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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
1794 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
(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
Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1795
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
1794 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
(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
Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1795
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
1794 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
(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
Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1795
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
1794 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
(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
Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1795
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
1794 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
(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
Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1795
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
(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
Schirrmeister et al PNAS | January 29 2013 | vol 110 | no 5 | 1795
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
1796 | wwwpnasorgcgidoi101073pnas1209927110 Schirrmeister et al
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
5 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
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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 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
45 Alfaro ME et al (2009) Nine exceptional radiations plus high turnover explain speciesdiversity in jawed vertebrates Proc Natl Acad Sci USA 106(32)13410ndash13414
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 2 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 3 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 4 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 5 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 6 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 7 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
distribution(offsetmea
n)LN
lognorm
aldistribution(offsetmea
nSD
)mdashnotap
plicab
le
Truncatedat
38Bya
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 8 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 9 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 10 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 11 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
4 Node 3 153 161E-7 2164 Node 3 19 089Node 33 07 097 Node 34 094 100Root 017 142E-7 Root 029 540E-7 3681
5 Node 3 168 322E-8 2135 Node 3 188 09Node 33 086 097 Node 34 106 100Root 016 124E-6 Root 027 349E-7 3679
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
8 Node 3 147 183E-7 2163 Node 3 194 085Node 33 074 097 Node 34 089 100
UCED rates of evolutionRoot 048 366E-7 Root 028 996E-7 3713
1 Node 4 194 288E-2 2119 Node 3 166 096Node 33 1 097 Node 34 029 100Root 039 102E-8 Root 023 196E-8 3777
2 Node 4 167 707E-2 2226 Node 3 136 096Node 33 064 098 Node 34 018 100Root 05 176E-8 Root 03 902E-7 3671
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
5 Node 4 202 012 2092 Node 3 172 096Node 33 102 097 Node 34 023 100Root 046 367E-7 Root 027 887E-7 3804
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
7 Node 4 195 107E-5 2119 Node 3 167 096Node 33 091 097 Node 34 019 100Root 042 249E-7 Root 025 646E-7 3815
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 12 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 13 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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)
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 14 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15
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
Schirrmeister et al wwwpnasorgcgicontentshort1209927110 15 of 15