This article is protected bycopyright.All rights reserved version andthe VersionofRecord.Please cite thisarticle as copyediting, typesetting, paginationand proofreadingprocess,whichmay leadtodifferences betweenthis This article hasbeenaccepted forpublication andundergone fullpeer review buthasnotbeenthroughthe Article type : Perspective DR. JONATHANPZEHR(OrcidID: 0000-0002-5691-5408) Department ofBiochemistry andMicrobiology,Rutgers University,Jersey, NewBrunswick, Center forFundamental andApplied Microbiomics& SchoolofLifeSciences, ArizonaState AcceptedDepartment ofOceanSciences, UniversityofCalifornia, SantaCruz,California, USA95064 Article Department ofBiology,Washington 63130 University,St. Louis,Missouri,USA What’s inaname?Thecaseofcyanobacteria University, Tempe, Arizona,USA85287 Debashish Bhattacharya, and Himadri B.Pakrasi Ferran Garcia-Pichel Jonathan P.Zehr USA 08901 USA doi: 10.1111/JPY.12934 2 , This article is protected bycopyright.All rights reserved Sericytochromatia, Margulisbacteria, Saganbacteria. Keywords: phylogenomics,oxygenic photosynthesis, blue-green algae,Melainabacteria, organisms.” reduce carbon dioxideasasource ofcarbon,orthosesecondarily evolvedfromsuch domainbacteria able tocarryout oxygenicphotosynthesiswithwater asanelectron donorandto distantly relatednon-phototrophic lineages. Wedefine Cyanobacteria hereas“Organismsinthe A redefinition ofthecyanobacterial lineagehasbeen proposedbasedonphylogenomic analysisof ABSTRACT Editorial Responsibility: R.Wetherbee (AssociateEditor) Running title: DefiningCyanobacteria Fax: 831-459-4882 Phone: 831-459-4009 Email: [email protected] 2 Accepted Article Corresponding author This article is protected bycopyright.All rights reserved Acceptedbenefit ofthisconcordance isthatidentification ofcyanobacterial sequencesina molecularsurvey functionality, phylogenyand nomenclature isacherished, very usefulrarityamong bacteria.Adirect are clearly descendants ofoxygenicphototrophs.This level ofconcordance betweenbiological lostPSII,RuBisCOandphotopigmentknown asUCYN-A) genes(Tripp Castenholz some, 1990).And suchasthesymbiont photosynthetic modewhenPhotosystemII(PSII) is poisonedbyhydrogensulfide (Garcia-Pichel and oxygenic. True, photosynthesis. Ofmorethan 500cyanobacterial genomesthat havebeen sequenced,all butafeware intellectual propertydocumentshave usedittodescribe prokaryotescapable ofoxygenic by Stanierand colleagues(Stanier 1977),morethan 23,000publishedpapersandnumerous maintains thetwoasseparate Phyla.Andyet,sincethe timewhenthe termcyanobacteria coined was recognizes thecloserelationship between Melainabacteria andCyanobacteria in aranklessgroup,but conceptualized taxonomy hasalready madeitswayinto widelyuseddatabaseslike Silva,whileNCBI correspondence betweensystematics andfundamental biological functionality. Thisnewly a newinformal definition ofthePhylumCyanobacteria, the proposalsdoawaywiththe non-photosynthetic bacteria (Melainabacteria and Sericytochromatia) belongingto siblingcladesinto ArticleCyanobacteriota asmore recentlymodified (Soo do notsharethe mainmetabolic characteristics ofcyanobacteria shouldbenamed cyanobacteria --or them, butwhatcyanobacteria infact of markergenes commontomostbacteria and archaea,have challenged notjust fundamentally different, deeperconundrum.Recent proposalsbasedonphylogenetic studiesofa set the recognition of“cyanobacteria” asabacterial clade attherank ofPhylum,weareconfrontedwith a microorganisms. Butjustwhenthingsseemed tohavesettled downinthelast thirtyyearsorsowith that havebeenusedtoname thisevolutionarily distinct, easilyrecognizable groupofphotosynthetic cyanophyceae, blue-green algae,oxyphotobacteria, andcyanoprokaryotes aresome oftheepithets microbiologists (andtheir respectivetaxonomic codes)foralmost twocenturies: myxophyceae, studies. They have alsobeenthe subjectoflively nomenclatural discussionsinvolvingbotanists and important microorganisms onEarth,andthe subjectofmanybiological, ecological, and evolutionary The cyanobacteria, previouslyknownasblue-greenalgae, havebeen, andstillare, oneofthemost

a fewareable tocarryoutanoxygenic photosynthesistemporarily asthe main are . Sooetal.(2014)argued thatanentire groupoflineages that

et al. 2019).Byincludinglittle-known, butclearly Candidatus

Atelocyanobacterium thalassa Atelocyanobacterium

et al.2010).Buteven these what weoughttocall (also This article is protected bycopyright.All rights reserved Accepted thus is It 2003). al. et (Garcia-Pichel tons metric million thousand a a with surpassing lands, arid biomass of global expanses vast in and oceans the in productivity primary of drivers major the of one remain cyanobacteria today, Even cross-talk. evolutionary this of because only exist Botany and Phycology 2010). (Keeling forms plant and algal to symbioses through rise gave cyanobacteria CO of reduction the for donor electron of source unrestricted virtually a into tap to cyanobacteria allowed that planet our of history evolutionary the in feat momentous a photosynthesis, oxygenic of development the by marked is transition major This group. distinct a as evolved cyanobacteria the which within context evolutionary the and provides 2013) it because al. underestimated, be cannot findings et supported robustly its Rienzi and study genomic elegant Di this 2005, al. et of importance The cyanobacteria. with ancestor (Ley common a share 2014) al. et (Soo Melainabacteria Sericytochromatia that concluded They 1). cyanobacteria to (Fig. clades sibling deeply-branching are that Melainabacteria, and Sericytochromatia wider biological context canoften besubjective. a in data novel these of interpretation results, phylogenetic of implementation routine a being from far However, 2016). al. et (Hug organisms of lineages diverging early suggest and deep are which of some branches, new with replete Articlenow is and changed, dramatically has life of tree The genomics. cell their and discovered been single- including techniques, cultivation-independent using have determined been have sequences genome microorganisms, uncultivated including organisms, new years, evolutionary and similarities few past of the In hierarchies. taxonomic inferring to approach pragmatic a enabling thereby relatedness, identification facilitated It taxa. of classification and description groups ofheterotrophs. formal re-definitionto redressthesituation whileleaving roomtoaccommodate the newlydiscovered relationships wereathandand wouldhaveconstituted amoreparsimonious approach.Weproposea concordance betweengenomics andecological functionand informationonthe evolutionary and arelikely tocause muchconfusion,particularly whenalternatives that preserveboththe being studied. immediately provides stronginferencepowerabout thenature ofprimaryproduction intheecosystem o e al et Soo the on effect profound a had has approaches phylogenetic molecular in revolution The

We contendhere thatthese recentnomenclatural changeswereunnecessary,ill-timed, ( . 2014 ) eotd hlgnmc nlss f el ietfe bacteria, identified newly of analyses phylogenomic reported 2 : water. Early on during the diversification of the phylum, the of diversification the during on Early water. :

This article is protected bycopyright.All rights reserved Acceptedgroups canbegleaned fromatree oftheribosomal (Fig.1),whichreproduce 16S rRNA thosearrived Margulisbacteria andSaganbacteria (Matheus Carnevali into asingle lineofdescent. Twootherlikely sibling clades havebeensubsequently described: sequences ofasubsetshared genesplacedSericytochromatia, Melainabacteria and Cyanobacteria clade tocyanobacteria. Sooetal. (Soo al.2014)demonstrated thatphylogenies obtained from and cytochrome that they lackall genesforthylakoid membrane-bound electron transfercomplexes suchasPSI,PSII taxonomic worldwide has alga import thatissupportedbytheir uniquecell andgenomebiology. red term The 2018). al. et (Bhattacharya organelle photosynthetic phycobilisome-containing a and bodies, basal few and flagella with of inventories absence phagotrophy, gene of nuclear lack introns, reduced highly namely, algae, red of attributes major the lack algae they because Rhodophyta heterotrophic red as considered be scheme taxonomic sensible any in not would to group sister (Gawryluk a Archaeplastida form the within and (Rhodophyta) plastid primary genome-lacking a contain that of flagellates finding recent the is example analogous An use. Articlepractical little of be to consider we that the taxon order high heterogenous and biologically a of changes creation nomenclatural unnecessary avoid would that biologically- approach a alternative provide explicit to based, wish expanded we Here the bacteria. With blue-green colorless appearance. have blue-green would one a meaning, them give often that phycobiliproteins, pigments, The 2017). (Blankenship as antenna photosynthetic of presence the to refers fact in cyanobacteria in “cyano” root the of etimology such cyanobacteria’ terminology ‘nonphotosynthetic awkward and of cyanobacteria’ use the ‘photosynthetic requires nomenclature proposed The phylogenetically groups. three distinct these of treatment systematic a for options alternative obviating and Class, a to Phylum the demoting 2015), (Castenholz cyanobacteria to synonym a considered is and use little had has that term a oxyphotobacteria, to cyanobacteria traditional the for epithet the changing 2019), al. et (Soo Cyanobacteriota recently, more again once or, Cyanobacteria group entire the rename to on went (2014) al. et Soo however, regrettably, opinion our In bacteria. fermentative anaerobic, likely of one place: took first event evolutionary this which within context biological the delineate to exciting Early studiesreportingMelainabacteria (Leyetal. 2005,DiRienziet al.2013)demonstrated b 6 f , aswell asthoseinvolvedinaerobic respiration, and treated themasasibling Rhodelphis:

t l 21) Ti nvl eeorpi lineage heterotrophic novel This 2019). al. et

et al.2019).Therelationships amongthese ee n ito rc, phagotrophic rich, intron and gene This article is protected bycopyright.All rights reserved Accepted2013 foranaccount ofwhatallthis modeoflife entails) bearsnoweight inthemulti-gene machinery andphotoautotrophic modeoflife ofcyanobacteria (seeOvermann andGarcia-Pichel are related tooxygenic photosynthesis.Andyetthe evolutionary investment indeveloping the all proteins ofcyanobacteria byweight,and typically 20-30% ofallORFsincyanobacterial genomes the Calvin-Benson cycle forcarbonfixation. Butphotosynthetic proteinsmakeupmore than50%of rooted sistergroupsare completely missinggenesfor the photosyntheticapparatus aswellthosefor major innovationanditssisterThis groups. isclearlythe caseforcyanobacteria. All ofitsdeeply- will provide underestimates ofthetrueevolutionary distance betweenthe groupthatunderwentthe encode the novelprocesseswill,bydefinition, beexcluded fromthe analyses.Hence,the comparisons fundamental metabolic innovation takesplace withintheorganismal set,the molecular sequencesthat marker genes thatareuniversally presentamong theorganismstobe compared.Incaseswhere a for ourdiscussion,isthefact thatmolecular phylogenetic studiesare basedonthecomparison of perhaps presentin someyettobediscovered member organisms.Second,andperhapsmostrelevant to provebecause allphotosynthesisgenes aresofarmissingin Melainabacteria mighthaveshared aphotosynthetic ancestorwithcyanobacteria, a pointnotpossible complex, withmetabolic diversitybeing inherited frombothaerobesandanaerobes andthat Articlecurrently knownfeatures.Anetworkanalysisapproachthat shows theevolution ofcyanobacteria was more intricate relationship ortowardsamore robustfunctional separationthat isconsistentwiththe current picture mightchange soon asmoremembers orlineagesare described,tipping thescalestoa provide onlybest guessesastotheirmetabolism orrolesin theenvironment. Itisconceivable thatthe come fromsingle-cell genomesofenvironmental samplesormetagenome-assembled genomes,which of isolateshave beenstudied, andmanyofthesequences andgenomesusedinthese typesofstudies often indirectly obtained frominventorying knowngenerepertoires intheirgenomes. Onlyahandful cyanobacteria. First,ourknowledge ofthenewsiblinggroupstocyanobacteria isquitemeager, and to pointout severalfactorsthat mayresult inabiaseddepiction oftheir relatednesswith Although wedonotcontest therobustnessofphylogenetic and phylogenomicanalyses, wewish cyanobacteria, however,possessthegenetic components toallowphototrophyorautotrophy. (Battistuzzi andHedges2008)in theDomainBacteria. Noneofthesesibling cladesother thanthe at usingphylogenomics. Allofthesegroupsare embeddedwithin thelarger(rankless) Terrabacteria Melainabacter (Harelet al.2015)but This article is protected bycopyright.All rights reserved Acceptedbetween Gammaproteobacteria andAcidithiobacilli) and 0.6(forexample between nearest neighbor hoversaround0.4-0.5substitutions persite, butitvaries between0.1(forexample, exhaustive surveybyHugetal.(2016),the typicaldistance betweenan establishedphylum andits plasticity oftheconcept onthebasisofribosomal RNA sequences,andusingdatafrom the recognized (includingsome candidate phylawithfew cultivated representatives). Inlookingat the of thephylogenetic distance sufficient fornamingnew phyla, ofwhichalargenumber arecurrently (Article 3;Turlandet al.2018).Thismodeoforganic growthhasresulted inapragmatic, softconcept al. 2015),although itdoesinthe International CodeofNomenclature forAlgae,FungiandPlants finds noofficial placein theInternational CodeofNomenclature ofProkaryotes(Rule 5b;Parker accumulated, soastoaccommodate clades that weresufficiently well-delimited. Therank“phylum” standards buthasrather evolvedwiththe fieldasourknowledge oftheextant microbial diversity diversification amongits phototrophicandnon-phototrophic clades. evidence thatleads toanexciting hypothesis,onethat likelyunderestimates the levelof analyzed. Insum,thephylogenetic relationships depicted inFig.1constitute aninteresting pieceof challenging to“prove” suchdeeprelationships, becausetheancestors, longgone,cannever be Articlethe event canbeconsidered underanymodel ofevolutiontobe ofancientderivation. Itisthushighly “protocyanobacteria” varybymorethan onebillionyears (Shih recent estimatesofthe timingofthe splitofcyanobacteria from non-photosynthetic use fundamentally different powersourcesandaremechanistically worldsapart.Finally, although chariot, whenonelooksatsomeofthe majorsharedparts (2axlesand4wheels),even thoughthey among these lineages.Asananalogy, achild’scarriage ismoresimilar toanautomobile than toa interpreted asreconstructing aconsensusphylogenyoftheshared non-photosynthetic“chassis” for thetwophyla withinthelarger contextofnitrogenase phylogeny.The treesarethusbest are only distantly related totheir homologsinMelainabacteria, yieldingapolyphyletic relationship estimate variance (Garcia-Pichel etal.2019).Forexample, thenitrogenase genesofCyanobacteria element, whichmaynot becoincident, resultingin estimatesofevolutionary relationship that neglect concatenated molecules alsotend toaverageestimates ofthe evolutionarydistance recordedin each phylogenies that useasubsetofthegenome information ofcommonoccurrence. Analysesof Traditional usageofthe rankofphylumwithin thebacteria isnotbasedonconventional

et al.2017,Garcia-Pichel etal. 2019), Deinococcus

et This article is protected bycopyright.All rights reserved Phylum underthe International CodeforNomenclature ofAlgae,FungiandPlants: the situation isparamount. Inordertoenable thecorrection, weprovideaformal definition ofthe congruency isa definiteasset. Inthisregard,thewillingness ofsequencedatabases totimely correct practitioners ofscience inallotherrelevant basic andapplied disciplines, forwhichsimplicity and not onlythose familiar withtheintricacies andsubtleties ofsystematics, butalsothemore casual we refrainhere fromdoingso.Wehave tobemindful that nomenclature shouldpragmatically serve will be neededtofindthe answer.Temperance isthusonthesideofeventual taxonomic stability, and relevance isifoneshouldinclude Margulisbacteria andSaganbacteria inthat definition. Morework confusion ifthingsare leftasis.Inview oftheexpandingdynamics ofdiscovery,aquestion of phototrophic trait,anda newepithetbe coinedforthe largergroupofcladesto avoidfurther maintained forthelineage containing the evolutionarily relatedorganisms thatacquired theoxygenic 2015), itwouldbeprudent tobeconservative. Wethusproposethat thename Cyanobacteria be Regardless ofthe ancestralrelationships, whichare clearlydeep anddifficult toprove(Harelet al. a widespread,established useinreferring toaphysiologically andecologically coherent group. after diverging fromtheancestral lineages, butwhethersuchrelationships justifyusinga termthat has phototrophic ornon-phototrophicancestor, orwhethercyanobacteria acquired photosynthesisgenes 2005, DiRienzi etal.2013,Matheus Carnevaliet al.2019). no suchneedandtreat each asadistinctgroup,keeping thephylum rankforcyanobacteria (Leyet al. clades.Several researchteams workingonthephylogeny ofcyanobacteria anditssibling cladessaw these distances likelyunderestimate thetrueevolutionary difference betweenfunctionally divergent with their sisterclade intoasinglephylum. Thisisparticularly thecase, asdiscussedabove,when necessary reasonto demotethe traditional cyanobacteria toaclass rank,ortolumpthem together Cyanobacteria (thetwomost similarsibling cladesamongthose discussedhere)doesnotconstitute a between Synergistes andThermotogae. Clearlythe degreeofrelatedness ofMelainabacteria and and Gemmatimonatides, orthatseparating Betaproteobacteria fromGammaproteobacteria, orthat clade, theMelainabacteria, inthesamedataset isabout0.3,not unlikethat separatingActinobacteria / Accepted Article Thermus The issueat stakeisthusnotsomuchwhether thegroupsevolvedfrom acommon andThermodesulfobacteria). The distancebetween Cyanobacteria anditsclosest sister This article is protected bycopyright.All rights reserved Accepted ArticleSynonyms: blue-greenalgae, cyanophyta,oxyphotobacteria, oxychlorobacteria, myxophyceae carbon dioxide asasourceofcarbon,orthosesecondarilyevolved fromsuchorganisms. bacteria able tocarryoutoxygenic photosynthesiswith waterasanelectrondonorandtoreduce staff orcane,denoting anelongated shape).Aphylogenetic lineageinthe oforganisms domain imparted tomanyofits speciesbyphycocyanin, andfromlatinizedGreek Bacterion[ Phylum Cyanobacteria : (latinized fromGreekKyanos[  , blue-green,after thecoloration  , This article is protected bycopyright.All rights reserved AcceptedHug, L.A.,Baker,B.J.,Anantharaman, C.T.,A.J.,Castelle, K.,Brown, Probst, C.J.,Butterfield, C. Harel, A.,Karkar,S.,Cheng,Falkowski,P.G. &Bhattacharya, D.2015.Decipheringprimordial Gawryluk, R.M.,Tikhonenkov,D.V.,Hehenberger,E.,F.,Mylnikov,A.P.&Keeling, Husnik, P. J. 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This article is protected bycopyright.All rights reserved bootstrap values lessthan0.4. maximal andminimal distances fromtheirrespective nodes.Nodeswithoutcolor-coding have ( 16S rRNA sequencesavailable intheCydrasil curateddatabase Figure 1.Maximum likelihoodphylogenetic treeoftheCyanobacteria and siblingcladesbased on Accepted Articlehttps://github.com/FGPLab/cydrasil). Entries withineachclade arecollapsed triangles denoting Accepted Article