalprobabilityonthat.The existence of Parakaryon myojinensis might be encouraging for some – multiple origins ofcomplexity on earth means that complex life might be more common elsewhere in the universe.Maybe. What I would argue with more certainty is that, for energetic reasons, the evolution ofcomplex life requiresanendosymbiosisbetween twoprokaryotes,and that isa rare randomevent,disturbingly close to a freak accident,made all themore difficult by the ensuing intimate conflict

betweencells.Afterthat,wearebacktostandardnaturalselection.We’veseenthatmanypropertiessharedbyeukaryotes,fromthenucleustosex,arepredictablefromfirstprinciples.Wecangomuchfurther.Theevolutionoftwosexes,thegermline–somadistinction,programmedcelldeath,mosaicmitochondria,andthetrade-offsbetweenaerobicfitnessandfertility,adaptabilityanddisease,ageinganddeath,allthesetraitsemerge,predictably,fromthestartingpointthatisacellwithinacell.Woulditallhappenoveragain?I thinkthatmuchof itwould.Incorporatingenergyintoevolutionis longoverdue,andbeginstolayamorepredictivebasistonaturalselection.

Energy is far less forgiving than genes. Look around you. This wonderful world reflects thepowerofmutations and recombination,genetic change– thebasis fornatural selection.You sharesomeofyourgeneswiththetreethroughthewindow,butyouandthattreepartedcompanyveryearlyin eukaryotic evolution, 1.5 billion years ago, each following a different course permitted bydifferentgenes,theproductofmutations,recombination,andnaturalselection.Yourunaround,andIhopestillclimbtreesoccasionally;theybendgentlyinthebreezeandconverttheairintomoretrees,themagic trick toend themall.Allof thosedifferencesarewritten in thegenes,genes thatderivefromyourcommonancestorbuthavenowmostlydivergedbeyondrecognition.All thosechangeswere permitted, selected, in the long course of evolution. Genes are almost infinitely permissive:anythingthatcanhappenwillhappen.

But that tree has mitochondria too, which work in much the same way as its chloroplasts,endlessly transferring electrons down its trillions upon trillions of respiratory chains, pumpingprotonsacrossmembranesastheyalwaysdid.Asyoualwaysdid.Thesesameshuttlingelectronsandprotonshavesustainedyoufromthewomb:youpump1021protonspersecond,everysecond,withoutpause.Yourmitochondriawerepassedonfromyourmother,inhereggcell,hermostpreciousgift,thegiftoflivingthatgoesbackunbroken,unceasing,generationongeneration,tothefirststirringsof life inhydrothermalvents,4billionyearsago.Tamperwith this reactionatyourperil.Cyanidewillstemtheflowofelectronsandprotons,andbringyourlifetoanabruptend.Ageingwilldothesame,butslowly,gently.Deathis theceasingofelectronandprotonflux, thesettlingofmembranepotential,theendofthatunbrokenflame.Iflifeisnothingbutanelectronlookingforaplacetorest,deathisnothingbutthatelectroncometorest.

This energy flux is astonishing and unforgiving. Any change over seconds or minutes couldbring thewhole experiment to an end.Spores canpull it off, descending intometabolicdormancyfromwhich theymust feel lucky toemerge.But for therestofus…wearesustainedby thesameprocesses thatpowered the first livingcells.Theseprocesseshaveneverchanged ina fundamentalway; how could they?Life is for the living.Living needs an unceasing flux of energy. It’s hardlysurprisingthatenergyfluxputsmajorconstraintsonthepathofevolution,definingwhatispossible.It’snotsurprisingthatbacteriakeepdoingwhatbacteriado,unabletotinkerinanyseriouswaywiththeflamethatkeepsthemgrowing,dividing,conquering.It’snotsurprisingthattheoneaccidentthatdidwork out, that singular endosymbiosis between prokaryotes, did not tinkerwith the flame, butigniteditinmanycopiesineachandeveryeukaryoticcell,finallygivingrisetoallcomplexlife.It’snotsurprisingthatkeepingthisflamealiveisvitaltoourphysiologyandevolution,explainingmanyquirksof ourpast andour lives today.How lucky that ourminds, themost improbablebiologicalmachinesintheuniverse,arenowaconduitforthisrestlessflowofenergy,thatwecanthinkaboutwhylifeisthewayitis.Maytheproton-motiveforcebewithyou!

Footnotes1TheendosymbiontsinParakaryonmyojinensisarefoundinsidewhattheauthorsdescribeasphagosomes(vacuolesinthecell)despitethepresenceofanintactcellwall.Theyconcludethatthehostcellmustoncehavebeenaphagocyte,butlaterlostthiscapacity.That’snotnecessarilythecase.LookagainatFigure25.Theseintracellularbacteriaareenclosedbyverysimilar‘vacuoles’,butinthiscase

thehostcellisrecognisablyacyanobacterium,andthereforenotphagocytic.DanWujekascribesthesevacuolessurroundingtheendosymbiontstoshrinkageduringpreparationforelectronmicroscopy,andI’dguessthe‘phagosomes’inParakaryonmyojinensisarealsoanartefactofshrinkage,andhavenothingtodowithphagocytosis.Ifso,there’snoreasontothinkthattheancestralhostcellwasamorecomplexphagocyte.2DatafromthespacetelescopeKeplersuggeststhat1in5sun-likestarsinthegalaxyhavean‘earth-sized’planetinthehabitablezone,givingthatprojectedtotalof40billionsuitableplanetsintheMilkyWay.

GLOSSARY

aerobicrespiration–ourownformofrespiration,inwhichenergyfromthereactionbetweenfoodandoxygenisharnessedtopowerwork;bacteriacanalso‘burn’mineralsorgaseswithoxygen.Seealsoanaerobicrespirationandrespiration.

alkalinehydrothermalvent–atypeofvent,usuallyontheseafloor,thatemitswarmalkalinefluidsrichinhydrogengas;probablyplayedamajorroleintheoriginoflife.

allele–oneparticularformofageneinapopulation.aminoacid–oneof20distinctmolecularbuildingblocksthatarelinkedtogetherinachaintoforma

protein(oftencontaininghundredsofaminoacids).anaerobicrespiration–anyoneofmanyalternativeformsofrespiration,commoninbacteria,in

whichmoleculesotherthanoxygen(suchasnitrateorsulphate)areusedto‘burn’(oxidise)food,mineralsorgases.Anaerobesareorganismsthatlivewithoutoxygen.Seealsoaerobicrespirationandrespiration.

ångström(Å)–aunitofdistance,roughlythescaleofanatom,technicallyoneten-billionthofametre(10–10m);ananometreis10timesthatlength,one-billionthofametre(10–9m).

antiporter–aprotein‘turnstile’thattypicallyexchangesonechargedatom(ion)foranotheracrossamembrane,forexampleaproton(H+)forasodiumion(Na+).

apoptosis–‘programmed’celldeath,anenergy-consumingprocessencodedbygenes,inwhichacelldismantlesitself.

archaea–oneofthethreegreatdomainsoflife,theothertwobeingthebacteriaandeukaryotes(suchasourselves);archaeaareprokaryotes,lackinganucleustostoretheirDNA,andmostotherelaboratestructuresfoundincomplexeukaryotes.

archezoa–don’tmixtheseupwitharchaea!Archezoaaresimple,single-celledeukaryotes,oncemistakenforevolutionary‘missinglinks’betweenbacteriaandmorecomplexeukaryoticcells.

ATP–adenosinetriphosphate,thebiologicalenergy‘currency’usedbyallknowncells.ADP(adenosinediphosphate)isthebreakdownproductformedwhenATPis‘spent’;theenergyofrespirationisusedtojoinaphosphate(PO4

3–)backontoADPtoreformATP.Acetylphosphateisasimple(two-carbon)biologicalenergy‘currency’thatworksabitlikeATP,whichcouldhavebeenformedbygeologicalprocessesontheearlyearth.

ATPsynthase–aremarkablerotatingmotorprotein,ananoturbinethatsitsinthemembraneandusestheflowofprotonstopowerthesynthesisofATP.

bacteria–oneofthethreegreatdomainsoflife,theothertwobeingthearchaeaandeukaryotes(likeus);alongwitharchaea,bacteriaareprokaryotes,whichlackanucleustostoretheirDNA,aswellasmostotherelaboratestructuresfoundincomplexeukaryotes.

chemiosmoticcoupling–thewayinwhichenergyfromrespirationisusedtopumpprotonsacrossamembrane;thefluxofprotonsbackthroughproteinturbinesinthemembrane(ATPsynthase)thendrivestheformationofATP.Sorespirationis‘coupled’toATPsynthesisbyaprotongradient.

chloroplast–aspecialisedcompartmentinplantcellsandalgaewherephotosynthesistakesplace;originallyderivedfromphotosyntheticbacteriacalledcyanobacteria.

chromosome–atubularstructurecomposedofDNAtightlywrappedinproteins,visibleduringcelldivision;humanshave23pairsofdistinctchromosomescontainingtwocopiesofallourgenes.Afluidchromosomeundergoesrecombination,givingdifferentcombinationsofgenes(alleles).

cytoplasm–thegel-likesubstanceofcells,excludingthenucleus;thecytosolisthewaterysolutionsurroundinginternalcompartmentssuchasmitochondria.Thecytoskeletonisthedynamicproteinscaffoldinsidecells,whichcanformandreformascellschangeshape.

disequilibrium–apotentiallyreactivestateinwhichmoleculesthat‘want’toreactwitheachotherhaveyettodoso.Organicmatterandoxygenareindisequilibrium–giventheopportunity(strikingamatch)organicmatterwillburn.

dissipativestructure–astablephysicalstructurethattakesacharacteristicform,asinawhirlpool,hurricane,orthejetstream,sustainedbyacontinuousfluxofenergy.

DNA–deoxyribonucleicacid,thehereditarymaterial,whichtakestheformofadoublehelix;parasiticDNAisDNAthatcancopyitselfselfishly,attheexpenseoftheindividualorganism.

electron–asubatomicparticlethatcarriesanegativeelectricalcharge.Anelectronacceptorisanatomormoleculethatgainsoneormoreelectrons;anelectrondonorloseselectrons.

endergonic–areactionthatrequiresaninputoffreeenergy(‘work’,notheat)toproceed.Anendothermicreactionrequiresaninputofheattoproceed.

endosymbiosis–amutualrelationship(usuallyatradeofmetabolicsubstances)betweentwocells,inwhichonepartnerphysicallylivesinsidetheother.

entropy–astateofmoleculardisordertendingtowardschaos.enzyme–aproteinthatcatalysesaparticularchemicalreaction,oftenincreasingitsratebymillions

oftimestheuncatalysedrate.eukaryote–anyorganismcomposedofoneormorecellscontaininganucleusandotherspecialised

structureslikemitochondria;allcomplexlifeforms,includingplants,animals,fungi,algae,andprotistssuchastheamoeba,aremadeofeukaryoticcells.Eukaryotesformoneofthethreegreatdomainsoflife,theothertwobeingthesimplerprokaryoticdomainsofbacteriaandarchaea.

exergonic–areactionthatreleasesfreeenergy,whichcanpowerwork.Anexothermicreactionreleasesheat.

fattyacid–along-chainhydrocarbon,typicallywith15–20linkedcarbonatoms,usedinthefatty(lipid)membranesofbacteriaandeukaryotes;alwayshasanacidgroupatoneend.

fermentation–thisisnotanaerobicrespiration!FermentationisapurelychemicalprocessofgeneratingATP,whichdoesnotinvolveprotongradientsacrossmembranesortheATPsynthase.Differentorganismshaveslightlydifferentpathways;weproducelacticacidasawasteproduct,yeastsformalcohol.

FeScluster–iron–sulphurcluster,asmallmineral-likecrystalcomposedofalatticeofironandsulphuratoms(usuallythecompoundFe2S2orFe4S4)foundattheheartofmanyimportantproteins,includingsomeofthoseusedinrespiration.

fixation–whenoneparticularformofagene(allele)isfoundinallindividualsinapopulation.freeenergy–energythatisfreetopowerwork(notheat).freeradical–anatomormoleculewithanunpairedelectron(whichtendstomakeitunstableand

reactive);oxygenfreeradicalsescapingfromrespirationmayplayaroleinageinganddisease.

gene–astretchofDNAencodingaprotein(orotherproductsuchasregulatoryRNA).Thegenomeisthetotalcompendiumofgenesinanorganism.

germline–thespecialisedsexcellsinanimals(suchasspermandeggcells),whichalonepassonthegenesthatgiverisetonewindividualsineachgeneration.

intron–a‘spacer ’sequencewithinagene,whichdoesnotcodeforaproteinandisusuallyremovedfromthecode-scriptbeforetheproteinismade.Mobileintronsaregeneticparasitesthatcancopythemselvesrepeatedlywithinagenome;eukaryoticintronsapparentlyderivefromaproliferationofmobilebacterialintronsearlyineukaryoticevolution,followedbymutationaldecay.

lateralgenetransfer–thetransferof(usually)asmallnumberofgenesfromonecelltoanotherortheuptakeofnakedDNAfromtheenvironment.Lateralgenetransferisatradeingeneswithinthesamegeneration;inverticalinheritance,theentiregenomeiscopiedandpassedontothedaughtercellsatcelldivision.

LUCA–thelastuniversalcommonancestorofallcellslivingtoday,whosehypotheticalpropertiescanbereconstructedbycomparingthepropertiesofmoderncells.

meiosis–theprocessofreductivecelldivisioninsex,toformgameteswhichhaveasinglecompletesetofchromosomes(makingithaploid)ratherthanthetwosetsfoundintheparentcells(diploid).Mitosisisthenormalformofcelldivisionineukaryotes,inwhichchromosomesaredoubled,thenseparatedintotwodaughtercellsonamicrotubularspindle.

membrane–averythinfattylayersurroundingcells(alsofoundinsidecells);composedofa‘lipidbilayer ’withahydrophobic(water-hating)interiorandhydrophilic(water-loving)head-groupsoneitherside.Membranepotentialistheelectricalcharge(potentialdifference)betweenoppositesidesofthemembrane.

metabolism–thesetoflife-sustainingchemicalreactionswithinlivingcells.mitochondria–thediscrete‘powerhouses’ineukaryoticcells,whichderivefromα-proteobacteria,

andwhichretainatinybuthugelyimportantgenomeoftheirown.Mitochondrialgenesarethosephysicallylocatedwithinthemitochondria.Mitochondrialbiogenesisisthereplication,orgrowth,ofnewmitochondria,whichalsorequiresgenesinthenucleus.

monophyleticradiation–thedivergenceofmultiplespeciesfromasinglecommonancestor(orasinglephylum)likethespokesofawheelradiatingfromacentralhub.

mutation–usuallyreferstoachangeinthespecificsequenceofagene,butcanalsoincludeothergeneticchanges,suchasrandomdeletionsorduplicationsofDNA.

nucleus–the‘controlcentre’ofcomplex(eukaryotic)cells,whichcontainsmostofthecell’sgenes(somearefoundinmitochondria).

nucleotide–oneofthebuildingblockslinkedtogetherinachaintoformRNAandDNA;therearescoresofrelatednucleotidesthatactascofactorsinenzymes,catalysingspecificreactions.

ortholog–thesamegenewiththesamefunctionfoundindifferentspecies,allofwhichinheriteditfromacommonancestor.

oxidation–theremovalofoneormoreelectronsfromasubstance,renderingitoxidised.paralog–amemberofafamilyofgenes,formedbygeneduplicationswithinthesamegenome;

equivalentgenefamiliescanalsobefoundindifferentspecies,inheritedfromacommonancestor.

pH–ameasureofacidity,specificallytheconcentrationofprotons:acidshaveahighconcentrationofprotons(givingthemalowpH,below7);alkalishavealowconcentrationofprotons,givingthemahighpH(7–14);purewaterhasaneutralpH(7).

phagocytosis–thephysicalengulfingofonecellbyanother,swallowingitupintoa‘food’vacuoletobedigestedinternally.Osmotrophyistheexternaldigestionoffood,followedbyabsorptionof

smallcompounds,aspractisedbyfungi.photosynthesis–theconversionofcarbondioxideintoorganicmatter,usingsolarenergytoextract

electronsfromwater(orothersubstances)andultimatelyattachthemontocarbondioxide.plasmid–asmallringofparasiticDNAthattransmitsselfishlyfromonecelltoanother;plasmids

canalsoprovideusefulgenesfortheirhostcells(suchasgenesconferringantibioticresistance).polyphyleticradiation–thedivergenceofmultiplespeciesfromanumberofevolutionarilydistinct

ancestors(differentphyla)likethespokesofmultiplewheelsradiatingfrommultiplehubs.prokaryote–ageneraltermconnotingsimplecellsthatlackanucleus(literally‘beforethenucleus’)

andincludingbothbacteriaandarchaea,twoofthethreedomainsoflife.protein–achainofaminoacidslinkedtogetherinapreciseorderspecifiedbythesequenceofDNA

lettersinagene;apolypeptideisashorterchainofaminoacids,whoseorderneednotbespecified.

protist–anysingle-celledeukaryote,someofwhichcanbeverycomplex,withasmanyas40,000genesandanaveragesizeatleast15,000timeslargerthanbacteria;protozoaisavividbutdefunctterm(meaning‘firstanimals’)whichreferredtoprotistssuchastheamoebathatbehavelikeanimals.

proton–asubatomicparticlewithpositivecharge;ahydrogenatomiscomposedofasingleprotonandasingleelectron;lossoftheelectronleavesthehydrogennucleus,thepositivelychargedprotondenotedH+.

protongradient–adifferenceintheconcentrationofprotonsonoppositesidesofamembrane;theproton-motiveforceistheelectrochemicalforceresultingfromthecombineddifferenceinelectricalchargeandconcentrationofH+acrossamembrane.

recombination–theexchangeofonepieceofDNAforanequivalentpiecefromanothersource,givingrisetodifferentcombinationsofgenes(specificallyalleles)on‘fluid’chromosomes.

redox–thecombinedprocessofreductionandoxidation,whichamountstoatransferofelectronsfromadonortoanacceptor.Aredoxcoupleisaspecificelectrondonorwithaspecificacceptor;aredoxcentrereceivesanelectronbeforepassingiton,makingitbothanacceptorandadonor.

reduction–theadditionofoneormoreelectronstoasubstance,renderingitreduced.replication–theduplicationofacellormolecule(typicallyDNA)togivetwodaughtercopies.respiration–theprocessbywhichnutrientsare‘burned’(oxidised)togenerateenergyintheformof

ATP.Electronsarestrippedfromfoodorotherelectrondonors(suchashydrogen)andpassedontooxygenorotheroxidants(suchasnitrate)viaaseriesofstepscalledtherespiratorychain.Theenergyreleasedisusedtopumpprotonsacrossamembrane,generatingaproton-motiveforcethatinturndrivesATPsynthesis.Seealsoanaerobicrespirationandaerobicrespiration.

ribosome–protein-building‘factories’foundinallcells,whichconverttheRNAcode-script(copiedfromDNA)intoaproteinwiththecorrectsequenceofaminoacidbuildingblocks.

RNA–ribonucleicacid;aclosecousinofDNA,butwithtwotinychemicalalterationsthattransformitsstructureandproperties.RNAisfoundinthreemainforms:messengerRNA(acode-scriptcopiedfromDNA);transferRNA(whichdeliversaminoacidsaccordingtothegeneticcode);andribosomalRNA(whichactsas‘machineparts’inribosomes).

RNAworld–ahypotheticalearlystageofevolutioninwhichRNAactssimultaneouslyasatemplateforitsownreplication(inplaceofDNA)andacatalystthatspeedsupreactions(inplaceofproteins).

selectivesweep–strongselectionforaparticulargeneticvariant(allele),eventuallydisplacingallothervariantsfromapopulation.

selfishconflict–ametaphoricalclashbetweentheinterestsoftwodistinctentities,forexamplebetweenendosymbiontsorplasmidsandahostcell.

serpentinisation–achemicalreactionbetweencertainrocks(mineralsrichinmagnesiumandiron,suchasolivine)andwater,givingrisetostronglyalkalinefluidssaturatedinhydrogengas.

sex–areproductivecycle,involvingthedivisionofcellsbymeiosistoformgametes,eachwithhalfthenormalquotaofchromosomes,followedbythefusionofgametestoproduceafertilisedegg.

sexdetermination–theprocessescontrollingmaleorfemaledevelopment.snowballearth–aglobalfreeze,withglaciersencroachingtosealevelattheequator;thoughtto

haveoccurredonseveraloccasionsinearth’shistory.substrate–substancesrequiredforcellgrowth,convertedbyenzymesintobiologicalmolecules.thermodynamics–abranchofphysicsdealingwithheat,energyandwork;thermodynamicsgoverns

thereactionsthatcouldoccurunderaparticularsetofconditions;kineticsdefinestherateatwhichsuchreactionsactuallydotakeplace.

thermophoresis–theconcentrationoforganicsbythermalgradientsorconvectioncurrents.transcription–theformationofashortRNAcode-script(calledmessengerRNA)fromDNA,asthe

firststepinmakinganewprotein.translation–thephysicalassemblyofanewprotein(onaribosome)inwhichtheprecisesequence

ofaminoacidsisspecifiedbyanRNAcode-script(messengerRNA).uniparentalinheritance–thesystematicinheritanceofmitochondriafromonlyoneoftwoparents,

typicallyfromtheeggbutnotthesperm;biparentalinheritanceistheinheritanceofmitochondriafrombothparents.

variance–ameasureofspreadinasetofnumbers;ifvarianceiszero,allthevaluesareidentical;ifvarianceissmall,thevaluesallfallclosetothemean;highvarianceindicatesawiderangeofvalues.

T

ACKNOWLEDGEMENTS

hisbookmarkstheendofalongpersonaljourney,andthebeginningofanewjourney.Thefirstjourney began as I was writing an earlier book, Power, Sex, Suicide: Mitochondria and the

MeaningofLife,publishedbyOUPin2005.ThatwaswhenIfirstbegantograpplewiththequestionsIdealwithhere–theoriginsofcomplexlife.IwasstronglyinfluencedbytheextraordinaryworkofBill Martin on the origin of the eukaryotic cell, and his equally radical work with pioneeringgeochemistMikeRussellontheoriginoflifeandtheveryearlydivergenceofarchaeaandbacteria.Everything in that (and this) book is grounded in the context laid out by these two colossi ofevolutionarybiology.But someof the ideasdevelopedhereareoriginal.Writingbooksgivesonescopetothink,andformethisisanunmatchablepleasureofwritingforageneralaudience:Ihavetothinkclearly,totrytoexpressmyselfinawaythat,firstofall,Icanunderstandmyself.Thatbringsme face to face with the things I don’t understand, some of which, thrillingly, turn out to reflectuniversalignorance.SoPower,Sex,Suicidewasmoreorlessobligedtoputforwardafeworiginalideas,whichIhavebeenlivingwitheversince.

Ipresentedtheseideasatconferencesanduniversitiesaroundtheworld,andgrewusedtodealingwithastutecriticisms.Myideasbecamerefined,asdidmyoverallconceptionof the importanceofenergyinevolution;andafewcherishedthoughtswerethrownawayaswrong.Buthowevergoodanideamaybe,itonlybecomesrealsciencewhenitisframedandtestedasarigoroushypothesis.Thatseemed a pipe dream until 2008, when University College London announced a new prize for‘ambitious thinkers’ toexploreparadigm-shifting ideas.TheProvost’sVentureResearchPrizewasthe brainchild of ProfessorDonBraben, a dynamo of aman,who has long fought for ‘scientificfreedom’.Scienceisfundamentallyunpredictable,Brabenargues,andcan’tbeconstrainedtoorder,however much society might wish to prioritise the spending of tax-payers’ money. Genuinelytransformative ideasalmostalwayscome from the leastexpectedquarters; thatalonecanbe reliedupon.Suchideasaretransformativenotonlyintheirscience,butalsotothewidereconomy,whichisfuelledby scientific advances. It’s therefore in thebest interestsof society to fund scientistson thestrengthof their ideas alone, however intangible thesemight seem, rather than trying to target theperceivedbenefitstohumanity.Thatrarelyworksbecauseradicallynewinsightsusuallycomefromoutsideafieldaltogether;naturehasnorespectforhumanboundaries.1

Luckily, Iwas eligible to apply to theUCL scheme. I had a book full of ideas that desperatelyneeded testing,and thankfully,DonBrabenwaseventuallypersuaded.While the impetusbehind theprize came from Don, to whom I am immensely indebted, I owe as much to the generosity andscientificvisionofProfessorDavidPrice,ViceProvostforResearchatUCL,andthethenProvost,ProfessorMalcolmGrant, for supporting both the scheme andme personally. I’m also extremely

gratefultoProfessorSteveJonesforhissupport,andforwelcomingmetothedepartmentheheadedatthetime:Genetics,EvolutionandEnvironment,naturalhomefortheresearchIwastoundertake.

Thatwassixyearsago.Sincethen,Ihavebeenattackingasmanyoftheproblems,fromasmanyangles,as Ican.TheVentureResearchfunding itself lasted threeyears,enough tosetmydirectionandgivemeafightingchanceofwinningfundingfromothersourcestocontinue.InthisIamverygrateful to theLeverhulmeTrust,whohave supportedmyworkon the origin of life over the lastthreeyears.Notmanyorganisationsarewillingtobackagenuinelynewexperimentalapproach,withall the teething problems that entails. Thankfully, our little bench-top origin-of-life reactor is nowbeginningtoproduceexcitingresults,noneofwhichwouldhavebeenpossiblewithouttheirsupport.Thisbookisadistillationofthefirstmeaningfromthesestudies,thebeginningofanewjourney.

Ofcourse,noneofthisworkhasbeendonealone.IhavebouncedmanyideasbackandforthwithBill Martin, Professor of Molecular Evolution at the University of Düsseldorf, who is alwaysgenerous with his time, energy and ideas, while never hesitating to demolish poor reasoning, orignorance.IthasbeenagenuineprivilegetowriteseveralpaperswithBill,whichI’dliketothinkarenoteworthycontributions to the field.Certainly, fewexperiences in life canmatch the intensityandpleasureofwritingapaperwithBill.I’velearntanotherimportantlessonfromBill–nevercluttertheproblemwithpossibilitiesthatcanbeimagined,butareunknownintherealworld;focusalwaysonwhatlifeasweknowitactuallydoes,andthenaskwhy.

I’mequallygrateful toAndrewPomiankowski,ProfessorofGeneticsatUCL,morecommonlyknownasPOM.POMisanevolutionarygeneticiststeeped in the intellectual traditionsof thefield,having worked with legendary figures such as John Maynard Smith and Bill Hamilton. POMcombines their rigour with an eye for the unsolved problems of biology. If I have succeeded inpersuadinghimthattheoriginofcomplexcellsisjustsuchaproblem,hehasintroducedmetotheabstractbutpowerfulworldofpopulationgenetics.Approaching theoriginsof complex life fromsuchcontrastingpointsofviewhasbeenasteeplearningcurve,andgreatfun.

Another good friend at UCL, with boundless ideas, enthusiasm and expertise to drive theseprojectsforward, isProfessorFinnWerner.Finnbringsanothersharplycontrastingbackgroundtothe samequestions, that of structural biology, and specifically themolecular structure of theRNApolymerase enzyme, one of themost ancient andmagnificentmolecularmachines,which in itselfgivesinsightsintotheearlyevolutionoflife.EveryconversationandlunchwithFinnisinvigorating,andIreturnreadyforchallengesnew.

I’vealsobeenprivilegedtoworkwithanumberofgiftedPhDstudentsandpost-docs,whohavedrivenmuchofthisworkforward.Thesefallintotwogroups,thoseworkingontherealdown-dirtychemistry of the reactor, and those bringing theirmathematical skills to bear on the evolution ofeukaryotictraits.Inparticular,IthankDrBarryHerschy,AlexandraWhicherandEloiCamprubifortheirskillsinmakingdifficultchemistryactuallyhappeninthelab,andfortheirsharedvision;andDrLewisDartnell,whoattheoutsethelpedbuildtheprototypereactorandsettheseexperimentsinmotion.Inthisendeavour,I’malsogratefultoJulianEvansandJohnWard,ProfessorsofMaterialsChemistryandMicrobiology,respectively,whohaveofferedfreelytheirtime,skillsandlaboratoryresourcestothereactorprojectandtheco-supervisionofstudents.Theyhavebeenmycomradesinarmsthroughthisadventure.

Thesecondgroupofstudentsandpost-docs,workingonmathematicalmodelling,haveselectedthemselvesfromanunparalleleddoctoral trainingprogrammeatUCL,fundeduntil recentlyby theEngineeringandPhysicalSciencesResearchCouncil.Thisprogrammegoesby thesmartacronymCOMPLEX,standingimprobablyfortheCentreforMathematicsandPhysicsintheLifeSciencesandExperimental Biology. COMPLEX students working with POM and me include Dr ZenaHadjivasiliou,Victor Sojo,ArunasRadzvilavicius, JezOwen, and recently,DrsBramKuijper and

LaurelFogarty.Allstartedoutwithrathervagueideas,andturnedthemintorigorousmathematicalmodelsthatgivestrikinginsightsintohowbiologyreallyworks.Ithasbeenanexcitingride,andI’vegiven up trying to predict the outcome. This work began with the inspirational Professor RobSeymour,who knewmore biology thanmost biologists,while being a formidablemathematician.Tragically,Robdiedof cancer in2012at the ageof sixty-seven.Hewas lovedbyagenerationofstudents.

WhilethisbookisgroundedintheworkI’vepublishedoverthelastsixyearswiththiswidearrayofresearchers(sometwenty-fivepapersinall;seeFurtherReading),itreflectsamuchlongerperiodof thinking anddiscussion at conferences and seminars, by email and in the pub, all ofwhich hassculptedmyviews.Inparticular,ImustthankProfessorMikeRussell,whoserevolutionaryideasontheoriginof life inspireda risinggeneration,andwhose tenacity inadversity isamodel tousall.LikewiseI thankProfessorJohnAllen,whosehypothesesonevolutionarybiochemistryhave lituptheway.Johnhasalsobeenanoutspokendefenderofacademicfreedom,whichhasrecentlycosthimdearly.IthankProfessorFrankHarold,whosesynthesisofbioenergetics,cellstructureandevolutionislaidoutinseveralwonderfulbooks,andwhoseopen-mindedscepticismhasconstantlychallengedmetogoalittlefurther;ProfessorDougWallace,whoseconceptionofmitochondrialenergeticsasthecentraldriverofageinganddiseaseisvisionaryandinspirational;andProfessorGustavoBarja,whoseessoclearly throughthedense thicketsofmisunderstandingaboutfreeradicalsandageing,thatIalwaysturnfirsttohisview.Notleast,ImustthankDrGrahamGoddard,whoseencouragementandplainspeakingmanyyearsagochangedthecourseofmylife.

Thesefriendsandcolleaguesareofcoursebutthetipofaniceberg.Ican’t thankall thosewhohave shaped my thinking in detail, but I am indebted to them all. In random order: ChristopheDessimoz,PeterRich,AmandineMarechal,SirSalvadorMoncada,MaryCollins,BuzzBaum,UrsulaMittwoch,MichaelDuchen,GyuriSzabadkai,GrahamShields,DominicPapineau, JoSantini, JürgBähler,DanJaffares,PeterCoveney,MattPowner,IanScott,AnjaliGoswami,AstridWingler,MarkThomas,RazanJawdatandSiobanSenGupta,allatUCL;SirJohnWalker,MikeMurphyandGuyBrown (Cambridge); Erich Gnaiger (Innsbruck); Filipa Sousa, Tal Dagan and Fritz Boege(Dusseldorf); Paul Falkowski (Rutgers); Eugene Koonin (NIH); Dianne Newman and John Doyle(Caltech);JamesMcInerney(Maynooth);FordDoolittleandJohnArchibald(Dalhousie);WolfgangNitschke(Marseilles);MartinEmbley(Newcastle);MarkvanderGiezenandTomRichards(Exeter);Neil Blackstone (Northern Illinois); Ron Burton (Scripps); Rolf Thauer (Marburg); Dieter Braun(Munich); Tonio Enríquez (Madrid); Terry Kee (Leeds); Masashi Tanaka (Tokyo); MasashiYamaguchi,Chiba;GeoffHill (Auburn);KenNealson and JanAmend (SouthernCalifornia);TomMcCollom (Colorado);ChrisLeaver andLeeSweetlove (Oxford);MarkusSchwarzländer (Bonn);John Ellis (Warwick); Dan Mishmar (Ben Gurion); Matthew Cobb and Brian Cox (Manchester);RobertoandRobertaMotterlini(Paris);andSteveIscoe(Queens,Kingston).Thankyou,all.

I’malsoverygratefultoahandfuloffriendsandfamilywhohavereadandcommentedonparts(orall)ofthisbook.Inparticular,myfather,ThomasLane,whohassacrificedtimefromwritinghisown books on history to readmuch of this book, finessingmy use of language throughout; JonTurney, who has likewise been generous with his time and comments, especially on pitch, whileengagedonhisownwritingprojects;MarkusSchwarzländer,whoseenthusiasmbuoyedmethroughdifficult periods; and Mike Carter, who alone among my friends has read and commented, withtrenchantwit, on every chapter of every book I havewritten, occasionally even persuadingme tochangetack.Andalthoughnoneofthemhavereadthisbook(yet),IhavetothankIanAckland-Snow,AdamRutherfordandKevinFongfor lunchesandgoodconversations in thepub.Theyknowwellhowimportantthatisforsanity.

I need hardly say that this book has benefited greatly from the expertise of my agent and

publishers.I’mverygratefultoCarolineDawnayatUnitedAgents,forbelievinginthisprojectfromtheoutset;toAndrewFranklinatProfile,whoseeditorialcommentscutstraighttothequick,makingthebookmuchharderhitting;toBrendanCurryatNorton,whopointedastutelytopassageslackingclarity;andEddieMizzi,whosesensitivecopyeditingagainreflectshisfinejudgementandeclecticlearning.HisinterventionshavesparedmoreblushesthanIcaretoadmit.Manythanks,too,toPennyDaniel,SarahHull,ValentinaZancaand the teamatProfile, formarshalling thisbooktopressandbeyond.

AndfinallyIturntomyfamily.Mywife,DrAnaHidalgo,haslivedandbreathedthisbookwithme,readingeverychapteratleasttwice,andalwaysilluminatingthewayahead.Itrustherjudgementand knowledgemore thanmy own, andwhat good there is inmywriting has evolved under hernatural selection. I can thinkofnobetterway to spendmy life than trying tounderstand life,but Ialreadyknowthatmyownmeaningand joystemsfromAna, fromourwonderfulsonsEnekoandHugo,andfromourwiderfamilyinSpain,EnglandandItaly.Thisbookwaswritteninthehappiestoftimes.

Footnote1Ifyouwanttoknowmore,Brabenhasframedhisargumentsinseveralcompellingbooks,thelatestofwhichistitledPromotingthePlanckClub:HowDefiantYouth,IrreverentResearchersandLiberatedUniversitiesCanFosterProsperityIndefinitely(Wiley,2014).

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INDEX

Aacetogens

asancestralbacteria152electronbifurcationin151,153relianceontheacetylCoApathway131–2,149–52sodiumionpumping148n

acetylCoAasactivatedacetate132incarbonandenergymetabolism133Echandproductionof142asathioesterintermediate79

acetylCoApathway130–32,134,136differencesinarchaeaandbacteria131,149–50,152

acetylphosphateasATPequivalent98,102n,133,292catalysisofpeptideformation101,102nfrommethylthioacetate134–5

acidityseepHactinfilaments184activity

asbeneficial232,277free-radicalleakageandlifespan274–6andmitochondrialmutation232

adaptabilitytrade-offwithdiseaserisk264–5,267adaptivechangesandgradualism194ADP(adenosinediphosphate)190,292ADP–ATPtransporter190aerobicrespiration30,291age-relateddisease

canceras277deaththresholdand268andmetabolicdemand256–8

ageingelectronicinterpretation232freeradicaltheoryof268–79generegulationchanges276originsof232roleofmitochondrialgenes186,276utilityofantioxidants269

algaeChlamydomonas220effectsoffreeradicalleakage219nEuglena35,178genomesize159

alkalinehydrothermalvents291

biochemistryofmethanogensandacetogensand132–3CO2reductiontoformate119comparedwithblacksmokers106–8compositionchanges112onexoplanets121,287hydrogenin108–9,112ironsulphidesin108,113,117–19,134,136‘LostCity’example107,109–13andLUCAcharacteristics130LUCAemergenceenvisaged135primordialacetylCoApathwayin134,136protocellformation134–5protongradientsin115–21,134–6,138

alleles213–16,224,291Allen,John187,231‘amazingdisappearingtree’126–7Amend,Jan113aminoacids291

entropyofproteinsand59fromMiller-Ureyexperiments91

amoebaeAmoebaproteus178endosymbiotichostcomparedto10energypergene176,178aseukaryotes30,293genomiccomplexity22–3mitochondrialnumbers184Naegleria44asphagocytotic32,37asprotists2,297

anaerobesandoxidationevents32,47anaerobicrespiration276,291ångström(Å)unit69,241,291anisogamy229–31antioxidants

absorption273adverseeffects270–3freeradicaltheoryofageing269–70ineffectiveness277

antiporters291–2modificationbymethanogens153aspreadaptations146,149preconditionofescapefromvents287andprotocelldependencyonhydrothermalvents143–8

apoptosis(programmedcelldeath)292incytoplasmicmalesterility259deaththresholdand265–7discoveryof247inembryonicdevelopment248–9,262–3freeradicalinvolvement188,190,245–6andthefreeradicaltheoryofageing271asfunctionalselection248,256,264metabolicdemandand255–7protongradientinvolvement170roleofmitochondriain233,245–7,259roleofprotongradients71nsenescencealternative276–7interminallydifferentiatedtissues256

archaea292biochemicaldistinctionfrombacteria83–5,129,153aschemiosmotic129discovery8eukaryotesimilaritieswith8–9

eukaryoticgenesderivedfrom162exampleswithoutcellwalls159membranereplacement209methanogensasancestral152mitochondrialacquisition10morphologicalsimplicity11,157–8originsofdistinctionfrombacteria148–54asprokaryotes10,126nprotongradientsanddivergencefrombacteria147–8protongradientsandstructure13

Archaeopteryx48archezoa292

distinguishedfromarchaea48–9asecologicalintermediates47–8,197aseukaryotes48–9,163Giardiaas37–8,199hydrogenosomesandmitosomesin188notevolutionaryintermediates38–9,160,163,284origins42survival163

asexualreproductionseecloningATP(adenosinetriphosphate)292

abioticprecursors102nacetylphosphateasequivalent98consequencesofnon-consumption190formationusingprotongradients68inpolymerisationreactions98requirementforflight262requirementforpeptidebondformation172synthesisandfreeradicals270turnoverrates63asuniversalenergycurrency63

ATPspilling190nATPsynthase292

asconservedacrossalllife73,129incytoplasmicmalesterility259drivenbyprotonflux72,142LUCAuseof149,152inmitochondrialmembranes240,259asaproteinmachine73–4structure74,76

autotrophy108,168

Bbacteria292

absenceofeukaryotictraits46absenceofevidenceforextinctions47acetogensasancestral152BuchneraandWolbachia259nCarsonella183aschemiosmotic129aschimeric125cyanobacteria181–2,185,188distinction/divergencefromarchaea83–5,129,147–54E.coli125,178,181,198–9elsewhereintheuniverse287endosymbiosisbetween181–2,288eubacteria12,49eukaryoticgenesderivedfrom162failuretoevolvecomplexity157–8genescontributingtoeukaryoticgenome163giantbacteriaexamples176intronsin202L-form99,159,218

Leeuwenhoek’sobservationof4lossofcellwalls158–9metabolicversatility4,29,80,157mitochondriaandchloroplastsasderivedfrom6morphologicalsimplicity2,11,157–8Planctomycetes44asprokaryotes126nprotongradientsand13,147–8speciesdefinitionin123streamliningofgenomes174–6,181,201,208YellowstoneNationalPark102

banded-ironformations27–8,81n,112Barja,Gustavo268Baross,John103Barton,Nick216Battley,Ted56biochemistry

bacteriaandcomplexcells5catalysisofthebeginningsof100–1relationshiptogeochemistry27varietyofbacterial29

biomassproductionasexergonic114fromrespiration,comparedwithwaste90

biparentalinheritance230–1,299birds

colorationofmales261flightandmitonuclearcoadaptation262–5,267free-radicalleakage268n,278Haldane’srulein252,260–1heterogamicsexin252,261longevity274,278migration265pigeons246,266,268selfishconflictin259n

blackholemetaphor1–3,34–45,192‘blacksmokers’

comparedwithalkalinehydrothermalvents106–9discovery103intheearlyocean104,112andtheoriginoflife104asasourceofiron112

Blackstone,Neil219blebs247‘boringbillion’period31‘bronzecontrol’analogy187Buchnera259nbudding(celldivision)99Burton,Ron249–50Buss,Leo227n

CCairns-Smith,Graham97Calvincycle131Cambrianexplosion31,33,196Camprubi,Eloi119cancers

aspredominantlyage-related277andselfishconflict221

carbon,asbasisforalllife78,96carbondioxide(CO2)

electronacceptancegenerally78electronacceptanceinphotosynthesis28

Hadeanperiodlevels113plantlifedependenceon54stepwisereductionof114

carbondioxide(CO2)reactionwithH2intheacetylCoApathway131energeticsof150–1,169andthehydrogeneconomy114kineticsofmethaneproduction113intheoriginsoflife104,108inpredecessorsofeukaryotes11reactiondrivenbyprotongradients115–21reactioninacetogensandmethanogens152

carbonfixation131,132,151carbonflux

inalkalinehydrothermalvents109,148drivingmetabolicprocesses135andLUCA129neededbyprimitivecells99,101–2,120,142,286

carbonisotopes25carbonmetabolism,separationfromenergymetabolism78–9carbonmonoxide(CO)106,134,141cardiolipin245–6Carsonella183caspases247catalysts,inorganicandorganic100–1

seealsoenzymesCavalier-Smith,Tom37,39,159celldeath,programmedseeapoptosiscelldivision

meiosis,asreductive199,211–12,295mitosis42,182,218,295nuclearmembrane210surface-to-volumeratiosand99

cellformationpropertiesoflivingcells96–102protocells134–5

cellfusion218–19cellstructures

energyfluxdrivingcreation95sizelimits99–100,171

cellvolumes,ofeukaryotesandprokaryotes171–3cellwalls

inbacteriaandarchaea83,129,154,159,218lossof,inmosteukaryotes158–9Parakaryonmyojinensis281,283

cellularevolutionasaneglectedbranchofbiology3requirements96–102

ChanceandNecessity,byJacquesMonod20chemiosmoticcoupling292

controlrequiringmitochondrialgenes187evolutionof83,85–6asinescapableinprokaryotes167–71astranscendingchemistry169universality82–3,170,286–8

chemiosmotichypothesis75–7chemiosmoticorganisms128,154chemotaxis225nchimeras,bacteriaas125chimericproteins,nucleus210chimpanzees125,244,259n,279Chlamydomonas220chlorophyll81

chloroplasts292absentfromeukaryoticcommonancestor40derivedfrombacteria6generetentionby187originsasendosymbionts5,36predatedbymitochondria9–10

ChristPantocratoriconography237–9chromosomes292–3

advantagesofsmallbacterialchromosomes159–60cross-overpoints211‘fluidchromosomes’198,213–16,293mechanismofsegregation218sexchromosomes213,220,252–4,261straightchromosomes42,47,158–60,217–18

citratesynthase205claymineralsasreplicators97cloning

mitochondrialpopulationsasclonal222,248sexcontrastedwith198–9,213–17

coadaptationseemitonuclearcoadaptationcoenzymeAseeacetylCoAcoenzymeQ(ubiquinone)67,72colorationinmalebirds261commonancestryseelastcommonancestorcompartmentalisationinlivingcells96,98–9,101complexcellsseeeukaryoticcellscomplexlife

chimericorigins161–7,180commonancestry33,40elsewhereintheuniverse191,288–9andendothermy62nimportanceofmitochondria186–91mitochondrialacquisitionpreceding11,179–80needforapoptosis247originsinendosymbiosis10oxygenasenvironmentaltrigger29–34traitscommontoall1,14,191seealsomulticellularity

complexes,respiratorychaincomplexI66–7,69,72,76,238,246complexII70,72complexesIIIandIV70,72,240effectsofreduction190,245–7,256–7,264,271nandfree-radicalsignalling271respirationandfree-radicalleakage274

concentrationofatmosphericoxygen31–2leadingtoself-organisation102organicsinseawater93bythermophoresis110–11

conservedfeaturesacrossalllife52,73,83–4,101,205–6eukaryotes40–2,196,247,254

conservedgenes123n,124,126–7,161conservedproteins

acrossalllife129,135,149acrosseukaryotes194–5

convergentevolution20,45,151copepods249–51Copley,Shelley136corals113,226–7,230,232cost-benefittrade-offs

adaptabilitywithdiseaserisk264–5,267bacterialsizeand179

fertilitywithfitness14,249–50,261,264–5,267fertilitywithlifespan277–9intronnumbersand202uniparentalinheritance223

Crick,Francisanticipationofmolecularphylogenetics6–7geneticcodeinferredby22Miller-Ureyexperimentsascontemporary90–1Mitchellascontemporary75onpanspermia94n

Crow,James231cyanamide92,97cyanide

andearlylife79,92–3asarespiratorypoison137,290

cyanobacteriaendosymbiosisinvolving181–2genomecopies188respiratorymembranes185

cybrids(hybridcells)259,260n,272cytochromec72,245–7,256,259,264cytoplasm293cytoplasmicmalesterility258–9cytoskeleton,dynamic184,190,293

DDarwin,Charles

advocacyinOriginofSpecies14onevolutionaryintermediates45treeoflifeconcept122‘warmponds’speculation102

deDuve,Christian133death

electronicinterpretation232origins232seealsoapoptosis(celldeath)

deaththreshold264–8,271,278dehydrations,polymerisationreactionsas98,133dietandmitochondrialdiseases277ndifferentiation

absentfromearlymulticellularanimals227terminallydifferentiatedtissues256

dinucleotides136diseaseriskandadaptability264–5,267disequilibria13,18,103,135,293disorderseeentropy‘dissipativestructures’293

hydrothermalventsfavourableto102,106,110non-biologicalexamples94–5

disulphidebonds141DNA293

DAPIstaining177‘junk’DNA22n,204n,215non-coding42,200,203Parakaryonmyojinensis283parasitic23,201,203,293

DNAreplicationinbacteriaandarchaea83,130,153–4,159energyexpenditureon172inprokaryotesandeukaryotes159

Dobzhansky,Theodosius248domains

archaeaasthirddomainoflife8prokaryotic48–9

treeofliferepresentation124Doolittle,Ford200–1Drosophila251,259–60

EE.coli(Escherichiacoli)

genomicvariationin125metabolicrate178metagenomesize181,198–9seealsobacteria

Earth,earlyconditionson24,50,91n,98,103seealsoprimordial

Ech(energy-convertinghydrogenase)archaea-bacteriadifferences147,151–3leakymembranesand140–2LUCAuseof149inmethanogenbiochemistry136–7Na+/H+promiscuity144

Ediacarans31effectivepopulationsize214eggcells

mitochondriain230sizedifferencefromsperm230

electricalchargeandprotongradients138electrochemicalpotential,mitochondrialmembranes71,73electronacceptors293

characteristicsof80–1nitrateas30n,64,176oxygenas103,188

electronbifurcation150–1,153,169electrondonors293

inchemiosmoticcoupling169ferrousironas28,78–9,81hydrogensulphideas28,78–9,81–2,102–3inphotosynthesis28inredoxreactions64,78–80

electronfluxalternativepathwayand246nandexercise274freeradicalsand245,247,274membranepotentialand243mitonuclearincompatibilityand245requiredtosustainflight262–3

Ellobiuslutescens213Embley,Martin166–7embryonicdevelopment248–9,262–3endergonicreactions150,293endosymbiosis293

complexlifeoriginsin10,180genelossinbacterialendosymbionts181metabolicsyntrophyin190originofintrons201,207originsofmitochondriaandchloroplasts5,36Parakaryonmyojinensisand282,283,285phagocytosisnotrequiredfor166–7,181,182betweenprokaryotes,examples181,288serialendosymbiosistheory6,11,34–9,43,164singular,asgivingrisetoeukaryotes10–11,86,183n,191,193,279

endothermicreactions293endothermy

andaerobiccapacity279ncomplexityand62n

andlifespan278inplants46n

energybarrierstoproteinfolding60energybudgets

bioenergeticsandoccultmiscarriage267eukaryotes172andgeneloss183–4methanogens140–1

energycurrency,ATPas63,65energyflux

drivingcreationofcellstructures95driving‘dissipativestructures’94–5environmentalreactivityand62–3,80asfundamental290neededbyprimitivecells142,286–7andtheoriginsoflife13promotingtheself-organisationofmatter94

energygenerationdependenceonprotongradients13,169energymetabolism,separationfromcarbonmetabolism78–9

seealsofreeenergyenergypergene170–9

prokaryotesandeukaryotes171,178,191nscaled-upbacteria175–6

energyspilling190nenergyuse,bythehumanbody64Enriques,Antonio270–1entropy293

consideringtheenvironment56–63lifeasageneratorof65,80,95ofmembraneandcellformation57–8,98–9,113–14ofproteinfolding59relationtofreeenergy61andthesecondlaw22andspores56–7,59

environmentexplanationforapparentcharacteristicsofLUCA130genomesizeand23importanceofmembranesand77interplayofgenesand29–34neglectedindefinitionsoflife55possibleinfluenceonmitochondria264–5reactivityandenergyflux62–3,80

enzymes293environmentalsodiumand144–5functioninmetabolism89–90,101metal-sulphidecofactors100–1seealsoATPsynthase;Ech

eocytehypothesis166Epulopiscium176–9Errington,Jeff209‘errorcatastrophe’proposal269Euglena35eukaryotes293–4

archaeasimilaritieswith8–9,124archezoaas48bacteriasimilaritieswith124commontraits51,86,160,193,215conservedfeatures41–2,247endosymbiosisinphagocyticeukaryotes181energypergene171evolutionofthenucleus194–6excludingParakaryonmyojinensis283–4extremophile43genesasfragmented199–200

genesderivedbyendosymbiosis164,185genesderivedfromarchaeaandbacteria162geneticinstabilityofearlyforms197–8,207–8lackingmitochondria37asmonophyletic40,49–50morphologicalcomplexityandmorphologicalsimilarities34–6origins26,34,161–9,179–85signaturegenes162–3signatureproteins43straightchromosomesin42,47,158–60,217–18supergroupswithin40–1ubiquityofsexamong42,199,215,220seealsolasteukaryoticcommonancestor

eukaryoticcellschimericorigins12defined6morphologicalsimilarity1,34–6serialendosymbiosistheory6sizes158transportsystemswithin189–90

evolutioncellularevolution3,96–102ofchemiosmoticcoupling83,85convergentevolution20,45,151energeticconstraintson21ofeyes,flightandmulticellularity45–6gradualismin194‘byjerksandcreeps’162asnotpredictive21,221ofprotocells135reductiveevolution39timeline26seealsomutations;naturalselection

evolutionaryintermediatesabsence2,44–5,160,197archezoaproposedas39genuineexamples48losspredictedbynaturalselection45

evolutionaryrecapitulation285–6exergonicreactions114,131,150,294exoplanets,earth-like287exothermicreactions108,113,294extinctionrisk,asexualreproduction214–15extraterrestriallifeseelifeelsewhereextremophiles,eukaryotic43eyes

evolutionof45,196mosaicanalogy238

Ffattyacids294Fawcett,Don195fermentation294

alternativetochemiosmoticcoupling168alternativetorespiration79inbacteriaandarchaea10,83energeticsofrespirationand30nevolutionofrespirationand168andsenescence276,277nofwine100n

ferredoxin105,112,136,141,151–3fertility

balancewithfitness14,249–50,261,264–5,267balancewithlongevity277–9

FeSclusters/mineralsseeironFisher,SirRonald219–20fitness

balancewithfertility14,249–50,261,264–5,267‘hybridbreakdown’and249–50,252,258,265improvingformales242–3individualtissues228–30mitochondrialvarianceand228–30uniparentalinheritanceand223–5ofwholechromosomes213

fixation,carbon131,132,151fixation,mutations214,221,223,294fleas,bigandlittle192,201flight

evolutionof46mitochondrialperformanceand262–3

flowreactors,hydrothermalventsas102–9,112‘fluidchromosomes’198,213–16,293,297fluorescentdyes111,120Ford,Henry278formaldehyde

reductionpotential116–19fromstepwisereductionofCO2114,119

formatereductionpotential116–19fromstepwisereductionofCO2114

fossilrecord26–7,31,201freeenergy294

fromaprotongradient140requirementoflivingcells96,97–8Schrödinger’sviewsand52,61andspontaneousreactions61–2

freeradicals294inembryonicdevelopment263nrisktomitochondrialgenome243signalling270–2,276–7specieslifespanand273–4superoxideradical245–6,269,271n,275

free-radicalleakageandapoptosis245–7,256,264,266inbirdsandrodents266,268incybrids259–60nexerciseandlifespan274–6,278andmembranefusion219nandmitochondrialbiogenesis271nitricoxideand263nROS(reactiveoxygenspecies)and272

freeradicaltheoryofageing268–79FreeRadicalsinBiologyandMedicine,by

HalliwellandGutteridge270fungi

cellwalls159,281endosymbiosisin181matingtypes220osmotrophy32,34,296spores35asunikonts40

Ggametes

asproductsofmeiosis298sizedifference230

Garner,David133

genes294alleles213–16,224,291fragmentationofeukaryotic199–200interplayofenvironmentand29–34lossamongendosymbionts181–3lossinParakaryonmyojinensisendosymbionts286lossofandreplicationspeed180–1,183,242numbersinbacteriaandeukaryotes172–3orthologsandparalogs205–6phylogenyandlateraltransfer123recombinationineukaryotes200,211

geneticcodeenvisagedbySchrödinger22inferredbyCrickandWatson22origin135–6

geneticrecombination297ineukaryotes200,211immunesystemand200sexandwholegenomerecombination297

genomes294consequencesofmismatchedgenomes244,248–51,259ndualityandhybridbreakdown251,258,265dualityandmechanismofapoptosis247–8dualityandmetabolicrate261–3,267,279mitonuclearcoadaptation222–3,244,248,251,261–2,265asnotself-replicating180polyploidyincyanobacteria188polyploidyingiantbacteria176–8,188–9retentioninmitochondria186streamliningofbacterial174–6,180–1,201,208variationamongbacteria125seealsomitochondrialDNA/genome

‘genomeofEden’129–30genomesequencing

fullgenomecomparisons9,161interpretationproblems3wholegenomesequencing201

genomesizesendosymbionts181–3energyrequirementand172eukaryotes158–9humangenomecomparisons22–3mitochondria242mutationratesand216Parakaryonmyojinensis286

geochemistryrelationtobiochemistry27–8germcellaccumulationofmutations228germlines294

differentiationand227,256evolutionof231,233removalofmitochondrialmutations265sequestrationof227–8,230–232

gerontologyseeageingGerschman,Rebeca269Giardia37–8,199Gibbs,J.Willard61

seealsofreeenergyglaciations,snowballearth26,28,31,50,158,298glycerolstereoisomers84–5,153Goodsell,David74gorillas244,279gradualism194greatapes

chimpanzees125,244,259n,279

gorillas244,279lifespanofhumansand279

GreatOxidationEventapproximatetimingof26,28,34evolutionarysignificance28–31,36,47,50

Greenland25,27growthratesinmales253–4Gutteridge,John270

H‘Hadean’period

compositionofhydrothermalvents112likelyCO2levels113asawaterworld24,98,103

Hadjivasiliou,Zena222Haeckel,Ernst4haemoglobingenefamily206Haldane,JBS251–2Haldane’srule252–61Halliwell,Barry270Hamilton,Bill221Harman,Denham269Harold,Frank172heatlossbylivingthings62hereditarymaterial96

seealsoDNA;RNAhermaphroditism220,254n,258,259nHerschy,Barry119heterogameticsex252,260–1Hill,Geoff261homogameticsexchromosomes252homologousgenes162–3Hooke,Robert4horizontalgenetransferseelateralgenetransferhotbloodseeendothermyHuber,Claudia134humanbody

ATPturnover63energyuse64numbersandareaofmitochondria71numbersofcells238protonthroughput290

‘hybridbreakdown’249–50,252,258,261,265hybridcells(cybrids)259,260n,272hydrogen(H2)

inalkalinehydrothermalvents108–9,112alternativeviewofredoxreactions65asanelectrondonor79,102reductionpotential117seealsoprotongradients

hydrogen(H2)reactionwithCO2seecarbondioxidehydrogenbonding241,244hydrogeneconomy114hydrogenelectrode,standard116hydrogenhypothesis10–11,190hydrogensulphide

asananaerobicpreservative60nin‘blacksmokers’103aselectrondonor28,78–9,81–2,102–3

hydrogenases105,141seealsoEch

hydrogenosomes39,188,281,283hydroniumions(H3O

+)74,144

hydrophobicity57–9,84,186hydrothermalvents

alkalineventscomparedwithblacksmokers106–9discovery103discoveryofParakaryonmyojinensis281asdissipativestructures102asflowreactors102–9andtemperature92n,106,109–10seealsoalkaline;blacksmokers

hydroxideions(OH–)139–40hydroxylradicals(OH•)269hypotheses

chemiosmotichypothesis75–7definedanddefended14eocytehypothesis166hydrogenhypothesis10–11,190‘intronsearly’hypothesis200necessaryandtrivial186–7‘RNAworld’hypothesis92,96,100,298venerableeukaryotehypothesis162

Iimmunesystemandgeneticrecombination200information

genomesviewedas23,32lifeviewedas51,91–2

informationalgenes166inheritanceseebiparental;uniparentalinorganiccatalysts,metalsulphidesas100–1,106,132interbreeding198introns294

conservedpositions205–6endosymbioticorigins201,233mobileintrons201–3,206–8,217–18,286,294asnon-codingDNA42,200andtheoriginsofstraightchromosomes217–18andtheoriginsofthenucleus205–10andParakaryonmyojinensis285

introninvasionproposal204–8,216–17‘intronsearly’hypothesis200ionpumps

energetic144,146andevolutionofmembranes153,287seealsoprotonpumps;sodium

ironbanded-ironformations27–8,81n,112isotopicfractionation27,29inolivine108

iron,ferrousinearlyoceans106,108,112aselectrondonor28,78–9,81iniron-sulphurminerals104

iron-nickelsulphurclusters105,132,150–1ironsulphides

inalkalinehydrothermalvents112–13in‘blacksmokers’104,112‘pyritespulling’proposal104–6,115

iron-sulphurclusters(FeSclusters)294intheacetylCoApathway132inelectronbifurcation150npeptidebinding101inrespiratoryenzymes104–5,133n,246–7asrespiratoryintermediates65–7

iron-sulphurmineralsinalkalinehydrothermalvents108,117–19,134,287greigiteandmackinawite104–5polypeptideformation101

iron-sulphurproteinsalkalineventmodels112Echas136ferredoxin105,112,136,141,151–3

‘iron-sulphurworld’104–6isotopicfractionation25,27–9

JJacob,François221Japan281jargon15jumpinggenes55,192,201,215‘junkDNA’

arisingfromsexualreproduction215possiblefunction22n,204nseealsointrons

Jupiter,GreatRedSpot95

KKeplerspacetelescope287nkineticbarriers80,114,287kineticenergyandthermophoresis110kinetics,interplaywiththermodynamics113–14,298Kluyver,Albert4,5Koonin,Eugene41,204,206,208Kuhn,Thomas76

LL-formbacteria99,159,218Lane,Nick,previousbooks109,136,187n,270,279nlasteukaryoticcommonancestor(LECA)

ancientandrecentparalogs206endosymbiosisasessentialto193monphyleticradiationfrom39–41,51nuclearporesin195asaphylogenetic‘eventhorizon’43–4,160possiblesourcesofsharedtraits51,160,193sexualreproductionin215

lastuniversalcommonancestor(LUCA)295ofbacteriaandarchaea83–5,128–9aschemiosmotic287lateralgenetransferand128–30possibledevelopmentof131–7,149treesoflifeand124–6

lateralgenetransfer295aftertheendosymbiosisevent164,167andthebacterialmetagenome181chromosomesizeand159–60contrastedwithsexualreproduction46,198–9,215–17eukaryoticgenome164fromfailedendosymbionts207‘fluidchromosomes’from215leavingarchaeaandbacteriadistinct130,151protectingbacteriaagainstextinction47ofrespiratoryproteins82underminingthetreeoflifeconcept123–6,128

leakycells140,142–3leakymembranes138–40,142,145–6,287

divergenceofbacteriaandarchaea154Leaver,Chris259

Leber’shereditaryopticneuropathy255,258LECAseelasteukaryoticcommonancestorLeeuwenhoek,Antonyvan3–4LifeAscending,byNickLane136,187n,279nlifeonEarth

conflictingviewsofhistory24,50–2earliestevidence25energeticsandtheoriginsof90hydrothermalventsand103,106–8inorganicandorganiccatalysts100–1timeline26seealsolivingthings

lifeelsewherecellularevolutionandspeculationabout2complexityasimprobable191,288dependenceonchemiosmoticcoupling170dependenceonprotongradients154hydrothermalventsand121,287likelihoodofpossessingmitochondria288,289numberofearth-likeplanets287predictability86redoxchemistryand82,120whethercarbon-based78

lifespanbalancewithfertility277–9birds266,274,278andfreeradicals273–6,278rodents266

lightningasanenergysource81,91–2proton-motiveforceequivalent73,187,243

Linnaeus,Carl4lipids

evolutionofmodernmembranes143,153,209freeradicalvulnerabilityof246internalprecipitationasmembranes209inmembranestructures58spontaneousbilayerformation57,98–9seealsomembranes

livingfossils284livingthings

defining53–5entropyand56,59propertiesofallcells96–102,129

longevityseelifespan‘LostCity’alkalinehydrothermalvent107,109–13Lovelock,James6LUCAseelastuniversalcommonancestor

Mmales

colorationinbirds261costof215fitnessof242–3genomicmismatchinDrosophila259–60growthrate253–4mitochondrialdiseaseprevalence258selectivekilling259nvisibilitytoselection252

mammalsmitochondrialmutationrate262–3paralogousgenes206sexualdevelopment252–3

Margulis,Lynn

oxygen‘holocaust’30serialendosymbiosistheory5–6,34,36–7,39treeoflifeand9

Mars98Martin,Bill

author’scollaborationwith14,137,143,171,172ncollaborationwithEugeneKoonin204,208collaborationwithMikeRussell105,108collaborationwithMiklósMüller10–11,190hydrogenhypothesis10–11,190onhydrothermalvents108,115noniron-sulphurmineralsandclusters105,108treesoflife12,126–8,164–5onuniversalityofmitochondriaineukaryotes39

massextinctions30–1,47matingtypes220,225–6MaynardSmith,John160McCollom,Tom113McInerney,James163Medawar,Peter14,54meiosis199,211–12,295membranes295

inbacteriaandarchaea83–4,129,147–8,153‘coupling’144andtheenvironment77eukaryoticasabacterialreplacement209evolutionofmodernmembranes143,147–8,153free-radicalinducedfusion219nmosaicmembranes58,209respiratory,potentialinternalisation185siteofbacterialATPsynthesis173–5spontaneouslipidbilayerformation57,98–9structureoflipidmembranes58seealsoprotongradients

membranebioenergeticsseechemiosmoticcouplingmembranepermeability

effectsofprotonpumps146–7leakymembranerequirement138–40,142,145–6,287toprotons85,137–48tosodiumions143–4

membranepotentialsconsequencesofcollapse137,188,246–7needforlocalisedcontrol243

membraneproteinsarrangement58Echas136promiscuityin144seealsoATPsynthase

Mendel’sdemon,byMarkRidley217metabolism,defined295metabolicactivity

acetylCoAincarbonandenergymetabolism133catalysisofthebeginningsof100asnecessaryattributeoflife53–4precedenceofreplicationor96–7protongradientsincarbonandenergymetabolism120separationofcarbonandenergymetabolism78–9

metabolicpathwaysaschannels89–90metabolicrate

andcytoplasmicmalesterility259giantbacteria178–9andlifespan268,270,273–6metabolicdemandsand255–8,260nmitochondrialdiseasesand255

andsexdetermination253–4sexdifferences274nseealsoenergypergene

metabolicsyntrophy190metabolicvirtuosityofbacteria4,29,80metagenomes181,198metalsulphides

asinorganiccatalysts100–1seealsoiron;nickel

Metazoa41seealsomulticellularity

methanekineticsofproduction113oxidationandglaciations28andprimordialatmospheres24–5,28n,79fromreductionofCO2113–14,141

methanogensasancestralarchaea152biochemistryandtheventscenario136determiningageof126,128earlyevidence28nelectronbifurcationin150,152,169energybudgets140–1membraneproteinsof144relianceontheacetylCoApathway131sodiumionpumpingby148nsubsistenceonH2andCO2115useofantiporters143useofproteingradients137,140

methanol114–15methylthioacetate134–5,148microtubulesinplasmids218Miller,Stanley90–1,104nMiller–Ureyexperiment90–1mineralclustersseeiron;metalsulphides;nickelmiscarriages248,267–8Mitchell,Peter13,75–7mitochondria295

electronmicrographof72eukaryoteslacking37–9hydrogenosomesandmitosomesderivedfrom39importancetocomplexlife186–91,238–9inheritanceof220–1iron-sulphurclustersin133n,246numbersandareainhumanbody71numbersinamoebae184originsasbacterialendosymbionts5–6,36,167andplumageofmalebird261predatingchloroplasts9–10promotingcellfusion219replicationrate221–2rolesof233,245–7assiteofcellularrespiration68–9structure72,238–9timeofacquisition9–10,38

mitochondrialbiogenesis271,295mitochondrialdiseases

antioxidantsand270anddiet277nembryonicdevelopmentand248metabolicrateand256–7mosaicanalogy238mutationeffectsontissues227–8,256–7

prevalenceinmales258weakselectionagainst265

mitochondrialDNA/genome295andaviancoloration261contributiontorespiratoryproteins239cybridexperiments259,260n,272digestionofoneset220–1genelossandretentionin184–7,189,242influenceoftheenvironment264–5mutationrateandactivity263–4mutationrateandvariance226–7,230–1,240mutationratecomparedwithnucleargenes226,241–2,251mutationsandageing276mutationsandgermlinesequestration232mutationsin,andfitness221–2transfertothenucleus185,207

mitonuclearbreakdown251seealsohybridbreakdown

mitonuclearcoadaptation222–3,244,248,251,261–2,265mitonuclearincompatibilities258,261,265,267–8,276mitosis42,182,218,295mitosomes39,188mitoticspindle218Mittwoch,Ursula254,260,274nmobileintrons201,203,294modelling,mathematical

evolutionoftheeye196protongradients147sexualreproduction147

MolecularBiologyoftheCell186Monod,Jacques20monophyleticradiation295

eukaryotesasmonophyletic40,49–50predictedunderstructure-determinedevolution33

Moon,origins24Morowitz,Harold136morphologicalsimplicityofprokaryotes2,11,157mosaicanalogy237–8mosaicmembranes58,209mosaicmitochondria244,279,289mosaicrespiratorychain239–40,243,262–3movementandmitochondrialmutation232Mulkidjanian,Armen145nMüller,Miklós10–11,190multicellularity

apoptosisin247multicellulareukaryotesasdiploid211multicellularorganismsasclones221originsandevolutionof33,46spongesandcoralsasmodels226–7,230,232traitsdeterminedbymitochondrialvariation231universalityofuniparentalinheritance225seealsocomplexlife

mutations295accumulationbygermcells228fixation214,221,223,294highrates,favouringsexualreproduction216–17loadfavouringuniparentalinheritance223,230lowrates,inbirds263sexualcycleeffectson212–13

mutations,mitochondrialseemitochondrialDNA/genomeMyojinKnoll281,283

N

NaseesodiumNADHusebyacetogens152Naegleria44nanometreunits(nm)69n,291NASA(NationalAeronauticsandSpaceAdministration)55naturalselection

achievements289antiportersandselectiveadvantage145apoptosishonedby247–8,256atchromosomelevel213–14complexityfrom,requiringendosymbiosis193andearlierformsofselection135gametevisibilityto213immunityofmultiplegenomes180lossofevolutionaryintermediates45,197formosaicrespiratorychains243asopposingchange196ofuniversallyconservedsystems149

‘naturevs.nurture’influences32necessaryhypothesis186–7neurons

theeyeasaneuronalmosaic238asirreplaceable256

NewScientist122nickel

inalkalinevents112iron-nickelsulphurclusters105,132,150–1volcanicsource106

Nietzsche,Friedrich99nitrateandnitriterespiration32,79,82nitricoxide115n,263nNitschke,Wolfgang82,115nnuclei296

asabsentfrombacteria47chimericproteins210asconservedfeaturesofeukaryotes41–2evolution194–6,210intronsandtheoriginsof205–10structure194transferofmitochondrialgenesto185,207

nucleargenomemitochondrialmembraneproteinsand239–41mitochondrialmutationsand244

nuclearmembrane42,194,208–10Parakaryonmyojinensis283

nuclearporecomplexes194–5,210,283nucleotides296

catalyticdinucleotides136concentrationbythermophoresis111energeticsofpolymerisation172origins92,97

numts(nuclearmitochondrialsequences)207

OOccam’srazor97n,167oceans,ontheearlyEarth24,93,113olivine108,121,286opticnerve255organelles

claimedasendosymbionts36mitochondriaandchloroplastsas6

organics,concentrationbythermophoresis110,119Orgel,Leslie68TheOriginofSpecies14,45,122

originsoflifeseelifeonEarthorthologs25,296osmotrophy32–4,296ostricheggs100Otto,Sally216outbreeding219,225noutcrossing250,258Owen,Jez216‘oxphoswars’75,82oxidation296

aselectrontransfer64–5offerredoxin152oftopoisomerase-1271

oxidisedstatesearlyearth25GreatOxidationEvent28–9,31,34,36,47,50

oxygenabsencefromtheprimordialatmosphere25,112alternativesto,forrespiration79consumptioninprokaryotesandeukaryotes171asenvironmentaltriggerforcomplexity29–34freeradicalformation269andmethaneproduction113stabilityofspores,seedsandvirusesin60

Oxygen,byNickLane109,270‘oxygenholocaust’proposal30,47

PPacificOcean,MyojinKnoll281,283panspermia,irrelevanceof94nParakaryonmyojinensis284,285n,286–9‘parakaryotes’284paralogs206,296paramecium173,176–7parasites

geneticparasites208,217plantsandanimalsviewedas54–5selectivekillingofmalehosts259nvulnerabilityofclonalpopulation214n,217

Parkinson’sdisease256Pauling,Linus269Pearl,Raymond273,277Penny,David199peptides

ATPrequirementofbonds172,183formation,acetylphosphatecatalysis101,102n

pH296andreductionpotentials116–18,120

pHgradientsseeprotongradientsphagocytosis296

asabsentfrombacteria47,159inevolutionaryenvironments32notrequiredforendosymbiontevent166–7,181n,182Parakaryonmyojinensisvacuolesand285nsuggestedinearlyeukaryotes37–9

pheromones225nphotosynthesis296

anoxygenic81,102carbonfixationby131evidenceforearlyforms28originsofoxygenic26,28–9respirationasbasisof81

phylogeneticseukaryoticcommonancestorand40,160

seealsotreesoflifephylogeneticrevolution6–9phylogenomics42–3picoeukaryotes43pigeons246,266,268Planctomycetes44planets,evidenceforlife29plants

cytoplasmicmalesterility258–9thermoregulationin46nviewedasparasites54–5

plasmids296chromosomesegregationand218andthedefinitionoflivingthings55potentialforenlargingprokaryotes189,191

platetectonics5,104,108poltergeists,energeticsof57,59polychaeteworms,microbesof281polymerisationreactions98,119polypeptides,distinguishedfromproteins102n,297polyphyleticradiation/origins296

oxygenconcentrationand196npredictedbyserialendosymbiosis36predictedbystandardselection33–4,36

polyploidy176–8,188–9Pomiankowski,Andrew14,140,216,222populationgenetics,mathematical216,225–6,302populationgrowthandsexualreproduction215porosity,alkalinehydrothermalvents109–12,138nPower,Sex,Suicide,byNickLane279npreadaptations,antiportersas146prebioticchemistry93pressures,hydrothermalvents115Prigogine,Ilya94primordialatmosphere24–5,92primordialbiochemistry130‘primordialsoup’

asdiscredited50failuretomeetcellformationrequirements102inabilitytodriveformationofdissipativestructures95Miller-Ureyexperimentand90–3Wächtershäuser’sviewof104nwasteremovalproblems99–100

prions59prokaryotes296

archaeaas10,126nbacteriaas126nchemiosmoticcouplingasinescapable167–71constraintsoncomplexityin49examplesofendosymbiosisbetween181internalisingrespiration185Parakaryonmyojinensisas284–6twodomainsof48–9Woese’srejectionoftheterm9

proteins296–7behaviourofhydrophobic57,59distinguishedfrompolypeptides102nenergeticsoffolding59–60energyexpenditureofsynthesis172inlipidmembranes58ribosomesinproteinsynthesis8

proteinmachinesATPsynthaseas73inrespiration68–70

spliceosomesas200a-proteobacteria36,163,184,202b-proteobacteria182g-proteobacteria182protists2,4,45,125,231,297

aseukaryotes30–1,40–1,293Paramecium173,176–7seealsoamoebae

protocellformation134–5protons(H+)297

membranepermeabilityto85,137–48transferredintherespiratorychain70

protongradients297additionalfunctions170inATPformation68,137collapseofandcelldeath170dependenceofenergygenerationon13andthedivergenceofbacteriaandarchaea147–8,154drivingH2reactionwithCO2115–21,132,137electrochemicalpotentialfrom71energystoragein71,77,86,169leakymembranerequirement138,140usebymethanogens137

protongradients,naturalinalkalinehydrothermalvents115–21,134–6,138,154drivinganantiporter141,145earlylifedependenceon86,109,120,287fromelectronbifurcation151independencefrom146LUCAdependenceon149,151–2inprotocells135–40,142–9

proton-motiveforce290,297collapsecausingcelldeath170asconservedacrossalllife73,286–7needforlocalcontrol187originalproposalof75–6

protonpumpsinacetogensandmethanogens151advantageouswithantiporters146disadvantageoustoleakyprotocells142–3electronbifurcationand150npoweredbysodiumgradients143throughputinthehumanbody290

protozoa297‘pyritespulling’proposal104–6,115pyrophosphate102n

Q‘quantumtunnelling67,69,239–41quinine111,120

Rradiotelescopes19–20Radzvilavicius,Arunas227rats260n,263–8,274,278reactivebiogenesis271nrecombinationseegeneticrecombinationredoxcentres297

respiratorychain69,239–41,244–5redoxcouples80–1,169,297redoxreactions297

advantages79universalityforenergygeneration64–5

universalityforprotongradientgeneration77–8reductionpotentials

changing,forH2andCO2136asmeasuringelectronaffinity115–16pHeffects116–18,120ofredoxcentresinrespiration239,246n

Reid,John187nreplication297

precedenceofmetabolicactivityor96–7seealsoDNAreplication

replicatorsclayminerals97RNAasthefirst91

replicons153,159reproduction

barriersbetweenspecies250seealsocloning;sexualreproduction

respiration297advantages79anaerobic32ATPregenerationby63–5inbacteria5,29asbasisofphotosynthesis81aschemiosmotic75evolutionoffermentationand168heatlossfrom62internalisingbyprokaryotes185locationinmitochondria68–9oxygenandenergyproduction30rateinprokaryotesandeukaryotes171regulationbymitochondria188,243requiringnuclearandmitochondrialgenes222,243wasteandbiomassfrom90

respiratorychaincomplexescomplexI66–7,69,72,76,238,246complexII70,72complexesIIIandIV70,72,240effectsofreduction190,245–7,256–7,264,271nandfree-radicalsignalling271,274

respiratorychains297alternativepathway246nconsequencesofmismatchedgenomes244,247–8mosaicrespiratorychain239–40,243,263asmosaics239–40,243,262–3overview68–70redoxcouplesfor81

respiratoryenzymes/proteinsironin65iron-sulphurclustersin104–5,133n,246nuclearandmitochondrialgenes239

retrotransposons55reverseengineering167reversetranscriptases201–3riboseanddeoxyribose,synthesis119ribosomes297–8

inbacteriaandeukaryotes172exclusionfromthenucleus209inParakaryonmyojinensisnucleus283–4,286inphylogeneticinvestigations7–8,123–4,164–5inproteinsynthesis8,172

Rich,Peter75nRickettsia183Ridley,Mark217

RNA298asthefirstreplicator91RNAscissors201–2,204,208

‘RNAworld’hypothesis92,96,100,298ROS(reactiveoxygenspecies)272Russell,Mike105,108–10,115n

Ssaltationandadaptation194samplesofone21Schrödinger,Erwin22,51–2,59,61seawater,organicsin93secondlawofthermodynamics22,56,96sedimentaryrockswithbiologicalorigins27–8seeds

survivalinanoxygen-richenvironment60survivalinextremeconditions55–6

segregationchromosomal218,228–9mitochondrialmutants228–9,255

selectiveinterference214,216–217selectivesweeps214,216,298self-organisationofmatter

concentrationand102,134energyfluxand94

selfishconflict221,223,231,259,298selfishDNA201senescencealternativetoapoptosis276–7serialendosymbiosistheory6,34–9,43,164

contrastedwiththehydrogenhypothesis11‘serpentinisation’108,112,121,149,298serpentinite108SETI(searchforextraterrestrialintelligence)19sewage,asanaerobicpreservative60nsexchromosomes

homogametic252sexdeterminationwithout253X,WandZchromosomes252,261Ychromosome213,220,252–4

sexdetermination298Haldane’sruleand252–61variedmechanisms253withoutsexchromosomes253

sexualreproduction298advantages213–14,216–18ascommontoalleukaryotes42,199,215contrastedwithcloning213–16contrastedwithlateralgenetransfer46,198–9,215–17dealingwithmutations212disadvantages215–16favouredbyhighmutationrates216–17infirsteukaryotes198–9Haldane’sruleand252–5originsof211–19andpopulationgrowth215reciprocityasdistinguishing211sexualcycle212twosexes43,219–26,241

Shock,Everett132Sicily237,238nsight

evolutionofeyes45,196andmetabolicrate255

‘signatureproteins,’eukaryotic43

silicon,asalternativetocarbon78singularendosymbioticevent10–11,86,183n,191,193,279Smith,Eric136‘snowballearth’glaciations26,28,31,50,158,298sodiumions(Na+)

enzymeoptimisation144–5membraneproteindiscrimination144pumpingbyantiporters143pumpingenergetics146–8n

Sojo,Victor140SPAPs(sodium-protonantiporters)147species

definitionof123,198,250genomecoadaptationin244–51lifespanandfreeradicals273–4mitochondrialchangeandspeciation251

spermsizedifferencefromeggcells230spliceosomes200,202–4,208,210sponges226–7,230,232spontaneousreactions

exergonicproductionoforganics113–14,131–2freeenergyrequirement61–2,95polypeptideformation102ninreactiveenvironments62,80

spontaneousvesicleformation57,59,98–9,110spores

entropyand56–7,59survivalinanoxygen-richenvironment60survivalinextremeconditions55–6

SRYgene253staining,DAPI177standardhydrogenelectrode116Stanier,Roger5stars,energeticsof57n,62statisticalprobability21sterility250,252,255

cytoplasmicmalesterility258–60straightchromosomes,ineukaryotes42,47,158–60,217–18stromatolites26–7structuralconstraintsonbacterialevolution158–60substrates298sulphateandsulphiterespiration32,79sun,energyreceivedfrom64supercomplexes238supergroups,eukaryotic40–1superoxidedismutase246superoxideradicals245–6,269,271n,275surface-to-volumeratios

enlargingbacteria173,185vesiclegrowthanddivision99

Sutherland,John92symbiosis5,36

seealsoendosymbiosisSzent-Györgyi,Albert28

Ttardigrades55–6temperature

aroundhydrothermalvents92n,106,109–10andfreeenergychange61andhybridbreakdown258andsexdetermination253–4

terminallydifferentiatedtissues256

terminology15,49ntestes254,255n,260,276Thauer,Rolf150thermodynamics298

interplaywithkinetics113–14,298secondlaw22,56,96

thermophoresis110–12,119,134,299thioesters79,133–4,148Thiomargarita176–8TheThirdMan(film)157Tigriopuscalifornicus249Tiktaalik48timelineofevolution26tissues

dysfunctional228–30metabolicrequirements255–6,260nnon-regenerating232,256

topoisomerase-1271trade-offsseecost-benefittraits

commontoallcomplexlife1,14commontoalleukaryotes51,86,160,193,215

transcriptionandtranslation299transcriptionfactors144,204n,271ntransportsystemsineukaryoticcells189–90treesoflife

‘amazingdisappearingtree’126–7archezoain38basedoninformationalgenes166BillMartin’sversion12,126–8,164–5CarlWoeseand7–8Darwinand122eukaryoticsupergroups40–1lateralgenetransferand123–6,128LynnMargulisand9singlegeneandfullgenome161seealsophylogenetics

Triboliumspp.258trivialhypothesis186twosexes43,219–26,241

Uubiquinone67,72unikonts40–1uniparentalinheritance299

advantagesanddisadvantage223–4ofmitochondria220–3origins230–1universalityinmulticellularanimals225,231varianceincreasedby222,225,227

unitsångströmsandnanometres69nofentropy56

universe,lifeelsewhereseelifeelsewhereUrey,Harold90–1UV(ultraviolet)radiation

asanenergysource65,81,93formationofcyanides79,92–3intheoriginoflife92–3

Vvacuoles296

ingiantbacteria175–7inmicrographsofeukaryotes35,38,44

inParakaryonmyojinensis285nvanNiel,Cornelis5variance299

increasedbyuniparentalinheritance222,225,227mitochondrial,andorganismfitness228

‘vectorialchemistry’77‘venerableeukaryotehypothesis’162ventobionts(Ediacarans)31verticalinheritance295

treesoflifeand122,125vesicleformation57,59,98–9,110viruses

infectioncausingprogrammedcelldeath170survivalinanoxygen-richenvironment60survivaloutsidehosts55–6whetheralive53–6

vitamins54–5,269,273

WWächtershäuser,Günter

onmethylthioacetate134‘pyritespulling’proposal104–6,115reductionpotentialsand117viewsofRussellandMartincompared108

Walker,John76Wallace,Doug251,264wasteproduction

characteristicoflivingcells96,99–100fromfermentation168fromrespiration90

wateraselectrondonor28,78,81inthe‘Hadean’period24,98,103passageofprotons70requirementsofpolymerisationin98

waterchannelanalogy89–90Watson,James

geneticcodeinferredby22Miller-Ureyexperimentsascontemporary90–1Mitchellascontemporary75

Welles,Orson157WhatisLife?,byErwinSchrödinger22,51Wheeler,JohnArchibald120Whicher,Alexandra119Woese,Carl7–9,123–4Wolbachia259n

YYchromosome213,220,252–4yeasts162,168,247,251,294YellowstoneNationalPark102

Zzincsulphidephotosynthesis145nzircons24–5,79,91n

NICKLANE isabiochemist in theDepartmentofGenetics,EvolutionandEnvironmentatUniversityCollege London and leads the UCL Origins of Life Programme. He was awarded the 2015BiochemicalSocietyAwardforhisoutstandingcontributiontothemolecularlifesciences.HeistheauthorofLifeAscending:TheTenGreatInventionsofEvolution,whichwonthe2010RoyalSocietyPrizeforScienceBooks,aswellasPower,Sex,Suicide:MitochondriaandtheMeaningofLifeandOxygen:TheMoleculethatMadetheWorld.

ALSOBYNICKLANE

LifeAscendingTheTenGreatInventionsofEvolution

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