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Zurich Open Repository andArchiveUniversity of ZurichMain LibraryStrickhofstrasse 39CH-8057 Zurichwwwzorauzhch

Year 2013

Evolution of multicellularity coincided with increased diversification ofcyanobacteria and the Great Oxidation Event

Schirrmeister Bettina E de Vos Jurriaan M Antonelli Alexandre Bagheri Homayoun C

Abstract Cyanobacteria are among the most diverse prokaryotic phyla with morphotypes ranging fromunicellular to multicellular filamentous forms including those able to terminally (ie irreversibly) dif-ferentiate in form and function It has been suggested that cyanobacteria raised oxygen levels in theatmosphere around 245-232 billion y ago during the Great Oxidation Event (GOE) hence dramaticallychanging life on the planet However little is known about the temporal evolution of cyanobacterial lin-eages and possible interplay between the origin of multicellularity diversification of cyanobacteria andthe rise of atmospheric oxygen We estimated divergence times of extant cyanobacterial lineages underBayesian relaxed clocks for a dataset of 16S rRNA sequences representing the entire known diversity ofthis phylum We tested whether the evolution of multicellularity overlaps with the GOE and whethermulticellularity is associated with significant shifts in diversification rates in cyanobacteria Our resultsindicate an origin of cyanobacteria before the rise of atmospheric oxygen The evolution of multicellularforms coincides with the onset of the GOE and an increase in diversification rates These results suggestthat multicellularity could have played a key role in triggering cyanobacterial evolution around the GOE

DOI httpsdoiorg101073pnas1209927110

Posted at the Zurich Open Repository and Archive University of ZurichZORA URL httpsdoiorg105167uzh-76883Journal ArticlePublished Version

Originally published atSchirrmeister Bettina E de Vos Jurriaan M Antonelli Alexandre Bagheri Homayoun C (2013) Evolu-tion of multicellularity coincided with increased diversification of cyanobacteria and the Great OxidationEvent Proceedings of the National Academy of Sciences of the United States of America 110(5)1791-1796DOI httpsdoiorg101073pnas1209927110

Evolution of multicellularity coincided with increaseddiversification of cyanobacteria and the GreatOxidation EventBettina E Schirrmeistera12 Jurriaan M de Vosb Alexandre Antonellic and Homayoun C Bagheria

aInstitute of Evolutionary Biology and Environmental Studies University of Zurich CH-8057 Zurich Switzerland bInstitute of Systematic Botany University ofZurich CH-8008 Zurich Switzerland and cGothenburg Botanical Garden and Department of Biological and Environmental Sciences University of GothenburgSE 405 30 Gothenburg Sweden

Edited by Stjepko Golubic Boston University Boston MA and accepted by the Editorial Board December 13 2012 (received for review June 15 2012)

Cyanobacteria are among the most diverse prokaryotic phyla withmorphotypes ranging from unicellular to multicellular filamentousforms including those able to terminally (ie irreversibly) differ-entiate in form and function It has been suggested that cyano-bacteria raised oxygen levels in the atmosphere around 245ndash232billion y ago during the Great Oxidation Event (GOE) hence dra-matically changing life on the planet However little is knownabout the temporal evolution of cyanobacterial lineages and pos-sible interplay between the origin of multicellularity diversifica-tion of cyanobacteria and the rise of atmospheric oxygen Weestimated divergence times of extant cyanobacterial lineages un-der Bayesian relaxed clocks for a dataset of 16S rRNA sequencesrepresenting the entire known diversity of this phylum We testedwhether the evolution of multicellularity overlaps with the GOE andwhether multicellularity is associated with significant shifts in di-versification rates in cyanobacteria Our results indicate an originof cyanobacteria before the rise of atmospheric oxygen The evo-lution of multicellular forms coincides with the onset of the GOEand an increase in diversification rates These results suggest thatmulticellularity could have played a key role in triggering cyano-bacterial evolution around the GOE

early life | major transitions | prokaryotic phylogenetics | molecular clock

Cyanobacteria are one of the morphologically most diversegroups of prokaryotic organisms Growth forms range from

uni- to multicellular and can include levels of reversible or ter-minal (ie irreversible) cell differentiation These diverse growthstrategies have enabled cyanobacteria to inhabit almost everyterrestrial and aquatic habitat on Earth Cyanobacteria havetraditionally been classified into five subsections according totheir morphology (1 2) where subsections I and II refer to uni-cellular species and subsections IIIndashV describe multicellular speciesSpecies belonging to subsections IV and V are able to produceterminally differentiated cells Despite the usefulness of thesesubsections molecular evidence shows that morphological andgenetic diversity do not always coincide Molecular phylogeniesindicate that probably none of the five subsections is mono-phyletic (3 4) and several transitions between uni- and multi-cellularity have taken place (5) According to the fossil recordvarious distinct morphotypes attributed to cyanobacteria werealready present over 2 billion y ago (Bya) (6 7) The phylum isthought to have existed as early as 245ndash232 Bya based on theassumption that cyanobacteria were responsible for the accu-mulation of atmospheric oxygen levels referred to as the GreatOxidation Event (GOE) (8ndash12) Despite the generally acceptedtime-frame for the rise of cyanobacteria surprisingly little isknown about when morphological innovations such as multi-cellularity first appeared It is also unclear what influence if anythese innovations may have had on the diversification of thephylum The assumed link between the rise of atmospheric ox-ygen and cyanobacteria is also poorly understood did the GOEclosely follow the first appearance of cyanobacteria or did it take

place considerably later in possible association with morpho-logical innovations of the phylumThere have been previous attempts to estimate the origin of

cyanobacteria and their morphotypes (13ndash16) However it islikely that a biased taxonomic choice especially missing earlybranches of the cyanobacterial phylogeny may have led to in-complete conclusions (17 18) Phylogenetic evidence indicatesthat multicellularity evolved very early in the history of cyano-bacteria challenging the view that multicellularity is a derivedcondition in the phylum (5) Nonetheless important questionsremain (i) When did cyanobacteria and their major cladesevolve (ii) When did multicellularity first appear (iii) How arethese transitions associated with the GOE around 245ndash232 ByaThe far-reaching impact of the GOE cannot be emphasized

enough it changed Earthrsquos history by enabling the evolution ofaerobic life Unlike other eubacterial phyla cyanobacteria ex-hibit a well-studied fossil record (6 7 19 20) However fossildata are often limited and present only minimum age estimatesof clades Therefore a combination of fossil data with molecularphylogenetic methods has been advocated (21ndash23) The use ofcarefully selected calibration priors for molecular-dating analysescan provide new insights into the temporal evolution of cyano-bacteria and the early history of life Presently available genomedata for cyanobacteria are biased toward unicellular taxa and donot sufficiently represent the known diversity of this phylumTherefore we reconstructed phylogenetic trees on the basis of16S rRNA sequences which have been carefully sampled basedon phylogenetic disparity as described previously (5) We furtherestimated divergence times of cyanobacteria and addresseddifferent interpretations of the fossil record as calibration priorsWe then evaluated whether the GOE coincided with the de-velopment of major cyanobacterial morphotypes present todayFinally we tested for shifts in diversification rates incorporatinginformation on 281 species and 4194 strains Our results supporttheories of an early cyanobacterial origin toward the end of theArchean Eon before 25 Bya Evolution of multicellularity co-incided with the onset of the GOE and corresponded to amarked increase of diversification in cyanobacteria

Author contributions BES JMdV AA and HCB designed research BES andJMdV analyzed data and BES JMdV AA and HCB wrote the paper

The authors declare no conflict of interest

This article is a PNAS Direct Submission SG is a guest editor invited by theEditorial Board

Freely available online through the PNAS open access option

Data deposition The sequences reported in this paper have been deposited in the Gen-Bank database (accession no JX069960)1Present address School of Earth Sciences University of Bristol Bristol BS8 1RJUnited Kingdom

2To whom correspondence should be addressed E-mail bettinaschirrmeisterbristolacuk

This article contains supporting information online at wwwpnasorglookupsuppldoi101073pnas1209927110-DCSupplemental

wwwpnasorgcgidoi101073pnas1209927110 PNAS | January 29 2013 | vol 110 | no 5 | 1791ndash1796

EVOLU

TION

ResultsPhylogenetic Analyses To infer the early evolution of cyanobac-teria we reconstructed Bayesian phylogenetic trees using 16SrRNA sequence data A phylogenomic approach would givemisleading results because available cyanobacterial genomesequences to date are heavily biased toward unicellular speciesMoreover the few multicellular species that have been fully se-quenced are phylogenetically closely related and a comparisonof these species is unlikely to provide any information on theancient origin of multicellularity in cyanobacteria (17) In aprevious study (5) a phylogenetic tree of 1220 cyanobacterialsequences was reconstructed from which a subset of taxa wassampled that represents the surveyed diversity of this phylumHere we used this subset plus one strain (G40) that representsa potentially unique distinct species isolated by our group Ourunconstrained phylogenetic results (Fig S1) agree with previousfindings (3 5 15 24 25) which reject monophyly of severalmorphological groups previously described (1 2) FurthermoreGloeobacter violaceus is resolved as the sister group of all othercyanobacteria Three major groups can be distinguished (cladesE1 E2 and group AC) (Fig 1 and Figs S1 and S2) togetherrepresenting the majority of cyanobacterial taxa living today All

groups have been defined previously (5) with clades E1 and E2(subclades of E) including species of all morphological sub-sections Species belonging to morphological subsections IV andV occur solely in E1 The group AC contains unicellular marinepico-phytoplankton (subsection I) as well as some undifferentiatedmulticellular species (subsection III)

Divergence Time Estimation Divergence times along the cyano-bacterial phylogeny were estimated under Bayesian relaxedmolecular clocks using two different models of uncorrelated rateevolution (26) A lognormal distribution of rates has been shownto outperform a model with exponential rate distribution (26)Therefore our first model assumed rates were lognormally dis-tributed (uncorrelated lognormal UCLN) Robustness of resultswas tested with a second model assuming exponentially distrib-uted rates (uncorrelated exponentially distributed UCED) (SIText) For each clock model a set of eight different analyses wereperformed to take a broad range of prior assumptions into ac-count and evaluate their influence on the results (Table 1 andTable S1) The Bayesian consensus tree of divergence-timeanalysis 7 is presented in Fig 1 including age estimates (95highest posterior densities HPD) of important nodes as given by

25

Fig 1 Time calibrated phylogeny of cyanobacteria displaying divergence time estimates Bayesian consensus tree (analysis 7) based on 16S rRNA data with 95highest posterior densities of the discussed node ages shown as green bars (analyses 1 3 5 and 7 overlapping) Morphological features of taxa are marked bycolored boxes and listed in the inset Full taxon names are displayed in Table S3 Branches with posterior probabilities gt09 in all analyses are presented as thicklines Gray circles mark points used for calibration of the tree Details of the prior age estimates used for calibration are presented in Table 1 A significant increasein diversification rate (yellow triangle) [966-fold (average of all analyses)] can be detected at node 3 and a minor decrease (red triangle) at 3334 The earlier shiftclose to node 3 coincides with the origin of multicellularity Schematic drawings of cyanobacterial fossils are provided under the timeline with the ones used forcalibration of the tree marked in red Our results indicate that multicellularity (green shade) originated before or at the beginning of the GOE

1792 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al

analyses 1 3 5 and 7 (Table 1) Median node ages (m~ ) areshown in Fig 2 and are provided with 95 HPD in Table 1(discussed nodes) and Table S2 (all nodes) Although ages ofcyanobacterial nodes varied with respect to the analyses ourmajor conclusions are robust to different calibration priors Allanalyses indicated that extant cyanobacteria originated beforethe GOE (245 Bya) Multicellularity most likely originated alongthe branch leading to node 3 (5) For this node analyses sug-gested a median age before or at the beginning of the GOE(before 236 Bya) (Table 1 and Table S1) The ancestor of thelineage leading to node 3 was also a calibration point in ouranalyses (Table 1) Fig 3 compares the implied prior probabilitydistributions of that calibration point to posterior probabilities ofnode 3 hence assessing the extent to which our prior assump-tions affected the outcome Although the prior assumptions puta higher probability on an age after the GOE around 22 Bya ourdata contained strong signals to counteract these priors and in-dicate instead an older median node age for node 3 between242ndash308 Bya (all analyses) (Fig 3 and Table 1) which is beforethe GOE Furthermore groups E1 E2 and AC are estimated tohave originated around the end of the GOE These groupscomprise the majority of living cyanobacteria (91 of 281 spe-cies and 88 of 4194 strains)

Shifts in Diversification Rates To identify whether the GOE ormulticellularity might have influenced the net diversification ofcyanobacteria we tested whether diversification rates have beenconstant among cyanobacterial lineages Because previous worksuggested that taxonomy of cyanobacteria needed revision (1)we ran analyses incorporating information on both species (281)and strains (4194) Clades containing many species also containmany strains (Table S3) Results from the diversification rateestimation showed similar patterns independent of whetherspecies numbers or strain numbers were used (Table S4) Twosignificant shifts in diversification rates were detected At node34 where multicellularity evolved the diversification rate in-creased on average 844-fold (SD = 176) for trees reconstructedwith a UCLN model and 524-fold (SD = 189) for trees recon-structed with a UCED model (averaged over all analyses) (TableS4) Subsequently at node 3334 the diversification rate decreasedby a factor of 055 (SD = 019) for trees reconstructed with aTa

ble

1Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCLN

distributedev

olutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgroup

No

No

Yes

Yes

Yes

Yes

No

No

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCLN

)ethm~

THORN(HPD

)forall

Node1

295

(25ndash36)

367

(279ndash

474

)299

(257ndash

355

)335

(274ndash

415

)287

(253ndash

330

)306

(266ndash

353

)295

(253ndash

355

)339

(287ndash

380

)Node3

254

(228ndash

298

)308

(242ndash

384

)242

(221ndash

273

)265

(228ndash

318

)238

(220ndash

262

)249

(226ndash

281

)254

(229ndash

297

)286

(243ndash

334

)Node6

204

(177ndash

235

)233

(189ndash

287

)202

(172ndash

228

)210

(178ndash

254

)199

(167ndash

222

)202

(170ndash

232

)204

(179ndash

235

)218

(186ndash

260

)Node31

177

(14ndash224

)216

(153ndash

256

)172

(134ndash

220

)198

(139ndash

234

)167

(128ndash

217

)175

(130ndash

223

)177

(141ndash

225

)212

(150ndash

241

)Node43

200

(156ndash

243

)235

(173ndash

303

)185

(146ndash

225

)197

(148ndash

250

)180

(138ndash

219

)186

(141ndash

230

)200

(157ndash

241

)218

(171ndash

272

)

Eightdifferentco

mbinationsofcalib

rationpriors

forthedivergen

cetimeestimationwereusedEx

pex

ponen

tial

distribution(offsetmea

n)LN

lognorm

aldistribution(offsetmea

nSD

)mdashcalib

rationnot

applicab

le

Truncatedat

38Bya

Fig 2 Median age estimates under eight analytical scenarios Median ageestimates of clades (Table 1) The origin of cyanobacteria (node 1) and theevolution of multicellularity (node 3) are estimated before or at the begin-ning of the GOE Relatively soon after the GOE the stem lineages of thethree major cyanobacterial clades originated containing unicellular cyano-bacteria (node 6) terminally differentiated taxa (node 31) and marinephycoplankton (node 43)

Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1793

EVOLU

TION

UCLN model and by a factor of 022 (SD = 013) for treesreconstructed with a UCED model (Fig 1 and Figs S3ndashS6)

DiscussionLimitations of a Single Gene The exchange of genetic materialacross species boundaries poses a challenge for the inference ofevolutionary histories of living organisms (27ndash29) Phylogeneticreconstructions incorporating multiple genes help to reduce thedanger to recover false signals from genes affected by horizontalgene transfer (HGT) (30 31) Nevertheless although genomedata are accumulating they do not nearly achieve the breadth ofmicrobial diversity represented by 16S rRNA (32) 16S rRNAhas been used as a reliable measure of phylogenetic relationshipbecause of its size and conservation (33 34 35) These facts incombination with a potentially smaller impact of HGT on ge-nome evolution than commonly assumed and even less on 16SrRNA (32 36 37) support the usefulness of the small ribosomalsubunit for phylogenetic applications Here we can neither ex-clude nor prove the possibility of 16S rRNA being affected byHGT between species No cases have been found in support ofHGT for 16S rRNA between cyanobacterial genera We rely on16S rRNA sequences in this study because a genomic approachwould be biased toward unicellular taxa would not cover thecomplete known diversity of this phylum and hence fail to re-construct the early evolution of cyanobacteria (17) Neverthelesswe strongly encourage genome-sequencing projects that will helpto recover the diversity indicated by 16S rRNA and improvereconstruction of a cyanobacterial phylogeny

Evolution of Multicellularity and Possible Consequences In prokar-yotes simple forms of multicellularity occur in different phyla InActino- and Myxobacteria multicellular growth formed via cellaggregation is part of their life cycle (38) In cyanobacteria chlor-oflexi and some proteobacteria (eg Beggiatoa) multicellularity is ina filamentous form This result is achieved through cell division andadhesion which results in filament elongation (39) Requirementsfor directed growth in filaments are cellular recognition of polarity(40) and cellular communication Filamentous cyanobacteria

including simple forms like Pseudanabaena and Leptolyngbya showdirectional growth where the plane of cell division depicts a rightangle to the growth direction (1) In addition intercellular com-munication and resource exchange has been found in cyanobacteria(41ndash43) providing an evolutionary basis for the division of labor andterminal cell differentiation to evolve (44ndash46)Our results suggest a concurrence of the origin of multicellu-

larity the onset of the GOE and an increased diversification rateof cyanobacteria in addition although their precise timingcannot be fully ascertained they can be linked by theoretical andempirical lines of evidence The transition to multicellularityrepresents an important change in organismic complexity (47)There are various advantages that multicellularity could confer(39 48) Among others filamentous growth can improve motility(49) and cooperation of cells may also increase fitness becauseof economies of scale Experimental studies have shown thatmulticellularity might evolve relatively fast given selective pres-sure (50) and can provide metabolic fitness advantages comparedwith single cells (51) Increased fitness of multicellular speciescould have led to a higher frequency and wider distribution ofcyanobacteria at the end of the Archean consequently enhancingoxygen production Accumulation of oxygen may have resultedin new ecological opportunities Increased diversification ratesaround the time when multicellularity evolved suggest that cya-nobacteria might have used and possibly contributed to createnew adaptive opportunities Subsequently at the end of theGOE three clades (E1 E2 and AC) evolved that led to themajority of cyanobacteria living today

Early Earth History and the Fossil Record Our finding that cyano-bacteria have existed for a longer time than previously anticipatedis congruent with reconstructions of early Earth history Theorigin of Earth is deduced to date back sim45 Bya (52) Sub-sequently the planet cooled down and eventually separated intocore mantle and crust (53) Permanent existence of life before42ndash38 Bya is unlikely considering that the young Earth wassubject to strong bombardment by asteroids (52 54) Fossil ev-idence does not predate sim345 Bya (55 56) Most of these pro-karyotic fossils from the early Archean Eon have been identifiedin two regions the Barberton Greenstone Belt (BGB) SouthAfrica (around 320ndash350 billion y old) and the Pilbara Craton(PC) Western Australia (around 290ndash360 billion y old) (55ndash60)The oldest fossils from these regions are spherical probablyhyperthermophilic microbes [BGB (56 59)] and filaments ofpossibly anoxygenic photosynthetic prokaryotes [East-PC (5556)] both around 345 billion y old Further evidence for lifeincludes 34 billion-y-old trace fossils (PC) (60) 342 billion-y-olddeformed microbial mats (BGB) (57) and 30 billion-y-old bio-films (PC) (58) The earliest unequivocal cyanobacterial fossilsdate back around 20 Bya and come from two localities theGunflint iron formation and the Belcher Subgroup (both inCanada) (19 20) Although differences in the microbial fossilcomposition have been recognized (19) both cherts include fil-amentous and coccoidal species Gunflintia grandis and Gun-flintia minuta have been identified as filamentous cyanobacterialfossils from the Gunflint iron formation and Halythrix sp hasbeen described as an oscillatorian fossil from the Belcher sub-group (7) (Fig 1) Cyanobacterial fossils younger than 2 billion yare more widely distributed (20) with various examples given inFig 1 Archean fossil findings may potentially depict remains ofcyanobacteria but cannot be assigned beyond doubt (20) ldquoPos-siblerdquo cyanobacterial fossils have been found in 252ndash255 billion-y-old cherts in South Africa (20 61) ldquoProbablerdquo unicellular andfilamentous cyanobacterial fossils are distributed in 26 billion-y-old (20 62ndash64) and 326 billion-y-old (64) cherts Although pre-viously described biomarkers that supported an existence of cya-nobacteria around 27 Bya (65 66) have been dismissed (67) recentevidence has been found in favor of an early cyanobacterial origin

Fig 3 Prior and posterior probability distributions of ages for node 3 Marginalprior probability distributions of analyses using narrow (analysis 5) and wide(analysis 6) prior distributions were conservatively biased toward younger agesstrongly favoring an origin of multicellularity after the GOE Even so posteriorprobabilities point to an origin of multicellularity before or at the beginning ofthe GOE indicating that this main result is based on a strong signal in the datarather than a bias from a-priori assumptions Marginal prior probability dis-tributions were estimated in analyses that only sampled from the prior


(68ndash70) Our molecular dating results place the origin of bothunicellular and multicellular cyanobacteria rather before theGOE and thus suggest that some of those fossils could indeedrepresent relatives of cyanobacterial lineagesRecent studies have suggested that oxygen accumulation oc-

curred sim200ndash300 million y before the GOE (68 69 71) Currentevidence from the fossil record geochemical findings and ourmolecular analyses together support an origin of cyanobacteriaclearly before the GOE The origin of multicellularity toward theGOE could have entailed fitness advantages leading to an in-crease in cyanobacterial diversity and abundance which in turnwould positively influence net oxygen production

ConclusionCyanobacteria are one of the morphologically most diverseprokaryotic phyla on this planet It is widely accepted that theycaused the GOE starting 245 Bya but debates about their originare still ongoing (67 72 73) Various lines of fossil and geochemicalevidence have accumulated supporting an origin of cyanobacteriabefore 245 Bya (20 62 64 68ndash70) Here we applied Bayesianphylogenetic analyses using relaxed molecular clocks and differentcombinations of calibration priors We estimated the origin of extantcyanobacteria and their dominant morphotypes with respect to theGOE Although resulting age estimates of the different analysesdiffer somewhat in their HPD robust statements regarding the or-igin of cyanobacteria and their morphotypes can nevertheless beformulated (i) cyanobacteria originated before the GOE (ii) mul-ticellularity coincides with the beginning of the rise of oxygen and(iii) three clades representing the majority of extant cyanobacteriaevolved shortly after the accumulation of atmospheric oxygen

Materials and MethodsTaxon Sampling Most sequences were downloaded from GenBank (74) (TableS3) Three eubacterial species were chosen as an outgroup Beggiatoa spChlamydia trachomatis and Spirochaeta thermophila A total of 58 cyano-bacterial species were chosen for the analyses Aside from strain G40 (SI Text) alltaxa were selected as described previously (5) The taxa chosen comprise allmorphological subsections described by Castenholz (1) and cover the morpho-logical and genetic diversity of this phylum (5) Nomenclature and identity statedon GenBank might be incorrect Therefore we evaluated morphotypes (multi-cellularunicellular) of each cyanobacterial strain by thoroughly examining theliterature (Table S5) and conducting BLAST analyses as described in SI TextFor most of those situations full genome data are not yet available (17)

Alignment and Divergence Time Estimation Sequence alignments were con-structed using the program MUSCLE (Dataset S1) (75) Analyses were per-formed on datasets with outgroups [(i) 61 taxa 1090 sites gaps excluded507 sites variable] and without outgroups [(ii) 58 taxa 1077 sites gaps ex-cluded 421 sites variable] Uncorrected and corrected Akaike InformationCriterion (76 77) implemented in jModelTest v011 (78) suggested a gen-eral time-reversible substitution model with γ-distributed rate variationamong sites (GTR+G) (79) as the most suitable model of sequence evolutionPhylogenetic analyses using Bayesian inference were conducted as describedin SI Text We applied relaxed clocks with UCLN and UCED rate distributions(Table 1 and Table S1) (80) The analyses were conducted with a combinationof three calibration points Additionally monophyly constraints were set forthree nodes that were supported by our previous Bayesian phylogeneticanalyses (Fig S1 and SI Text) (i) the phylum cyanobacteria (ii) cyanobac-teria excluding Gloeobacter and (iii) cyanobacteria excluding Synecho-coccus sp P1 and Gloeobacter (Fig 1) The phylum cyanobacterian (i) hasbeen extensively investigated and confirmed before [ie cyanobacteria as amonophyletic group within the Eubacteria (5)] For cyanobacteria excludingGloeobacter (ii) an early divergence of Gloeobacter has been supported inprevious analyses (5 17 24) Unlike other cyanobacteria G violaceus lacks

thylacoid membranes (81) and various differences in gene content com-pared with cyanobacteria have been found (82) For cyanobacteria excludingSynechococcus sp P1 and Gloeobacter (iii) Synechococcus sp P1 is a ther-mophilic unicellular cyanobacterium isolated from Octopus Spring in Yel-lowstone nationalpark (83) Its proximity to Gloeobacter and eubacterialoutgroups has been shown by genetic comparisons and phylogenetic analyses(5 17 24) Divergence time estimation was conducted using the softwareBEAST v162 (80) and run on the CIPRES Science Gateway v31 (84) For eachanalysis we ran six Markov chain Monte Carlo chains for 50-million generationssampling every 2000th generation (input files provided as Dataset S2) Althoughconvergence of all parameters was reached before 5 million generations weexcluded a conservative 25 initial burn-in Results are presented on a 50majority-rule consensus tree calculated with SumTrees v331 (85)

Calibration Points The root Stem lineage of cyanobacteria Four of the eightdivergence time analyses included an outgroup (Table 1 analyses 3 4 5 6)which enabled calibrating the cyanobacterial stem lineage The GOE datesback 232ndash245 billion y (9) and is assumed to be a result of cyanobacterialactivity We use the start of the GOE as the minimum date for the di-vergence of cyanobacterial stem lineage and the outgroup The possibility ofpermanently existing lifeforms is suggested to occur earliest around 38 Bya(52) which we used as earliest date (ie maximum age) of our root cali-bration See Table 1 for a detailed description of prior age probability dis-tributions For analyses 7 and 8 the age of the earliest split of cyanobacterianamely between Gloeobacter and the rest of cyanobacteria was accordinglyrestricted to 38ndash245 ByaNode 3 First multicellular cyanobacteria Node 3 in Fig 1 was estimated to be amulticellular ancestor of extant cyanobacteria as recovered previously (5)Fossil records indicate that terminally differentiated cyanobacteria (subsectionsIV and V) evolved before 21 Bya Such differentiation may only evolve in amulticellular setting (44) We therefore assume that the stem lineage of node 3must have been present before 21 Bya and use this as a hard minimum boundof a lognormal prior distribution We used a soft upper bound linking thedistribution of prior probabilities to the timing of the GOE Multicellularitymay have evolved as a consequence of new habitats that became availableafter the GOE 23 Bya or it could instead have triggered a rise of oxygen inthe atmosphere Therefore we distinguish two calibration scenarios one bysetting the probability of the age of node 3 to a lognormal distribution with95 being younger than 245 (Table 1 analyses 1 3 5) and the other bysetting the median age of the before 245 Bya (Table 1 analyses 2 4 6)Node 31 or 32 First terminally differentiated cyanobacteria Cyanobacteria be-longing to subsection IV and V share the property to form resting cells namedakinetes Fossilized remains of these akinetes have been identified at variouslocations throughout the Proterozoic (6 19 86) The oldest of these fossilizedakinetes are found in 21 billion-y-old rocks (6 13) and imply that cyanobacteriabelonging to subsection IV and V originated before 21 Bya Taxa of this groupare capable of terminal cell differentiation Oxygen sensitive nitrogen fixation isspatially separated from oxygenic photosynthesis and takes place in so calledheterocysts Oxygen levels providing a selective advantage for separation ofthese processes were reachedsim245 Bya (13) As a calibration for the divergencetime estimation we set the most recent common ancestor of taxa from sub-sections IV and V to 21 billion y as a hard minimum bound and specified 95of prior probabilities before 245 Bya using a lognormal distribution

Shifts in Diversification Rates To test whether the rate of lineage accumulationhas been constant throughout cyanobacterial evolution we used the functionMEDUSA from the geiger 13-1 package in R (87)We corrected for possible taxonsampling biases by including information on known numbers of extant speciesand strains which were collected from GenBank Details are given in SI Text andTable S3 MEDUSA was run based on 50 majority-rule consensus trees calcu-lated with SumTrees v331 (85) derived from the eight BEAST analyses (Table 1)

ACKNOWLEDGMENTS We thank Akos Dobay Valentina Rossetti ManuelaFilippini-Cattani the editor SG and three anonymous reviewers for helpfulcomments on the manuscript This work was supported in part by Canton ofZurich AA is supported by grants from the Swedish and the EuropeanResearch Councils BES is supported by the Swiss National Science Foundation

1 Boone DR Castenholz RW (2001) Bergeyrsquos Manual of Systematic Bacteriology The Ar-

chaea and the Deeply Branching and Phototropic Bacteria Cyanobacteria ed Garrity GM

(Springer New York)2 Rippka R Deruelles J Waterbury JB Herdman M Stanier RY (1979) Generic assignments

strain histories and properties of pure cultures of cyanobacteria J Genl MicrobioLogy 111

1ndash61

3 Giovannoni SJ et al (1988) Evolutionary relationships among cyanobacteria and

green chloroplasts J Bacteriol 170(8)3584ndash35924 Gugger MF Hoffmann L (2004) Polyphyly of true branching cyanobacteria (Stigone-

matales) Int J Syst Evol Microbiol 54(Pt 2)349ndash3575 Schirrmeister BE Antonelli A Bagheri HC (2011) The origin of multicellularity in cy-

anobacteria BMC Evol Biol 1145


EVOLU

TION

6 Amard B Bertrand-Sarfati J (1997) Microfossils in 2000 ma old cherty stromatolites ofthe Franceville group Gabon Precambrian Res 81(3ndash4)197ndash221

7 Hofmann HJ (1976) Precambrian Microflora Belcher islands CanadamdashSignificanceand systematics J Paleontol 50(6)1040ndash1073

8 Blankenship RE (2002)MolecularMechanisms of Photosynthesis (Blackwell Science Oxford)9 Bekker A et al (2004) Dating the rise of atmospheric oxygenNature 427(6970)117ndash12010 Kopp RE Kirschvink JL Hilburn IA Nash CZ (2005) The Paleoproterozoic snowball

Earth A climate disaster triggered by the evolution of oxygenic photosynthesis ProcNatl Acad Sci USA 102(32)11131ndash11136

11 Allen JF MartinW (2007) Evolutionary biology Out of thin airNature 445(7128)610ndash61212 Frei R Gaucher C Poulton SW Canfield DE (2009) Fluctuations in Precambrian at-

mospheric oxygenation recorded by chromium isotopes Nature 461(7261)250ndash25313 Tomitani A Knoll AH Cavanaugh CM Ohno T (2006) The evolutionary diversification

of cyanobacteria Molecular-phylogenetic and paleontological perspectives Proc NatlAcad Sci USA 103(14)5442ndash5447

14 Battistuzzi FU Hedges SB (2009) A major clade of prokaryotes with ancient adapta-tions to life on land Mol Biol Evol 26(2)335ndash343

15 Blank CE Saacutenchez-Baracaldo P (2010) Timing of morphological and ecological in-novations in the cyanobacteriamdashA key to understanding the rise in atmospheric ox-ygen Geobiology 8(1)1ndash23

16 Larsson J Nylander JAA Bergman B (2011) Genome fluctuations in cyanobacteriareflect evolutionary developmental and adaptive traits BMC Evol Biol 11187

17 Schirrmeister BE Anisimova M Antonelli A Bagheri HC (2011) Evolution of cyano-bacterial morphotypes Taxa required for improved phylogenomic approachesCommun Integr Biol 4(4)424ndash427

18 Wu DY et al (2009) A phylogeny-driven genomic encyclopaedia of Bacteria andArchaea Nature 462(7276)1056ndash1060

19 Golubic S Lee SJ (1999) Early cyanobacterial fossil record Preservation palae-oenvironments and identification Eur J Phycol 34(4)339ndash348

20 Sergeev VN Gerasimenko LM Zavarzin GA (2002) [Proterozoic history and presentstate of cyanobacteria] Mikrobiologiia 71(6)725ndash740

21 Benton MJ (2003) The quality of the fossil record Telling the Evolutionary Time MolecularClocks and the Fossil Record eds Donoghue PCJ Smith MP (Tayler amp Francis London) pp66ndash90

22 Reisz RR Muumlller J (2004) Molecular timescales and the fossil record A paleontologicalperspective Trends Genet 20(5)237ndash241

23 Donoghue PCJ Benton MJ (2007) Rocks and clocks Calibrating the Tree of Life usingfossils and molecules Trends Ecol Evol 22(8)424ndash431

24 Turner S Pryer KM Miao VPW Palmer JD (1999) Investigating deep phylogeneticrelationships among cyanobacteria and plastids by small subunit rRNA sequenceanalysis J Eukaryot Microbiol 46(4)327ndash338

25 Honda D Yokota A Sugiyama J (1999) Detection of seven major evolutionary line-ages in cyanobacteria based on the 16S rRNA gene sequence analysis with new se-quences of five marine Synechococcus strains J Mol Evol 48(6)723ndash739

26 Drummond AJ Ho SYW Phillips MJ Rambaut A (2006) Relaxed phylogenetics anddating with confidence PLoS Biol 4(5)e88

27 Doolittle WF (1999) Phylogenetic classification and the universal tree Science 284(5423)2124ndash2129

28 Gogarten JP Doolittle WF Lawrence JG (2002) Prokaryotic evolution in light of genetransfer Mol Biol Evol 19(12)2226ndash2238

29 Andam CP Gogarten JP (2011) Biased gene transfer in microbial evolution Nat RevMicrobiol 9(7)543ndash555

30 Suchard MA (2005) Stochastic models for horizontal gene transfer Taking a randomwalk through tree space Genetics 170(1)419ndash431

31 Lapierre P Lasek-Nesselquist E Gogarten JP (2012) The impact of HGT on phyloge-nomic reconstruction methods Brief Bioinform 101093bibbbs050

32 Yarza P et al (2008) The All-Species Living Tree project A 16S rRNA-based phylo-genetic tree of all sequenced type strains Syst Appl Microbiol 31(4)241ndash250

33 Woese CR (1987) Bacterial evolution Microbiol Rev 51(2)221ndash27134 Olsen GJ Woese CR (1993) Ribosomal RNA A key to phylogeny FASEB J 7(1)113ndash12335 Schirrmeister BE Dalquen DA Anisimova M Bagheri HC (2012) Gene copy number

variation and its significance in cyanobacterial phylogeny BMC Microbiol 12(1)17736 Snel B Bork P Huynen MA (2002) Genomes in flux The evolution of archaeal and

proteobacterial gene content Genome Res 12(1)17ndash2537 Kurland CG Canback B Berg OG (2003) Horizontal gene transfer A critical view Proc

Natl Acad Sci USA 100(17)9658ndash966238 Rokas A (2008) The molecular origins of multicellular transitions Curr Opin Genet Dev

18(6)472ndash47839 Rossetti V Filippini M Svercel M Barbour AD Bagheri HC (2011) Emergent multi-

cellular life cycles in filamentous bacteria owing to density-dependent populationdynamics J R Soc Interface 8(65)1772ndash1784

40 Knoll AH Javaux EJ Hewitt D Cohen P (2006) Eukaryotic organisms in Proterozoicoceans Philos Trans R Soc Lond B Biol Sci 361(1470)1023ndash1038

41 Giddings TH Staehelin LA (1981) Observation of Microplasmodesmata in both het-erocyst-forming and non-heterocyst forming filamentous Cyanobacteria by freeze-fracture electron microscopy Arch Microbiol 129(4)295ndash298

42 Flores E Herrero A Wolk CP Maldener I (2006) Is the periplasm continuous in fila-mentous multicellular cyanobacteria Trends Microbiol 14(10)439ndash443

43 Flores E Herrero A (2010) Compartmentalized function through cell differentiation infilamentous cyanobacteria Nat Rev Microbiol 8(1)39ndash50

44 Rossetti V Schirrmeister BE Bernasconi MV Bagheri HC (2010) The evolutionary path toterminal differentiation and division of labor in cyanobacteria J Theor Biol 262(1)23ndash34

45 Ispolatov I Ackermann M Doebeli M (2012) Division of labour and the evolution ofmulticellularity Proc Biol Sci 279(1734)1768ndash1776

46 Rossetti V Bagheri HC (2012) Advantages of the division of labour for the long-termpopulation dynamics of cyanobacteria at different latitudes Proc Biol Sci 279(1742)3457ndash3466

47 Maynard Smith J Szathmary E (1995) The Major Transitions in Evolution (OxfordUniversity Press Oxford)

48 Bonner J (1998) The origin of multicellularity Integr Biol 1(1)28ndash3649 Adams DG (1997) Cyanobacteria Bacteria as Multicellular Organism eds Shapiro JA

Dworkin M (Oxford Univ Press New York) pp 109ndash14850 Ratcliff WC Denison RF Borrello M Travisano M (2012) Experimental evolution of

multicellularity Proc Natl Acad Sci USA 109(5)1595ndash160051 Koschwanez JH Foster KR Murray AW (2011) Sucrose utilization in budding yeast as

a model for the origin of undifferentiated multicellularity PLoS Biol 9(8)e100112252 Nisbet EG Sleep NH (2001) The habitat and nature of early life Nature 409(6823)

1083ndash109153 Mojzsis SJ (2010) Early earth leftover lithosphere Nat Geosci 3148ndash14954 Sleep NH Zahnle KJ Kasting JF Morowitz HJ (1989) Annihilation of ecosystems by

large asteroid impacts on the early Earth Nature 342(6246)139ndash14255 Westall F et al (2006) The 3466 ga ldquoKittyrsquos gap chertrdquo an early Archean microbial

ecosystem Spec Pap Geol Soc Am 405105ndash13156 Wacey D (2009) Early Life on Earth A Practical Guide (Springer New York)57 Tice MM Lowe DR (2004) Photosynthetic microbial mats in the 3416-Myr-old ocean

Nature 431(7008)549ndash55258 Sugitani K et al (2007) Diverse microstructures from Archaean chert from the mount

Goldsworthy-mount grant area Pilbara Craton Western Australia Microfossils du-biofossils or pseudofossils Precambrian Res 158228ndash262

59 Glikson M et al (2008) Microbial remains in some earliest Earth rocks Comparisonwith a potential modern analogue Precambrian Res 164(3ndash4)187ndash200

60 Wacey D et al (2008) Use of nanosims in the search for early life on Earth Ambientinclusion trails in a c 3400 ma sandstone J Geol Soc London 165(1)43ndash53

61 Knoll AH (1996) Palynology Principles and ApplicationsndashArchean and Proterozoic Pale-ontology (American Association of Stratigraphic Palynologists Tulsa OK) pp 51ndash80

62 Altermann W Schopf JW (1995) Microfossils from the Neoarchean Campbell GroupGriqualand west sequence of the Transvaal Supergroup and their paleoenvir-onmental and evolutionary implications Precambrian Res 75(1ndash2)65ndash90

63 Kazmierczak J Altermann W (2002) Neoarchean biomineralization by benthic cya-nobacteria Science 298(5602)2351

64 Schopf JW (2009) Paleontology microbial Encyclopedia of Microbiology edsLederberg J Schaechter M (Elsevier Amsterdam) 3rd Ed pp 390ndashndash400

65 Brocks JJ Logan GA Buick R Summons RE (1999) Archean molecular fossils and theearly rise of eukaryotes Science 285(5430)1033ndash1036

66 Summons RE Jahnke LL Hope JM Logan GA (1999) 2-Methylhopanoids as bio-markers for cyanobacterial oxygenic photosynthesis Nature 400(6744)554ndash557

67 Rasmussen B Fletcher IR Brocks JJ Kilburn MR (2008) Reassessing the first appear-ance of eukaryotes and cyanobacteria Nature 455(7216)1101ndash1104

68 Lyons TW Reinhard CT (2011) Earth science Sea change for the rise of oxygen Nature478(7368)194ndash195

69 Gaillard F Scaillet B Arndt NT (2011) Atmospheric oxygenation caused by a change involcanic degassing pressure Nature 478(7368)229ndash232

70 Waldbauer JR Sherman LS Sumner DY Summons RE (2009) Late Archean molecularfossils from the Transvaal Supergroup record the antiquity of microbial diversity andaerobiosis Precambrian Res 169(1ndash4)28ndash47

71 Stuumleken EE Catling DC Buick R (2012) Contributions to late Archaean sulphur cyclingby life on land Nat Geosci 5(10)722ndashndash725

72 Schopf JW (1993) Microfossils of the Early Archean Apex chert New evidence of theantiquity of life Science 260(5108)640ndash646

73 Brasier M McLoughlin N Green O Wacey D (2006) A fresh look at the fossil evidencefor early Archaean cellular life Philos Trans R Soc Lond B Biol Sci 361(1470)887ndash902

74 Bilofsky HS Burks C (1988) The GenBank genetic sequence data bank Nucleic AcidsRes 16(5)1861ndash1863

75 Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and highthroughput Nucleic Acids Res 32(5)1792ndash1797

76 Akaike H (1974) New look at statistical-model identification IEEE Trans AutomatContr AC19(6)716ndash723

77 Hurvich CM Tsai CL (1989) Regression and time-series model selection in small sam-ples Biometrika 76(2)297ndash307

78 Posada D (2008) jModelTest Phylogenetic model averagingMol Biol Evol 25(7)1253ndash125679 Lanave C Preparata G Saccone C Serio G (1984) A new method for calculating

evolutionary substitution rates J Mol Evol 20(1)86ndash9380 Drummond AJ Rambaut A (2007) BEAST Bayesian evolutionary analysis by sampling

trees BMC Evol Biol 721481 Rippka R Waterbury J Cohenbazire G (1974) Cyanobacterium which lacks thylakoids

Arch Microbiol 100(1)419ndash43682 Nakamura Y et al (2003) Complete genome structure of Gloeobacter violaceus PCC

7421 a cyanobacterium that lacks thylakoids DNA Res 10(4)137ndash14583 Ferris MJ Ruff-Roberts AL Kopczynski ED Bateson MM Ward DM (1996) Enrichment

culture and microscopy conceal diverse thermophilic Synechococcus populations ina single hot spring microbial mat habitat Appl Environ Microbiol 62(3)1045ndash1050

84 Miller M et al (2009) The CIPRES portals CIPRES Available at wwwphyloorgsub_sectionsportal Accessed February 2012

85 Sukumaran J Holder MT (2010) DendroPy A Python library for phylogenetic com-puting Bioinformatics 26(12)1569ndash1571

86 Golubic S Sergeev VN Knoll AH (1995) Mesoproterozoic Archaeoellipsoides Akinetesof heterocystous cyanobacteria Lethaia 28285ndash298

87 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414


Supporting InformationSchirrmeister et al 101073pnas1209927110SI TextTaxon Sampling Strain ldquoG40rdquo (deposited in GenBank) is a yet-uncharacterized terminally differentiated multicellular isolatefrom the North Sea Its closest relative based on 16S rRNA se-quences is Nodularia Strain G40 was isolated from ponds at theshore of northwestern Ameland The Netherlands The strainwas then cultivated in ASN III seawater medium and kept at 15 degCin an environmental chamber at a constant daynight cycle of 6 hdarkness and 18 h light

Phylogenetic Analyses Phylogenetic relationships were estimatedusing MrBayes v312 (1) We used two Markov chain MonteCarlo runs each calculating six Metropolis-coupled chains for100 million generations sampling every 2000th generation De-fault priors were adequate and left unchanged but the temper-ature parameter was adjusted to 01 to ensure proper mixingConvergence between runs was achieved as the potential scalereduction factor had approached 100 and average SDs of splitfrequencies was lt001 Mixing and convergence of all parame-ters was further assessed using the software Tracer v15 (2) Wecombined runs after discarding the first 25 of samples as aconservative burn-in including only samples from the stationaryphase Effective sample sizes were large (gt3000) for the likeli-hood samples and all estimated parameters supporting a well-mixed analysis The Bayesian 50 majority-rule consensus treeis shown in Fig S1

Morphotype AssessmentTo ensure morphological character states(unicellularmulticellular) were assigned correctly for each cya-nobacterial taxon used in this study we carefully examinedoriginal publications describing the morphology of each strainFurthermore we conducted BLAST analyses (3) for each se-quence to reassure its identity In cases where the publicationcontaining the original description of a strain was not availablewe examined the closest 16S rRNA relative (identified from theBLAST results ge95 maximum identity) for which a publica-tion was available For each strain additional information foundin the literature (4ndash44) is listed in Table S5 Furthermore a closeBLAST result is given for each taxon including percentage of itsmaximum identity (Table S5)

Shifts in Diversification Rates The function MEDUSA from thegeiger 13-1 package in R (45) uses maximum likelihood to es-timate a birth-death model of diversification that includes theoptimal number of rate shifts but penalizes for excess parametersbased on Akaike Information Criterion (AIC) scores Phyloge-netic positions of unsampled species and strains in the cyano-bacterial phylum were estimated with help of a phylogenetic treeof 1220 taxa compiled in a previous study (46) Subsequentlynumbers of unsampled species and strains were assigned to taxasampled for the dating analyses of this study (Table S3) In-ferences based on maximum clade credibility trees gave qual-itatively similar results

1 Ronquist F Huelsenbeck JP (2003) MrBayes 3 Bayesian phylogenetic inference undermixed models Bioinformatics 19(12)1572ndash1574

2 Rambaut A Drummond AJ (2007) Tracer v14 Available at http treebioedacuksoftwaretracer Accessed January 2012

3 Altschul SF et al (1997) Gapped BLAST and PSI-BLAST A new generation of proteindatabase search programs Nucleic Acids Res 25(17)3389ndash3402

4 Cuzman OA et al (2010) Biodiversity of phototrophic biofilms dwelling onmonumental fountains Microb Ecol 60(1)81ndash95


6 Nakamura Y et al (2002) Complete genome structure of the thermophiliccyanobacterium Thermosynechococcus elongatus BP-1 DNA Res 9(4)123ndash130

7 Lyra C et al (2001) Molecular characterization of planktic cyanobacteria of AnabaenaAphanizomenon Microcystis and Planktothrix genera Int J Syst Evol Microbiol 51(Pt 2)513ndash526

8 Casamatta DA Johansen JR Vis ML Broadwater ST (2005) Molecular and morphologicalcharacterisation of ten polar and near-polar strains with the Oscillatoriales (cyanobacteria)J Phycol 41421ndash438

9 Ishida T Watanabe MM Sugiyama J Yokota A (2001) Evidence for polyphyletic originof the members of the orders of Oscillatoriales and Pleurocapsales as determined by16S rDNA analysis FEMS Microbiol Lett 201(1)79ndash82

10 Ishida T Yokota A Sugiyama J (1997) Phylogenetic relationships of filamentouscyanobacterial taxa inferred from 16S rRNA sequence divergence J Gen ApplMicrobiol 43(4)237ndash241

11 Janssen PJ et al (2010) Genome sequence of the edible cyanobacterium Arthrospirasp PCC 8005 J Bacteriol 192(9)2465ndash2466

12 Tomitani A Knoll AH Cavanaugh CM Ohno T (2006) The evolutionary diversificationof cyanobacteria Molecular-phylogenetic and paleontological perspectives Proc NatlAcad Sci USA 103(14)5442ndash5447

13 Fuller NJ et al (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal thepredominance of a single marine Synechococcus clade throughout a stratified watercolumn in the Red Sea Appl Environ Microbiol 69(5)2430ndash2443

14 Urbach E Scanlan DJ Distel DL Waterbury JB Chisholm SW (1998) Rapid diversificationof marine picophytoplankton with dissimilar light-harvesting structures inferred fromsequences of Prochlorococcus and Synechococcus (Cyanobacteria) J Mol Evol 46(2)188ndash201

15 Moore LR Rocap G Chisholm SW (1998) Physiology and molecular phylogeny ofcoexisting Prochlorococcus ecotypes Nature 393(6684)464ndash467

16 Ernst A Becker S Wollenzien UIA Postius C (2003) Ecosystem-dependent adaptiveradiations of picocyanobacteria inferred from 16S rRNA and ITS-1 sequence analysisMicrobiology 149(Pt 1)217ndash228

17 Sugita C et al (2007) Complete nucleotide sequence of the freshwater unicellularcyanobacterium Synechococcus elongatus PCC 6301 chromosome Gene content andorganization Photosynth Res 93(1ndash3)55ndash67

18 van Hannen EJ et al (1999) Changes in bacterial and eukaryotic community structureafter mass lysis of filamentous cyanobacteria associated with viruses Appl EnvironMicrobiol 65(2)795ndash801

19 Sihvonen LM et al (2007) Strains of the cyanobacterial genera Calothrix and Rivulariaisolated from the Baltic Sea display cryptic diversity and are distantly related toGloeotrichia and Tolypothrix FEMS Microbiol Ecol 61(1)74ndash84

20 Boone DR Castenholz RW (2001) Bergeyrsquos Manual of Systematic Bacteriology TheArchaea and the Deeply Branching and Phototropic Bacteria Cyanobacteria edGarrity GM (Springer New York)

21 Wilmotte A Auwera G DeWachter R (1992) Structure of the 16S ribosomal RNA ofthe thermophilic cyanobacterium Chlorogloeopsis HTF (lsquoMastigocladus laminosusHTFrsquo) strain PCC75 18 and phylogenetic analysis FEBS Lett 317(1ndash2)96ndash100

22 Pointing SB Warren-Rhodes KA Lacap DC Rhodes KL McKay CP (2007) Hypolithiccommunity shifts occur as a result of liquid water availability along environmentalgradients in Chinarsquos hot and cold hyperarid deserts Environ Microbiol 9(2)414ndash424

23 Nguyen VLA Tanabe Y Matsuura H Kaya K Watanabe MM (2012) Morphological bio-chemical and phylogenetic assessments of water-bloom-forming tropical morphospeciesof Microcystis (Chroococcales Cyanobacteria) Phycological Res 60208ndashndash222

24 Winder B Stal LJ Mur LR (1990) Crinalium epipsammum sp nov A filamentouscyanobacterium with trichomes composed of elliptical cells and containing poly-β-(14) glucan (cellulose) Microbiology 136(8)1645ndash1653

25 Turner S Huang TC Chaw SM (2001) Molecular phylogeny of nitrogen fixingunicellular cyanobacteria Bot Bull Acad Sin 42181ndash186

26 Nuumlbel U Garcia-Pichel F Muyzer G (1997) PCR primers to amplify 16S rRNA genesfrom cyanobacteria Appl Environ Microbiol 63(8)3327ndash3332

27 Fewer D Friedl T Buedel B (2002) Chroococcidiopsis and heterocyst-differentiatingcyanobacteria are each others closest living relatives Mol Phyl Evol 23(1)82ndash90

28 Nelissen B Van de Peer Y Wilmotte A De Wachter R (1995) An early origin of plastidswithin the cyanobacterial divergence is suggested by evolutionary trees based oncomplete 16S rRNA sequences Mol Biol Evol 12(6)1166ndash1173

29 Ionescu D Hindiyeh MY Malkawi HI Oren A (2010) Biogeography of thermophiliccyanobacteria Insights from the Zerka Marsquoin hot springs (Jordan) FEMS MicrobiolEcol 72(1)103ndash113

30 Oren A Ionescu D Hindiyeh M Malkawi H (2009) Morphological phylogenetic andphysiological diversity of cyanobacteria in the hot springs of Zerka Marsquoin JordanBioRisk 3(Special Issue)69ndash82

31 Lehtimaumlki J et al (2000) Characterization of Nodularia strains cyanobacteria frombrackish waters by genotypic and phenotypic methods Int J Syst Evol Microbiol50(Pt 3)1043ndash1053

Schirrmeister et al wwwpnasorgcgicontentshort1209927110 1 of 15

32 Voss JD Mills DK Myers JL Remily ER Richardson LL (2007) Black band diseasemicrobial community variation on corals in three regions of the wider CaribbeanMicrob Ecol 54(4)730ndash739

33 Nakamura Y et al (2003) Complete genome structure of Gloeobacter violaceus PCC7421 a cyanobacterium that lacks thylakoids DNA Res 10(4)137ndash145

34 Micheletti E et al (2008) Sheathless mutant of Cyanobacterium Gloeothece sp strainPCC 6909 with increased capacity to remove copper ions from aqueous solutions ApplEnviron Microbiol 74(9)2797ndash2804

35 Nuumlbel U Garcia-Pichel F Muyzer G (2000) The halotolerance and phylogeny ofcyanobacteria with tightly coiled trichomes (Spirulina Turpin) and the description ofHalospirulina tapeticola gen nov sp nov Int J Syst Evol Microbiol 50(Pt 3)1265ndash1277

36 Taton A et al (2006) Polyphasic study of antarctic cyanobacterial strains J Phycol42(6)1257ndash1270

37 Pomati F Sacchi S Rossetti C Giovannardi S (2000) The freshwater cyanobacteriumPlanktothrix sp FP1 Molecular Identification and detection of paralytic shellfishpoisoning toxins J Phycol 36(3)553ndash562

38 Marin B Nowack ECM Gloumlckner G Melkonian M (2007) The ancestor of the Paulinellachromatophore obtained a carboxysomal operon by horizontal gene transfer froma Nitrococcus-like γ-proteobacterium BMC Evol Biol 785

39 Ligon PJB Meyer KG Martin JA Curtis SE (1991) Nucleotide sequence of a 16S rRNAgene from Anabaena sp strain PCC 7120 Nucleic Acids Res 19(16)4553

40 El-Shehawy R Lugomela C Ernst A Bergman B (2003) Diurnal expression of hetR anddiazocyte development in the filamentous non-heterocystous cyanobacteriumTrichodesmium erythraeum Microbiology 149(Pt 5)1139ndash1146

41 Zwart G et al (2005) Molecular characterization of cyanobacterial diversity ina shallow eutrophic lake Environ Microbiol 7(3)365ndash377

42 Urbach E Robertson DL Chisholm SW (1992) Multiple evolutionary origins ofprochlorophytes within the cyanobacterial radiation Nature 355(6357)267ndash270

43 Kaneko T et al (1996) Sequence analysis of the genome of the unicellularcyanobacterium Synechocystis sp strain PCC6803 II Sequence determination of theentire genome and assignment of potential protein-coding regions DNA Res 3(3)109ndash136

44 Gugger MF Hoffmann L (2004) Polyphyly of true branching cyanobacteria(Stigonematales) Int J Syst Evol Microbiol 54(Pt 2)349ndash357


46 Schirrmeister BE Antonelli A Bagheri HC (2011) The origin of multicellularity incyanobacteria BMC Evol Biol 1145

Fig S1 Bayesian 50 majority-rule consensus phylogram based on MrBayes analysis Posterior probabilities shown at nodes when gt090 Unicellular cya-nobacteria belonging to sections I and II are marked by yellow and orange whereas multicellular cyanobacteria from sections III IV and V are marked bygreen blue and purple respectively Gloeobacter violaceus groups closest to the eubacterial outgroup


Fig S2 Bayesian consensus tree of BEAST analysis 7 Posterior probabilities and node numbers are presented at nodes Gray nodes were not recovered by allanalyses


Fig S3 Clade-specific diversification rates using species numbers (uncorrelated lognormal UCLN) Results of MEDUSA analyses indicating diversification rateshifts for the different consensus trees from the Bayesian analyses assuming uncorrelated lognormally distributed evolutionary rates


Fig S4 Clade-specific diversification rates using strain numbers (UCLN) Results of MEDUSA analyses indicating diversification rate shifts for the differentconsensus trees from the Bayesian analyses assuming UCLN distributed evolutionary rates


Fig S5 Clade-specific diversification rates using species numbers (uncorrelated exponentially distributed UCED) Results of MEDUSA analyses indicatingdiversification rate shifts for the different consensus trees from the Bayesian analyses assuming UCED evolutionary rates


Fig S6 Clade-specific diversification rates using strain numbers (UCED) Results of MEDUSA analyses indicating diversification rate shifts for the differentconsensus trees from the Bayesian analyses assuming UCED evolutionary rates


Table

S1

Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCED

evolutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgr

mdashmdash

Yes

Yes

Yes

Yes

mdashmdash

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCED

)eth~ m

THORN(HPD

)forall

Node1

295

(239ndash

3-99

)372

(262ndash

540

)281

(241ndash

336

)317

(258ndash

40)

282

(245ndash

330

)306

(260ndash

560

)293

(245ndash

360

)333

(278ndash

380

)Node3

244

(221ndash

280

)295

(231ndash

397

)237

(220ndash

260

)26(225ndash

313

)239

(220ndash

265

)255

(224ndash

293

)244

(223ndash

28)

275

(232ndash

325

)Node6

200

(152ndash

231

)221

(165ndash

291

)197

(148ndash

227

)204

(149ndash

250

)196

(143ndash

230

)202

(145ndash

244

)2(156ndash

225

)211

(163ndash

258

)Node31

182

(112ndash

228

)216

(143ndash

265

)176

(107ndash

224

)212

(124ndash

242

)185

(111ndash

227

)212

(12ndash24)

185

(2-229)

213

(127ndash

244

)Node43

191

(115ndash

243

)22(131ndash

311

)18(15ndash229

)194

(117ndash

26)

181

(111ndash

230

)19(117ndash

247

)191

(124ndash

24)

207

(132ndash

273

)

Expex

ponen

tial


n)LN

lognorm


nSD

)mdashnotap

plicab

le

Truncatedat

38Bya


Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95

highest-posteriorden

sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le


Table S3 Names of taxa and corresponding numbers of species and strains to which they can be assigned in thecyanobacterial phylum

Taxa No species No strains GenBank accession

Acaryochloris sp JJ8A6 (4) 5 14 AM710387Synechococcus lividus C1dagger (5) AF132772Thermosynechococcus elongatus BP-1dagger (6) BA000039Anabaena sp PCC 7108 (7) 28 459 AJ133162Arthronema gygaxiana UTCC 393 (8) 5 57 AF218370Pseudanabaena sp PCC 7304dagger (9) AB039019Pseudanabaena sp PCC 6802dagger (9) AB039016ldquoPhormidium mucicolardquo IAM M-221dagger (10) AB003165Arthrospira platensis PCC 8005 (11) 9 42 X70769Lyngbya aestuarii PCC 7419dagger (12) AB075989Synechococcus sp CC9605 (13) 13 567 AY172802Synechococcus sp WH8101dagger (14) AF001480Prochlorococcus sp MIT 9313dagger (15) AF053399Cyanobium sp JJ23-1dagger (16) AM710371Synechococcus elongatus PCC 6301dagger (17) AP008231Prochlorothrix hollandicadagger (18) AJ007907Calothrix sp PCC 7103 (19) 6 226 AM230700Chamaesiphon subglobosus PCC 7430 (20) 1 2 AY170472Chlorogloeopsis sp PCC 7518 (21) 18 136 X68780Fischerella muscicola PCC 7414dagger (12) AB075986Chroococcidiopsis sp CC2 (22) 3 51 DQ914864Chroococcus sp JJCM (4) 10 325 AM710384Microcystis aeruginosa strain 038dagger (23) DQ363254Crinalium magnum SAG 3487dagger (24) 2 2 AB115965Starria zimbabweensis SAG 7490dagger (20) AB115962Cyanothece sp PCC 8801 (25) 4 22 AF296873Dactylococcopsis salina PCC 8305 (26) 4 34 AJ000711Dermocarpa sp MBIC10768 (9) 8 41 AB058287Myxosarcina sp PCC 7312dagger (27) AJ344561Myxosarcina sp PCC 7325dagger (27) AJ344562Pleurocapsa sp CALU 1126dagger (27) DQ293994Pleurocapsa sp PCC 7516dagger (28) X78681Dermocarpella ldquoincrassatardquo SAG 2984dagger (27) AJ344559Filamentous thermophilic cyanobacterium tBTRCCn 301 (29 30) 6 17 DQ471441Synechococcus sp C9dagger (5) AF132773Cyanobacterium G40 (present study) 17 247 JX069960Nodularia ldquosphaerocarpardquo PCC 7804dagger (31) AJ133181Geitlerinema sp BBD HS217 (32) 8 97 EF110974Gloeobacter violaceus PCC 7421 (33) 1 1 BA000045Gloeothece sp PCC 6909 (34) 3 13 HE975018Halospirulina tapeticola CCC Baja-95 Cl2 (35) 2 6 NR_026510Leptolyngbya sp ANTL521 (36) 6 332 AY493584ldquoPlanktothrixrdquo sp FP1dagger (37) AF212922ldquoPlectonemardquo sp F3dagger (36) AF091110Microcoleus chthonoplastes PCC 7420 (38) 19 434 AM709630Symploca sp PCC 8002dagger (9) AB039021Nostoc sp PCC 7120 (39) 25 649 X59559Oscillatoria sancta PCC 7515 (5) 30 129 AF132933Trichodesmium erythraeum IMS 101dagger (40) AB075999ldquoOscillatoriardquo sp IW19 (41) 20 147 AJ133106Prochloron sp (42) 4 6 X63141Synechocystis sp PCC 6308dagger (43) AB039001Radiocystis sp JJ30-3 (44) 2 2 AM710389Synechocystis sp PCC 6803 (44) 4 51 NC_000911Scytonema sp U-3ndash3 (38) 4 45 AY069954Spirulina sp PCC 6313 (38) 6 29 X75045Symphyonema sp strain 1517 (45) 7 9 AJ544084Synechococcus sp P1 (5) 1 2 AF132774

Phylogenetic positions of taxa and the amount of speciesstrains they represent were estimated with help of a phylogenetic treecomprising 1220 cyanobacterial taxa presented in the study by Schirrmeister et al (46)Citation for a close relative identified by BLAST searchdaggerClades that have been pruned for the diversification rate analyses Taxa with their names in quotes have likely been misidentified inthe original publication considering their BLAST results


Table S4 Estimated clade-specific diversification rates based on trees from eight phylogenetic analyses

Analysis

Species Strains

Clade r e AICc Clade r e AICc

UCLN distributed rates of evolutionRoot 017 473E-8 Root 028 779E-7

1 Node 3 163 907E-9 2066 Node 3 239 08 3577Node 33 137 093 Node 34 108 100Root 038 114E-8 Root 023 380E-7 3712

2 Node 4 143 231E-9 2184 Node 3 191 083Node 33 099 095 Node 34 078 100Root 017 467E-7 Root 028 107E-6 3654

3 Node 3 163 229E-7 2126 Node 3 199 886E-001Node 33 09 097 Node 34 106 100Root 015 503E-7 Root 025 179E-7 3668



6 Node 3 167 409E-7 2124 Node 3 197 09Node 33 081 097 Node 34 101 100

7 Root 017 390E-10 Root 028 390E-7 3578Node 3 163 245E-7 2067 Node 3 239 08Node 33 138 093 Node 34 107 100Root 015 442E-7 Root 024 789E-7 3700


UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713



3 Node 4 192 018 2109 Node 3 16 097Node 33 093 098 Node 34 017 100Root 045 2178E-8 Root 026 494E-7 3728

4 Node 4 158 036 2477 Node 3 128 097node 33 007 099 Node 34 01 100Root 049 192E-7 Root 03 161E-6 369


6 Node 4 158 564E-2 2200 Node 3 126 097Node 33 112E-5 10 Node 34 002 100Root 048 107E-8 Root 029 178E-8 3716


8 Node 4 158 025 2216 Node 3 129 097Node 33 024 098 Node 34 024 100

Only nodes where shifts of diversification rates have occurred are presented In 31 of 32 trees two rate shifts were detected In 23 trees the first shift isestimated to occur at node 3 In the remaining nine trees the first shift is estimated to occur at node 4 The second shift occurs in 16 trees at node 33 and in 16trees at node 34 r speciation rates e extinction rates


Table S5 Additional information for each taxon (or close 16S rRNA relatives) that have been used in this study as found in the literature

Taxon Notes

Acaryochloris sp JJ8A6 BLAST result Aphanocapsa muscicola 5N-04 (97) is a unicellular cyanobacterium isolatedfrom monumental fountains in Florence (Italy) (4)

Anabaena sp PCC 7108 Strain PCC 7108 is a nontoxic filamentous cyanobacterium capable of forming heterocystsisolated from intertidal zone (United States) (7) BLAST result Anabaena sp KVSF7 (98)

Arthronema gygaxiana UTCC 393 Strain UTCC 393 is filamentous isolated from lake of Bays Twnsp Ontario (Canada) (8)BLAST result Pseudanabaena sp 1tu24s9 (98)

Arthrospira platensis PCC 8005 Strain PCC 8005 is a filamentous cyanobacteria not forming heterocysts usually found inalkaline lakes (11) BLAST result Spirulina maxima UTEX ldquoLB 2342rdquo (99)

Calothrix sp PCC 7103 Strain PCC 7103 is a filamentous cyanobacterium able to form heterocysts (basal end) andakinetes (19) BLAST result Calothrix desertica PCC 7102 (99)

Chamaesiphon subglobosus PCC 7430 Strain PCC 7430 has been described as unicellular cyanobacterium that reproduces bysuccessive unequal binary fission and occurs in freshwater (20) BLAST resultChamaesiphon minutus (98)

Chlorogloeopsis sp PCC 7518 Strain PCC 7518 is a multicellular thermophilic cyanobacterium that lacks branchingpatterns Additionally strain PCC 7518 has lost the ability to form heterocysts (21)BLAST result Chlorogloeopsis sp Greenland_2 (99)

Chroococcidiopsis sp CC2 Strain CC2 is a hypolithic cyanobacterium isolated from enrichment cultures whichhave been originally sampled from hyperarid deserts in China (22) BLAST resultChroococcidiopsis sp CC3 (99)

Chroococcus sp JJCM BLAST result Chroococcus sp 2T05h (97) is a unicellular bacterium isolated fromfountains in Italy and Spain (4)

Crinalium magnum SAG 3487 BLAST result Crinalium epipsammum SAG 2289 (100) is a filamentous cyanobacteriumwith elliptical trichomes (24)

Cyanobacterium G40 (present study) Strain G40 is a marine filamentous cyanobacterium with cell differentiation isolatedfrom North sea (The Netherlands) BLAST result Nodularia spumigena (95)

Cyanobium sp JJ23-1 BLAST result Synechococcus sp BO 8805 (99) is a unicellular phycocyanin rich isolatefrom Lake Constance (16)

Cyanothece sp PCC 8801 Strain PCC 8801 is a unicellular nitrogen-fixing cyanobacterium without sheath originallynamed ldquoSynechococcusrdquo RF-1 (25) BLAST result Cyanothece sp PCC 8802 (99)

Dactylococcopsis salina PCC 8305 Strain PCC 8305 is a unicellular halophil cyanobacterium with fusiform often elongatedcells growing (26) BLAST result Cyanothece sp 104 (99)

Dermocarpa sp MBIC10768 BLAST result Stanieria PCC 7301 (98) is a unicellular cyanobacterium able to formbaeocysts (9)

Dermocarpella ldquoincrassatardquo SAG 2984 Strain SAG 2984 (PCC 7326) is a unicellular marine cyanobacterium isolated from snail shellPuerto Penasco (Mexico) and the only axenic representative (27) BLAST resultMyxosarcina sp PCC 7312 (96)

Filamentous thermophilic cyanobacteriumtBTRCCn 301

Cyanobacterium tBTRCCn 301 is a filamentous cyanobacterium of unknown affiliationisolated from thermal springs close to the dead sea (Jordan) (29 30) BLAST resultSynechococcus sp C9 (92)

Fischerella muscicola PCC 7414 Strain PCC 7414 is a filamentous heterocystous cyanobacterium with uniserate branchescomposed of longer cells (12 20) BLAST result Fischerella muscicola (99)

Geitlerinema sp BBD HS217 Strain BBD HS217 is a gliding filamentous nonheterocystous cyanobacteria (32) Trichomesof Geitlerinema are generally described as straight or slightly sinuous (20) BLAST resultGeitlerinema sp BBD_P2b-1 (99)

Gloeobacter violaceus PCC 7421 Strain PCC 7421 is a rod-shaped unicellular cyanobacterium lacking thylacoid membranesisolated from calcareous rock Switzerland (33) BLAST result Gloeobacter violaceusVP3-01 (99)

Gloeothece sp PCC 6909 Strain PCC 6909 is a unicellular rod-shaped cyanobacterium capable of nitrogen fixationisolated from freshwater (20) BLAST result Gloeothece membranacea (99)

Halospirulina tapeticola CCC Baja-95 Cl2 Strain CCC Baja-95 Cl2 is a highly halotolerant filamentous cyanobacterium with helicallycoiled trichomes (35) BLAST result Halospirulina sp CCC Baja-95 Cl3 (99)

Leptolyngbya sp ANTL521 Strain ANTL521 is a filamentous cyanobacterium with sheath (36) BLAST resultPhormidium priestleyi ANTLPR26 (98)

Lyngbya aestuarii PCC 7419 Strain PCC 7419 is a filamentous cyanobacterium without cell differentiation that is capableof nitrogen fixation (12) BLAST result Lyngbya aestuarii (99)

Microcoleus chthonoplastes PCC 7420 Strain PCC 7420 is a filamentous nonheterocystous cyanobacterium collected from saltmarsh at Woods Hole MA Trichomes often parallel surrounded by common homogenoussheath (20 38) BLAST result Microcoleus chthonoplastes NCR (99)

Microcystis aeruginosa strain 038 BLAST result Microcystis aeruginosa VN441 (99) is a unicellular coccoid cyanobacteriumwhich produces mycrocystin isolated from ponds in Vietnam (23)

Myxosarcina sp PCC 7312 Strain PCC 7312 is a unicellular cyanobacterium morphologically indistinguishable fromChroococcidiopsis Isolated from a snail shell Puerto Penasco Mexico (27) BLAST resultPleurocapsa sp PCC 7314 (99)


Table S5 Cont

Taxon Notes

Myxosarcina sp PCC 7325 Description of PCC 7325 is the same as for Myxosarcina sp PCC 7312 (27) BLAST resultMyxosarcina sp PCC 7312 (96)

Nodularia ldquosphaerocarpardquo PCC 7804 Strain PCC 7804 is a toxic filamentous heterocystous strain isolated from thermalspring (southern France) (20 31) BLAST result Nodularia sphaerocarpa BECID36 (99)

Nostoc sp PCC 7120 Strain PCC 7120 is a filamentous cyanobacterium capable of forming heterocysts (39)BLAST result Anabaena sp CCAP 14034A (98)

Oscillatoria sancta PCC 7515 Strain PCC 7515 is a filamentous nonheterocystous cyanobacterium isolated from agreenhouse water tank Stockholm Sweden Capable of aerobic nitrogen fixation(5 20) BLAST result Oscillatoria sancta (99)

ldquoOscillatoriardquo sp IW19 Strain IW19 is a filamentous isolate from Lake Loosdrecht Originally termedldquoOscillatoria limnetica-likerdquo (41) BLAST result Leptolyngbya sp 0BB30S02 (97)

ldquoPhormidium mucicolardquo IAM M-221 Strain M-221 is a filamentous nonheterocystous cyanobacterium (10) BLAST resultPseudanabaena PCC7403 (99)

ldquoPlanktothrixrdquo sp FP1 Strain FP1 is a filamentous nonheterocystous cyanobacterium isolated from LakeVarese Italy (37) BLAST result Limnothrix redekei 2LT25S01 (99)

ldquoPlectonemardquo sp F3 Strain F3 is a filamentous cyanobacterium with false branching It is a marinenonpolar cyanobacterial strain (36) BLAST result Leptolyngbya sp ANTL521 (97)

Pleurocapsa sp CALU 1126 BLAST result Pleurocapsa minor SAG 499 (98) is a unicellular hypolithiccyanobacterium isolated from a quartz pebble Namib desert Namibia (27)

Pleurocapsa sp PCC 7516 Strain PCC 7516 is a unicellular marine cyanobacterium isolated from a rock chipMarseille France (20 28) BLAST result Myxosarcina sp PCC 7312 (97)

Prochlorococcus sp MIT9313 Strain MIT9313 is a low-light adapted unicellular cyanobacterium isolated from GulfStream water samples (15) BLAST result Prochlorococcus sp MIT9303 (99)

Prochloron sp Prochloron sp is a unicellular spherical shaped symbiont of marine ascidians lackingPhycobilisomes (42) BLAST result Cyanothece sp PCC 8802 (93)

Prochlorothrix hollandica This is a filamentous cyanobacterium isolated from Lake Loosdrecht Netherlands (18)BLAST result Prochlorothrix hollandica SAG 1089 (99)

Pseudanabaena sp PCC 7304 Strain PCC 7304 is a filamentous cyanobacterium belonging to the Pseudanabaenacluster (9) BLAST result Phormidium mucicola AM M-221 (99)

Pseudanabaena sp PCC 6802 Strain PCC 6802 is a filamentous cyanobacterium belonging to the Pseudanabaenacluster (9) BLAST result Pseudanabaena sp 1a-03 (95)

Radiocystis sp JJ30-3 BLAST result Synechocystis sp PCC 6803 (100) is a unicellular cyanobacterium isolatedfrom freshwater California (20 43)

Scytonema sp U-3-3 BLAST result Scytonema hofmanni PCC 7110 (95) is a filamentous heterocystouscyanobacterium with sheath isolated from limestone Crystal Cave Bermuda (38)

Spirulina sp PCC 6313 Strain PCC 6313 was isolated from brackish water California It is a filamentous looselycoiled cyanobacterium (20 38) BLAST result Spirulina major 0BB36S18 (99)

Starria zimbabweensis SAG 7490 Starria is a nonbranching filamentous cyanobacterium with triradiate trichomes (20)BLAST result Crinalium magnum SAG 3487 (97)

Symphyonema sp 1517 Strain 1517 is a filamentous heterocystous cyanobacterium showing mainly Y-branchingisolated from Soil Papua New Guinea (44) BLAST result Symphyonema sp1269ndash1(100)

Symploca sp PCC 8002 Strain PCC 8002 is a filamentous nonheterocystous cyanobacterium isolated from highintertidal zones North Wales (9 20) BLAST result Symploca sp HBC5 (97)

Synechococcus lividus C1 Strain C1 is a thermophilic unicellular cyanobacterium a part of theldquounicellular-thermophilicrdquo (UNIT) sequence group (5) BLAST result Synechococcus spStrain PCC 6717 (99)

Synechococcus sp WH8101 Strain WH8101 is a unicellular facultatively marine cyanobacterial strain that lacksphycoerythrin (14) BLAST result Synechococcus sp RS9909 (99)

Synechococcus sp CC9605 Strain CC9605 is a marine unicellular cyanobacterium isolated from California current(13) BLAST result Synechococcus sp WH 8109 (99)

Synechococcus sp C9 Strain C9 is a unicellular cyanobacterium part of the Gloeobacter (GBACT) groupestimated to be close to the root of cyanobacteria (5) BLAST result CandidatusGloeomargarita lithophora D10 (98)

Synechococcus elongatus PCC 6301 Strain PCC 6301 is a unicellular rod-shaped cyanobacterium that occurs in freshwater(17) BLAST result Synechococcus elongatus PCC 7942 (100)

Synechocystis sp PCC 6803 Strain PCC 6803 is a unicellular cyanobacterium isolated from freshwater California(20 43) BLAST result Synechocystis sp PCC 6714 (100)

Synechococcus sp P1 Strain P1 is a unicellular cyanobacterium part of the Gloeobacter (GBACT) group whichhas been estimated to be close to the root of the cyanobacterial line (5) BLAST resultSynechococcus sp P2 (100)


Dataset S1 Sequence alignment used for the phylogenetic-tree reconstruction

Dataset S1

Dataset S2 XML input files for the different analyses using a relaxed clock with lognormally distributed evolutionary rates

Dataset S2

Table S5 Cont

Taxon Notes

Synechocystis sp PCC 6308 Strain PCC 6308 is a unicellular cyanobacterium isolated from lake water WI (20) BLASTresult Synechocystis sp VNM-13ndash10 (98)

Thermosynechococcus elongatus BP-1 Strain BP-1 is a thermophilic rod-shaped unicellular cyanobacterium originally isolatedfrom a hot spring in Beppu (Japan) (6) BLAST result Synechococcus elongatus (100)

Trichodesmium erythraeum IMS 101 Strain IMS101 is a marine filamentous cyanobacterium capable of aerobic nitrogenfixation without heterocyst formation (40) BLAST result Trichodesmium sp (99)

For each taxon a BLAST result is shown with the maximum identity given in brackets Taxa with their names in quotes have likely been misidentified in theoriginal publication considering their BLAST resultsCitation for a close relative identified by BLAST search


Evolution of multicellularity coincided with increaseddiversification of cyanobacteria and the GreatOxidation EventBettina E Schirrmeistera12 Jurriaan M de Vosb Alexandre Antonellic and Homayoun C Bagheria

aInstitute of Evolutionary Biology and Environmental Studies University of Zurich CH-8057 Zurich Switzerland bInstitute of Systematic Botany University ofZurich CH-8008 Zurich Switzerland and cGothenburg Botanical Garden and Department of Biological and Environmental Sciences University of GothenburgSE 405 30 Gothenburg Sweden

Edited by Stjepko Golubic Boston University Boston MA and accepted by the Editorial Board December 13 2012 (received for review June 15 2012)

Cyanobacteria are among the most diverse prokaryotic phyla withmorphotypes ranging from unicellular to multicellular filamentousforms including those able to terminally (ie irreversibly) differ-entiate in form and function It has been suggested that cyano-bacteria raised oxygen levels in the atmosphere around 245ndash232billion y ago during the Great Oxidation Event (GOE) hence dra-matically changing life on the planet However little is knownabout the temporal evolution of cyanobacterial lineages and pos-sible interplay between the origin of multicellularity diversifica-tion of cyanobacteria and the rise of atmospheric oxygen Weestimated divergence times of extant cyanobacterial lineages un-der Bayesian relaxed clocks for a dataset of 16S rRNA sequencesrepresenting the entire known diversity of this phylum We testedwhether the evolution of multicellularity overlaps with the GOE andwhether multicellularity is associated with significant shifts in di-versification rates in cyanobacteria Our results indicate an originof cyanobacteria before the rise of atmospheric oxygen The evo-lution of multicellular forms coincides with the onset of the GOEand an increase in diversification rates These results suggest thatmulticellularity could have played a key role in triggering cyano-bacterial evolution around the GOE

early life | major transitions | prokaryotic phylogenetics | molecular clock

Cyanobacteria are one of the morphologically most diversegroups of prokaryotic organisms Growth forms range from

uni- to multicellular and can include levels of reversible or ter-minal (ie irreversible) cell differentiation These diverse growthstrategies have enabled cyanobacteria to inhabit almost everyterrestrial and aquatic habitat on Earth Cyanobacteria havetraditionally been classified into five subsections according totheir morphology (1 2) where subsections I and II refer to uni-cellular species and subsections IIIndashV describe multicellular speciesSpecies belonging to subsections IV and V are able to produceterminally differentiated cells Despite the usefulness of thesesubsections molecular evidence shows that morphological andgenetic diversity do not always coincide Molecular phylogeniesindicate that probably none of the five subsections is mono-phyletic (3 4) and several transitions between uni- and multi-cellularity have taken place (5) According to the fossil recordvarious distinct morphotypes attributed to cyanobacteria werealready present over 2 billion y ago (Bya) (6 7) The phylum isthought to have existed as early as 245ndash232 Bya based on theassumption that cyanobacteria were responsible for the accu-mulation of atmospheric oxygen levels referred to as the GreatOxidation Event (GOE) (8ndash12) Despite the generally acceptedtime-frame for the rise of cyanobacteria surprisingly little isknown about when morphological innovations such as multi-cellularity first appeared It is also unclear what influence if anythese innovations may have had on the diversification of thephylum The assumed link between the rise of atmospheric ox-ygen and cyanobacteria is also poorly understood did the GOEclosely follow the first appearance of cyanobacteria or did it take

place considerably later in possible association with morpho-logical innovations of the phylumThere have been previous attempts to estimate the origin of

cyanobacteria and their morphotypes (13ndash16) However it islikely that a biased taxonomic choice especially missing earlybranches of the cyanobacterial phylogeny may have led to in-complete conclusions (17 18) Phylogenetic evidence indicatesthat multicellularity evolved very early in the history of cyano-bacteria challenging the view that multicellularity is a derivedcondition in the phylum (5) Nonetheless important questionsremain (i) When did cyanobacteria and their major cladesevolve (ii) When did multicellularity first appear (iii) How arethese transitions associated with the GOE around 245ndash232 ByaThe far-reaching impact of the GOE cannot be emphasized

enough it changed Earthrsquos history by enabling the evolution ofaerobic life Unlike other eubacterial phyla cyanobacteria ex-hibit a well-studied fossil record (6 7 19 20) However fossildata are often limited and present only minimum age estimatesof clades Therefore a combination of fossil data with molecularphylogenetic methods has been advocated (21ndash23) The use ofcarefully selected calibration priors for molecular-dating analysescan provide new insights into the temporal evolution of cyano-bacteria and the early history of life Presently available genomedata for cyanobacteria are biased toward unicellular taxa and donot sufficiently represent the known diversity of this phylumTherefore we reconstructed phylogenetic trees on the basis of16S rRNA sequences which have been carefully sampled basedon phylogenetic disparity as described previously (5) We furtherestimated divergence times of cyanobacteria and addresseddifferent interpretations of the fossil record as calibration priorsWe then evaluated whether the GOE coincided with the de-velopment of major cyanobacterial morphotypes present todayFinally we tested for shifts in diversification rates incorporatinginformation on 281 species and 4194 strains Our results supporttheories of an early cyanobacterial origin toward the end of theArchean Eon before 25 Bya Evolution of multicellularity co-incided with the onset of the GOE and corresponded to amarked increase of diversification in cyanobacteria

Author contributions BES JMdV AA and HCB designed research BES andJMdV analyzed data and BES JMdV AA and HCB wrote the paper

The authors declare no conflict of interest

This article is a PNAS Direct Submission SG is a guest editor invited by theEditorial Board

Freely available online through the PNAS open access option

Data deposition The sequences reported in this paper have been deposited in the Gen-Bank database (accession no JX069960)1Present address School of Earth Sciences University of Bristol Bristol BS8 1RJUnited Kingdom

2To whom correspondence should be addressed E-mail bettinaschirrmeisterbristolacuk

This article contains supporting information online at wwwpnasorglookupsuppldoi101073pnas1209927110-DCSupplemental

wwwpnasorgcgidoi101073pnas1209927110 PNAS | January 29 2013 | vol 110 | no 5 | 1791ndash1796

EVOLU

TION




25





ble

1Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCLN

distributedev

olutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgroup

No

No

Yes

Yes

Yes

Yes

No

No

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCLN

)ethm~

THORN(HPD

)forall

Node1

295

(25ndash36)

367

(279ndash

474

)299

(257ndash

355

)335

(274ndash

415

)287

(253ndash

330

)306

(266ndash

353

)295

(253ndash

355

)339

(287ndash

380

)Node3

254

(228ndash

298

)308

(242ndash

384

)242

(221ndash

273

)265

(228ndash

318

)238

(220ndash

262

)249

(226ndash

281

)254

(229ndash

297

)286

(243ndash

334

)Node6

204

(177ndash

235

)233

(189ndash

287

)202

(172ndash

228

)210

(178ndash

254

)199

(167ndash

222

)202

(170ndash

232

)204

(179ndash

235

)218

(186ndash

260

)Node31

177

(14ndash224

)216

(153ndash

256

)172

(134ndash

220

)198

(139ndash

234

)167

(128ndash

217

)175

(130ndash

223

)177

(141ndash

225

)212

(150ndash

241

)Node43

200

(156ndash

243

)235

(173ndash

303

)185

(146ndash

225

)197

(148ndash

250

)180

(138ndash

219

)186

(141ndash

230

)200

(157ndash

241

)218

(171ndash

272

)

Eightdifferentco

mbinationsofcalib

rationpriors

forthedivergen


pex

ponen

tial


n)LN

lognorm


nSD

)mdashcalib

rationnot

applicab

le

Truncatedat

38Bya



EVOLU

TION






















1ndash61






EVOLU

TION















































































































































Table

S1

Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCED

evolutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgr

mdashmdash

Yes

Yes

Yes

Yes

mdashmdash

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCED

)eth~ m

THORN(HPD

)forall

Node1

295

(239ndash

3-99

)372

(262ndash

540

)281

(241ndash

336

)317

(258ndash

40)

282

(245ndash

330

)306

(260ndash

560

)293

(245ndash

360

)333

(278ndash

380

)Node3

244

(221ndash

280

)295

(231ndash

397

)237

(220ndash

260

)26(225ndash

313

)239

(220ndash

265

)255

(224ndash

293

)244

(223ndash

28)

275

(232ndash

325

)Node6

200

(152ndash

231

)221

(165ndash

291

)197

(148ndash

227

)204

(149ndash

250

)196

(143ndash

230

)202

(145ndash

244

)2(156ndash

225

)211

(163ndash

258

)Node31

182

(112ndash

228

)216

(143ndash

265

)176

(107ndash

224

)212

(124ndash

242

)185

(111ndash

227

)212

(12ndash24)

185

(2-229)

213

(127ndash

244

)Node43

191

(115ndash

243

)22(131ndash

311

)18(15ndash229

)194

(117ndash

26)

181

(111ndash

230

)19(117ndash

247

)191

(124ndash

24)

207

(132ndash

273

)

Expex

ponen

tial


n)LN

lognorm


nSD

)mdashnotap

plicab

le

Truncatedat

38Bya


Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes









25





ble

1Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCLN

distributedev

olutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgroup

No

No

Yes

Yes

Yes

Yes

No

No

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCLN

)ethm~

THORN(HPD

)forall

Node1

295

(25ndash36)

367

(279ndash

474

)299

(257ndash

355

)335

(274ndash

415

)287

(253ndash

330

)306

(266ndash

353

)295

(253ndash

355

)339

(287ndash

380

)Node3

254

(228ndash

298

)308

(242ndash

384

)242

(221ndash

273

)265

(228ndash

318

)238

(220ndash

262

)249

(226ndash

281

)254

(229ndash

297

)286

(243ndash

334

)Node6

204

(177ndash

235

)233

(189ndash

287

)202

(172ndash

228

)210

(178ndash

254

)199

(167ndash

222

)202

(170ndash

232

)204

(179ndash

235

)218

(186ndash

260

)Node31

177

(14ndash224

)216

(153ndash

256

)172

(134ndash

220

)198

(139ndash

234

)167

(128ndash

217

)175

(130ndash

223

)177

(141ndash

225

)212

(150ndash

241

)Node43

200

(156ndash

243

)235

(173ndash

303

)185

(146ndash

225

)197

(148ndash

250

)180

(138ndash

219

)186

(141ndash

230

)200

(157ndash

241

)218

(171ndash

272

)

Eightdifferentco

mbinationsofcalib

rationpriors

forthedivergen


pex

ponen

tial


n)LN

lognorm


nSD

)mdashcalib

rationnot

applicab

le

Truncatedat

38Bya



EVOLU

TION






















1ndash61






EVOLU

TION















































































































































Table

S1

Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCED

evolutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgr

mdashmdash

Yes

Yes

Yes

Yes

mdashmdash

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCED

)eth~ m

THORN(HPD

)forall

Node1

295

(239ndash

3-99

)372

(262ndash

540

)281

(241ndash

336

)317

(258ndash

40)

282

(245ndash

330

)306

(260ndash

560

)293

(245ndash

360

)333

(278ndash

380

)Node3

244

(221ndash

280

)295

(231ndash

397

)237

(220ndash

260

)26(225ndash

313

)239

(220ndash

265

)255

(224ndash

293

)244

(223ndash

28)

275

(232ndash

325

)Node6

200

(152ndash

231

)221

(165ndash

291

)197

(148ndash

227

)204

(149ndash

250

)196

(143ndash

230

)202

(145ndash

244

)2(156ndash

225

)211

(163ndash

258

)Node31

182

(112ndash

228

)216

(143ndash

265

)176

(107ndash

224

)212

(124ndash

242

)185

(111ndash

227

)212

(12ndash24)

185

(2-229)

213

(127ndash

244

)Node43

191

(115ndash

243

)22(131ndash

311

)18(15ndash229

)194

(117ndash

26)

181

(111ndash

230

)19(117ndash

247

)191

(124ndash

24)

207

(132ndash

273

)

Expex

ponen

tial


n)LN

lognorm


nSD

)mdashnotap

plicab

le

Truncatedat

38Bya


Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes








ble

1Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCLN

distributedev

olutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgroup

No

No

Yes

Yes

Yes

Yes

No

No

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCLN

)ethm~

THORN(HPD

)forall

Node1

295

(25ndash36)

367

(279ndash

474

)299

(257ndash

355

)335

(274ndash

415

)287

(253ndash

330

)306

(266ndash

353

)295

(253ndash

355

)339

(287ndash

380

)Node3

254

(228ndash

298

)308

(242ndash

384

)242

(221ndash

273

)265

(228ndash

318

)238

(220ndash

262

)249

(226ndash

281

)254

(229ndash

297

)286

(243ndash

334

)Node6

204

(177ndash

235

)233

(189ndash

287

)202

(172ndash

228

)210

(178ndash

254

)199

(167ndash

222

)202

(170ndash

232

)204

(179ndash

235

)218

(186ndash

260

)Node31

177

(14ndash224

)216

(153ndash

256

)172

(134ndash

220

)198

(139ndash

234

)167

(128ndash

217

)175

(130ndash

223

)177

(141ndash

225

)212

(150ndash

241

)Node43

200

(156ndash

243

)235

(173ndash

303

)185

(146ndash

225

)197

(148ndash

250

)180

(138ndash

219

)186

(141ndash

230

)200

(157ndash

241

)218

(171ndash

272

)

Eightdifferentco

mbinationsofcalib

rationpriors

forthedivergen


pex

ponen

tial


n)LN

lognorm


nSD

)mdashcalib

rationnot

applicab

le

Truncatedat

38Bya



EVOLU

TION






















1ndash61






EVOLU

TION















































































































































Table

S1

Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCED

evolutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgr

mdashmdash

Yes

Yes

Yes

Yes

mdashmdash

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCED

)eth~ m

THORN(HPD

)forall

Node1

295

(239ndash

3-99

)372

(262ndash

540

)281

(241ndash

336

)317

(258ndash

40)

282

(245ndash

330

)306

(260ndash

560

)293

(245ndash

360

)333

(278ndash

380

)Node3

244

(221ndash

280

)295

(231ndash

397

)237

(220ndash

260

)26(225ndash

313

)239

(220ndash

265

)255

(224ndash

293

)244

(223ndash

28)

275

(232ndash

325

)Node6

200

(152ndash

231

)221

(165ndash

291

)197

(148ndash

227

)204

(149ndash

250

)196

(143ndash

230

)202

(145ndash

244

)2(156ndash

225

)211

(163ndash

258

)Node31

182

(112ndash

228

)216

(143ndash

265

)176

(107ndash

224

)212

(124ndash

242

)185

(111ndash

227

)212

(12ndash24)

185

(2-229)

213

(127ndash

244

)Node43

191

(115ndash

243

)22(131ndash

311

)18(15ndash229

)194

(117ndash

26)

181

(111ndash

230

)19(117ndash

247

)191

(124ndash

24)

207

(132ndash

273

)

Expex

ponen

tial


n)LN

lognorm


nSD

)mdashnotap

plicab

le

Truncatedat

38Bya


Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes



























1ndash61






EVOLU

TION















































































































































Table

S1

Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCED

evolutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgr

mdashmdash

Yes

Yes

Yes

Yes

mdashmdash

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCED

)eth~ m

THORN(HPD

)forall

Node1

295

(239ndash

3-99

)372

(262ndash

540

)281

(241ndash

336

)317

(258ndash

40)

282

(245ndash

330

)306

(260ndash

560

)293

(245ndash

360

)333

(278ndash

380

)Node3

244

(221ndash

280

)295

(231ndash

397

)237

(220ndash

260

)26(225ndash

313

)239

(220ndash

265

)255

(224ndash

293

)244

(223ndash

28)

275

(232ndash

325

)Node6

200

(152ndash

231

)221

(165ndash

291

)197

(148ndash

227

)204

(149ndash

250

)196

(143ndash

230

)202

(145ndash

244

)2(156ndash

225

)211

(163ndash

258

)Node31

182

(112ndash

228

)216

(143ndash

265

)176

(107ndash

224

)212

(124ndash

242

)185

(111ndash

227

)212

(12ndash24)

185

(2-229)

213

(127ndash

244

)Node43

191

(115ndash

243

)22(131ndash

311

)18(15ndash229

)194

(117ndash

26)

181

(111ndash

230

)19(117ndash

247

)191

(124ndash

24)

207

(132ndash

273

)

Expex

ponen

tial


n)LN

lognorm


nSD

)mdashnotap

plicab

le

Truncatedat

38Bya


Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes




















































































































































Table

S1

Divergen

cetimes

forfive

importan

tnodes

estimated

usingarelaxe

dclock

withUCED

evolutionaryrates

Analysis

12

34

56

78

Model

assumptionsan

dcalib

rationpoints

Outgr

mdashmdash

Yes

Yes

Yes

Yes

mdashmdash

Root

mdashmdash

Exp(245281

6)Ex

p(245281

6)Ex

p(245281

6)

Exp(245281

6)

Node3

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

LN(2122705)

LN(2125808)

Node31

or32

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

LN(212131)

Resultsfordiscu

ssed

nodes

(UCED

)eth~ m

THORN(HPD

)forall

Node1

295

(239ndash

3-99

)372

(262ndash

540

)281

(241ndash

336

)317

(258ndash

40)

282

(245ndash

330

)306

(260ndash

560

)293

(245ndash

360

)333

(278ndash

380

)Node3

244

(221ndash

280

)295

(231ndash

397

)237

(220ndash

260

)26(225ndash

313

)239

(220ndash

265

)255

(224ndash

293

)244

(223ndash

28)

275

(232ndash

325

)Node6

200

(152ndash

231

)221

(165ndash

291

)197

(148ndash

227

)204

(149ndash

250

)196

(143ndash

230

)202

(145ndash

244

)2(156ndash

225

)211

(163ndash

258

)Node31

182

(112ndash

228

)216

(143ndash

265

)176

(107ndash

224

)212

(124ndash

242

)185

(111ndash

227

)212

(12ndash24)

185

(2-229)

213

(127ndash

244

)Node43

191

(115ndash

243

)22(131ndash

311

)18(15ndash229

)194

(117ndash

26)

181

(111ndash

230

)19(117ndash

247

)191

(124ndash

24)

207

(132ndash

273

)

Expex

ponen

tial


n)LN

lognorm


nSD

)mdashnotap

plicab

le

Truncatedat

38Bya


Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes






Table

S2

Estimated

Ages

ofnodes

foundin

theBay

esianco

nsensu

stree

s(reconstructed

withUCLN

rates)

forea

chan

alysesNd-nodenumber

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

1295

25

36

367

279

474

299

257

355

335

274

415

287

253

330

306

266

353

295

253

355

339

287

380

2277

242

329

347

267

440

263

235

298

296

249

361

256

233

284

275

244

314

277

243

328

322

272

372

3254

228

298

308

242

384

242

221

273

265

228

318

238

220

262

249

226

281

254

229

297

286

243

334

4233

214

27

276

221

339

224

212

247

240

214

284

222

212

239

228

213

254

233

214

268

258

221

301

5216

21

245

250

210

302

224

210

260

214

210

225

216

210

237

216

210

244

233

210

270

6204

177

235

233

189

287

202

172

228

210

178

254

199

167

222

202

170

232

204

179

235

218

186

260

7191

162

225

221

174

278

189

157

217

199

163

241

185

153

213

189

156

221

191

162

224

207

171

250

817

141

203

198

153

253

167

135

199

177

141

220

161

129

192

165

131

199

170

141

203

185

151

226

915

12

182

175

132

226

146

114

179

156

119

197

140

108

172

143

109

176

150

120

182

164

129

203

10131

1166

153

109

202

126

091

162

135

095

176

119

085

154

122

087

159

131

099

165

144

108

183

11064

043

088

075

048

107

058

038

084

063

039

091

056

034

081

057

036

083

064

043

088

070

047

098

12056

037

078

066

042

094

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

056

038

078

062

040

086

13048

031

067

056

034

081

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

047

031

067

052

033

074

14039

024

058

046

027

070

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

039

024

057

043

026

064

15025

013

041

029

015

049

026

012

045

028

012

048

024

010

043

025

011

044

025

013

040

027

014

044

16098

062

135

114

070

165

091

052

130

098

055

143

085

045

124

087

047

128

098

061

134

108

068

150

1713

099

162

151

110

199

125

093

158

134

098

174

119

087

152

122

089

156

129

099

161

142

108

180

18097

068

13

113

075

157

096

065

130

103

069

142

090

058

123

093

060

128

097

067

129

106

073

142

19087

058

118

101

064

142

083

052

115

089

057

127

077

047

109

080

049

113

086

058

118

095

062

129

20063

036

093

074

041

111

058

031

090

063

033

097

054

026

084

055

027

086

063

036

093

069

040

102

21113

078

149

132

086

180

105

068

141

112

072

155

099

062

136

101

065

139

113

078

149

124

086

164

22069

039

104

081

042

126

062

032

098

066

031

104

057

026

092

059

028

095

069

037

104

076

042

115

23147

115

182

170

125

225

142

107

177

152

111

194

136

097

170

139

101

176

147

114

181

159

121

200

24137

099

175

158

107

212

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

136

098

173

149

106

192

25111

068

152

127

075

185

106

060

151

113

062

163

099

053

146

101

054

152

110

067

151

120

073

168

26065

036

101

076

040

123

063

030

101

068

032

113

058

027

097

060

028

100

065

037

098

071

039

109

27129

066

182

147

075

218

116

053

175

124

056

189

112

050

174

117

049

180

128

067

182

139

073

201

28141

091

189

161

101

227

126

077

180

136

079

194

123

072

181

129

075

186

141

092

189

152

098

207

29066

03

111

076

034

130

059

024

106

064

026

113

057

022

106

059

023

109

066

031

112

072

033

120

3004

018

07

046

019

081

036

014

067

039

015

074

035

013

068

036

012

071

040

018

070

043

019

076

31177

14

224

216

153

256

172

134

220

198

139

234

167

128

217

175

130

223

177

141

225

212

150

241

32151

118

181

192

159

218

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

151

120

182

mdashmdash

mdash

33118

085

158

144

099

185

108

076

144

117

079

162

102

070

139

106

072

147

119

087

160

133

094

175

34067

041

1081

047

121

064

036

095

069

039

107

060

033

092

063

035

097

068

040

100

075

044

112

35049

024

079

057

027

095

043

019

074

047

021

082

040

016

071

042

017

074

049

024

080

054

026

088

36021

009

038

025

011

047

020

007

039

022

008

043

019

006

037

020

006

040

021

009

038

023

009

043

37092

062

127

110

072

151

082

052

116

090

055

128

077

047

112

080

050

117

093

062

127

103

068

142

38061

035

09

072

041

107

053

028

082

057

030

090

049

025

079

051

026

082

061

036

091

067

039

100

40034

015

06

040

017

072

029

012

056

032

012

062

027

009

054

029

010

058

034

015

060

037

016

067

4114

098

18

153

109

193

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash128

079

176

141

098

179

148

106

187

4211

066

156

120

072

165

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

110

065

154

116

071

162

432

156

243

235

173

303

185

146

225

197

148

250

180

138

219

186

141

230

200

157

241

218

171

272

44175

134

218

205

147

272

159

119

198

170

123

222

154

112

193

159

116

204

175

133

216

191

144

243

45158

119

198

185

132

247

142

105

179

151

107

200

136

098

174

140

102

184

158

120

197

171

130

221


Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes






Table

S2

Cont

Node

12

34

56

78

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

~ mLo

Up

46136

099

176

160

109

216

120

084

157

128

087

175

113

078

151

117

079

158

137

099

177

150

107

197

47095

065

131

112

072

160

085

055

119

091

057

131

079

050

115

082

050

118

096

064

131

105

070

146

48037

022

058

044

026

068

034

019

052

036

020

057

032

017

050

033

018

054

038

023

057

041

024

062

49017

007

031

020

008

037

015

006

028

017

006

031

014

005

027

015

005

029

017

007

031

019

008

034

5003

016

047

035

018

057

026

012

043

028

014

047

024

011

041

025

011

043

030

016

047

033

017

051

51134

089

178

157

100

220

119

076

161

127

078

178

112

069

154

116

070

162

134

089

177

146

097

197

52025

01

047

029

011

055

023

008

046

025

009

050

022

007

046

023

008

048

025

010

047

027

011

051

53138

071

199

165

083

248

123

060

183

133

062

202

116

051

177

120

055

187

139

073

202

152

080

226

54013

004

025

015

005

030

012

004

026

013

004

028

011

003

026

012

003

027

013

004

025

014

005

028

5514

083

202

165

094

250

127

071

193

139

076

214

123

064

190

127

068

200

139

084

200

154

091

227

56063

03

107

075

035

130

056

025

099

061

025

110

053

021

099

055

021

103

063

030

105

070

033

118

57004

001

011

005

001

013

004

001

011

005

001

012

004

001

011

004

001

011

004

001

011

005

001

012

Lolower

boundaryofthe95


sity~ mmed

iannodeag

eUpupper

boundaryofthe95

highest-probab

ility

den

sitymdashnotap

plicab

le








Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes












Analysis

Species Strains























Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes







Taxon Notes






























Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes






Table S5 Cont

Taxon Notes































Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes







Dataset S1


Dataset S2

Table S5 Cont

Taxon Notes






Evolution of multicellularity coincided with increased … · 2019-09-27 · atmosphere around...

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