Accepted Manuscript

The evolution of photosynthesis and aerobic respiration in the cyanobacteria

Rochelle M. Soo, James Hemp, Philip Hugenholtz

PII: S0891-5849(18)32300-1

DOI: https://doi.org/10.1016/j.freeradbiomed.2019.03.029

Reference: FRB 14214

To appear in: Free Radical Biology and Medicine

Received Date: 30 October 2018

Revised Date: 5 March 2019

Accepted Date: 26 March 2019

Please cite this article as: R.M. Soo, J. Hemp, P. Hugenholtz, The evolution of photosynthesis andaerobic respiration in the cyanobacteria, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/j.freeradbiomed.2019.03.029.

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x

Calvin cycle (Rubisco & PRK)Phototropy (PSI & PSII)

Loss of ETC

Complex III

bccomplexes

b6f

bc-related

bc1

ACIII

Vampirovibrionia (Melainabacteria)

Sericytochromatia (Blackallbacteria)

Obscuribacterales

SHAS531

V201-46

S15B-MN24

GL2-53

Vampirovibrio

Genus Order Class

Vampirovibrionales2-02-FULL-34-122-02-FULL-35-15

Cyanobacteriia (Oxyphotobacteria)

GCA-2770975UBA7694

UBA4093 UBA8530

Caenarcanum

Gastranaerophilales

Caenarcaniphilales

Obscuribacterales

S15B-MN24 UBA7694 (GL2-53)

Complex IV

High oxygenadapted

Low oxygenadapted

A-family oxygenreductase

C-family oxygenreductase

bd oxidase

Gloeobacterales Eurycoccales Pseudanabaenales Gloeomargaritales

Synechococcales_A

Limnotrichales Synechococcales Synechococcales_B Thermosynechococcales PCC-7407 Neosynechococcales Leptolyngbyales Psueudophormidiales Phormidesmiales

Cyanobacteriales

Gre

at O

xida

tion

Even

t (~2

.3 G

a)

1.00.4 0.5 0.6 0.7 0.8 0.90.30.2

Relative evolutionary divergence

≥90% ≥70% ≥50%

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The evolution of photosynthesis and aerobic respiration in the

Cyanobacteria

Rochelle M. Sooa, James Hempb, Philip Hugenholtza aAustralian Centre for Ecogenomics, School of Chemistry and Molecular

Biosciences, University of Queensland, St Lucia, Queensland, Australia 5

bDepartment of Internal Medicine, University of Utah, Salt Lake City, UT, USA

*Corresponding author: Rochelle M. Soo

Highlights 10

• The recent discovery of two major non-photosynthetic bacterial lineages (classes)

specifically related to oxygenic photosynthetic Cyanobacteria has reinvigorated

debate on the evolution of oxygenic photosynthesis.

• The three cyanobacterial classes use different sets of proteins to perform aerobic 15

respiration, suggesting that this trait was independently acquired in each class.

• Independent acquisition of aerobic respiration is consistent with oxygenic

photosynthesis and the rise of oxygen occurring after divergence of the primary

cyanobacterial lines of descent, although alternative scenarios cannot be ruled out 20

based on the currently available data.

Abstract

For well over a hundred years, members of the bacterial phylum Cyanobacteria have 25

been considered strictly photosynthetic microorganisms, reflected in their

classification as “blue-green algae” in the botanical code. Recently, genomes

recovered from environmental sequencing surveys representing two major

uncultured basal lineages (classes) of Cyanobacteria have been found to completely

lack photosynthetic and CO2 fixation genes. The most likely explanation for this 30

finding is that oxygenic photosynthesis was not an ancestral feature of the

Cyanobacteria, and rather originated following divergence of the primary lines of

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descent. Here we describe recent findings on the evolution of aerobic respiration in

the non-photosynthetic cyanobacterial classes, and how this has been interpreted by

researchers interested in the evolution of oxygenic photosynthesis. 35

Abbreviations

MAGs, metagenome assembled genomes; ETC, electron transport chain; ACIII,

Alternative complex III, RCs, Reaction centres; HGT, horizontal gene transfer; Ga,

billion years ago 40

Keywords

Cyanobacteria; oxygenic photosynthesis; aerobic respiration

1. Introduction 45

The origin of oxygenic photosynthesis was a key biological event in Earth’s history,

leading to the oxygenation of its surface environment and giving rise to complex

Eukaryotes [1]. Traditionally Cyanobacteria were thought to be strictly

photosynthetic, however this dogma was challenged when environmental 16S

ribosomal RNA gene surveys revealed at least two major basal cyanobacterial 50

lineages, 4C0d-2 and ML635J-21, in a range of aphotic habitats, including rumen [2],

drains [3], termite guts [4], human guts [5], groundwater [6], a hot-spring [7], rice

paddy soil [8] and a glacier [9].

The recent ability to extract draft genomes of individual microbial populations from 55

metagenomic datasets (so called metagenome-assembled genomes or MAGs) has

provided us with a new and exciting opportunity to examine the metabolic potential of

as-yet uncultured organisms [10], including basal cyanobacterial lineages. The first

non-photosynthetic Cyanobacteria MAG was reported in 2012 from an aphotic

anaerobic microbial community decomposing poplar wood chips. This MAG encoded 60

the ability for aerobic respiration, however it contained no genes associated with

photosynthesis. Unfortunately, the authors did not address its evolutionary

importance [11]. In 2013, Di Rienzi and colleagues reported the first MAG

representatives of 4C0d-2, which they obtained from human faeces and a

subsurface aquifer [12]. It was suggested that these MAGs represent a new 65

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candidate phylum sibling to the Cyanobacteria, for which they proposed the name

Melainabacteria, after the Greek nymph of dark waters [12]. Metabolic inference

indicated that the Melainabacteria were markedly different to the classical

Cyanobacteria in that they entirely lack the ability to perform photosynthesis, aerobic

respiration and CO2 fixation [12]. In 2014, additional MAGs belonging to this lineage 70

were obtained from koala and human faeces, an anaerobic sludge blanket and an

enhanced biological phosphorous removal reactor [13]. Based on this analysis, it

was proposed that the Melainabacteria should be reclassified as a class within the

phylum Cyanobacteria, due to robust monophyly with photosynthetic Cyanobacteria

and shared (inferred) ancestral traits, such as cell envelope components and 75

circadian rhythm and light-response regulators [13]. The latter classification has

been recently supported by a normalised taxonomy based on genome phylogeny

[14], and four orders are currently defined in GTDB release 03-RS86 (Fig. 1),

although MAGs representing at least two additional orders have been reported [15].

The 2014 study also found that MAG representatives of the Melainabacteria lack 80

genes for photosynthesis and CO2 fixation, but identified genes for aerobic

respiration in representatives of the Obscuribacterales, specifically Complex III and

IV [13].

In 2015, the first cultured representative of the Melainabacteria was identified 85

serendipitously, Vampirovibrio chlorellavorus [16], an obligate predator of the

microalga Chlorella vulgaris [17]. V. chlorellavorus had been isolated in co-culture

with its host in 1972, but erroneously classified as a member of the

Deltaproteobacteria [18]. Many years later, its 16S rRNA gene was sequenced by

the American Type Culture Collection as part of the Living Tree Project [19], alerting 90

researchers to its true identity. Shotgun sequencing of V. chlorellavorus directly from

lyophilised cells confirmed its phylogenetic affiliation (the sole representative of the

order Vampirovibrionales, Fig. 1) and revealed a genome devoid of photosynthetic

and CO2 fixation genes, consistent with all other known members of the

Melainabacteria. However, it had a complete set of electron transport chain (ETC) 95

genes, including a terminal oxidase (Complex IV), confirming its known ability to

aerobically respire based on cultivar studies [18], [20], [21].

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The recovery of three additional representatives of the Caenarcaniphilales in 2016

[22] identified genes encoding Complexes III and IV, leaving the Gastranaerophilales 100

as the only order lacking evidence for aerobic respiration. Recently Utami and

colleagues identified 16S rRNA genes belonging to multiple Gastranaerophilales

populations (Fig. 1) in the gut of a number of termite and cockroach species, where

they are estimated to represent up to 1.9% of the gut community [23]. A partially-

complete single-cell genome from one of the Gastranaerophilales populations was 105

obtained which lacked genes required for photosynthesis, CO2 fixation, and

respiratory metabolism. While habitat and physiology supports the inference of an

absence of these metabolic traits, it should be noted that the estimated genome

completeness was 61% meaning that the presence of these traits cannot be entirely

ruled out based on genomic information alone. 110

In 2017 [15], MAG representatives of the second basal cyanobacterial class

identified in 16S rRNA gene surveys (ML635J-21), were obtained from a coal bed

methane well [24], an algae-associated biofilm from a lab-scale bioreactor and

subsurface groundwater [25]. Comparative genome analysis confirmed their 115

affiliation with the Cyanobacteria in a lineage distinct from both classical

photosynthetic cyanobacteria and Melainabacteria, and absence of genes for

photosynthesis and CO2 fixation. As with the Melainabacteria, some members of the

group had genes for aerobic respiration that were inferred to have been acquired

well after the divergence of the primary lines of descent (Fig. 1 and see below). For 120

this reason the class Sericytochromatia was proposed, meaning “late cytochromes”

[15]. Two orders belonging to the Sericytochromatia are currently recognised in

GTDB release 03-RS86; S15B-MN24 and UBA7694 (also called GL2-53; [15]) (Fig

1). Recently, three additional MAGs belonging to a single species (>95% ANI identity

between genomes) in the order UBA7694, were reported from a deep terrestrial 125

aquifer, for which a new candidate phylum name was proposed, Blackallbacteria

[26].

Given the current taxonomic uncertainty of the Cyanobacteria (including the non-

trivial complication that they are still classified under the Botanical Code), we are 130

following the GTDB classification which uses a normalised genome-based

phylogenetic framework [14], combined with a recent proposal to formalise the rank

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of phylum [27]. Consequently, the phylum Cyanobacteria becomes Cyanobacteriota

[28], encompassing the classes Cyanobacteriia comprising all oxygenic phototrophs

(previously called Oxyphotobacteria, [13], [15]), Vampirovibrionia after the first 135

cultured representative and replacing the Candidatus name Melainabacteria, and

Candidatus Sericytochromatia proposed by Soo et al., [15], which predates

Candidatus Blackallbacteria [26], noting however, that both names lack a

nomenclature type which will need to be assigned [29] (Fig. 1).

140

The availability of basal Cyanobacteriota provides an opportunity to re-evaluate the

origin and evolution of oxygen associated metabolisms, oxygenic photosynthesis

and aerobic respiration, in this phylum. We first address the evolution of aerobic

respiration in these lineages and then discuss the ongoing debate on the evolution of

oxygenic photosynthesis within the phylum. 145

2. Evolution of aerobic respiration in Cyanobacteriota

ETC complexes III and IV can be used to provide insights into the evolution of

aerobic respiration in the Cyanobacteriota, as genes encoding these complexes are

present in all three classes (Fig. 1, Table 1). 150

2.1. Cyanobacteriia

All members of the Cyanobacteriia share a unique cytochrome b6f complex in

addition to photosystem I, photosystem II and an A-family oxygen reductase.

Approximately 11% of genomes in this class also contain one or more bd oxidases 155

used in low oxygen habitats (Fig. 1, Table 1). Phylogenetic analyses suggest that

the cytochrome b6f complex, the A-family oxygen reductase and potentially the bd

oxidase were acquired by the ancestor of the Cyanobacteriia after diverging from

Vampirovibrionia and Sericytochromatia. Following acquisition, the b6f gene has

been predominantly vertically inherited, whereas both the A-family oxygen reductase 160

and bd oxidases appear to have been extensively, but exclusively, transferred

horizontally within the Cyanobacteriia [15]. More recently, a C-family oxygen

reductase was acquired in the genus Synechococcus (Fig. 1, [30]) suggesting that

while aerobic respiration is well established and conserved in the Cyanobacteriia,

innovations are still occurring. 165

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2.2. Vampirovibrionia

Originally, it was thought that Vampirovibrionia were strictly fermentative as the first

set of MAGs described for this class lacked ETC genes [12]. However, subsequently

a number of Vampirovibrionia lineages were predicted to be capable of aerobic

respiration, based on the presence of near complete gene sets for Complexes III and 170

IV [15]. Of the four described Vampirovibrionia orders, three have representatives

predicted to be capable of aerobic respiration; Vampirovibrionales,

Obscuribacterales and Caenarcaniphilales (Fig. 1). It was originally thought that the

Caenarcaniphilales had lost the ability for aerobic respiration as they adapted to

anoxic environments [15] but the recovery of two additional MAGs [22] have 175

indicated that this ability has been retained in at least one member of the

Caenarcaniphilales, extracted from a well under high O2 conditions. The

Gastranaerophilales are found primarily in animal gastrointestinal tracts [12], [13]

and are inferred to gain energy strictly via fermentation. All aerobic Vampirovibrionia

have a unique fused complex III-IV operon consisting of a C-family oxygen reductase 180

(cbb3-type) and two cytochrome bc-related proteins, which are inferred to be

ancestral to known members of the class and subsequently lost in the

Gastranaerophilales and some members of the Caenarcaniphilales. The

Obscuribacterales has an additional fused complex III-IV operon consisting of a

cytochrome bc complex and a bd-like oxidase with a cytochrome c fused to the 185

periplasmic side. An unfused bd-like oxidase appears to have been independently

acquired in the Vampirovibrionales. As yet, no high oxygen-adapted reductases (A-

family oxygen reductases) have been identified in the Vampirovibrionia suggesting

that the aerobic members of the group are adapted to low-oxygen conditions [15].

However, it should be stressed that these inferences will need to be reassessed as 190

more MAG representatives are discovered, and as representatives are cultivated

and experimentally characterised, as activity of these partial and predicted

complexes has yet to be confirmed.

2.3. Sericytochromatia 195

Presently, the Sericytochromatia are represented by only seven MAGs but they

display a remarkable diversity of respiratory proteins compared to other

Cyanobacteriota, with an inferred ability to function in both high and low-oxygen

conditions (Fig. 1; [15]). Members of UBA8530 have a fused Complex III-IV operon

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comprised of a cytochrome bc1 and a highly modified A-family oxygen reductase that 200

is missing its proton channels, suggesting it is unable to pump protons [15]. This

genus also contains a second cytochrome bc1 and a second (unmodified) A-family

oxygen reductase only distantly related to the first, and an Alternative Complex III

(ACIII) and C- family oxygen reductase. All of these respiratory genes appear to

have been acquired after UBA4093 diverged from the family UBA8530 (Fig. 1), 205

although more representatives of these lineages are needed to refine this inference.

In the other major recognised branch of the class, order UBA7694, all members have

an ACIII and an A- and C-family oxygen reductase distinct from those found in

UBA8530. The genus GCA-002770975 also has an additional phylogenetically

distinct A-family oxygen reductase. The presence of numerous complex III and IV 210

genes in the Sericytochromatia with distantly related homologues in each gene

family (Fig. 1) points to multiple independent acquisitions of aerobic respiration in

this class [15]. Given the extremely limited genomic sampling of this class to date, it

is likely that we have only glimpsed the tip of the respiratory iceberg in the

Sericytochromatia. 215

2.4. Comparison of Cyanobacteriota ETCs

Comparison of potential for aerobic respiration in the Cyanobacteriia,

Vampirovibrionia and Sericytochromatia indicate that phylogenetically distinct

respiratory genes are used in each class, often involving novel combinations, 220

particularly instances of fused Complex III and IV genes (Fig. 1). The most

parsimonious interpretation of these data is that the ancestor of the Cyanobacteriota

was not capable of aerobic respiration and that this capability was independently

acquired in all three classes on multiple occasions following, and likely permitted by,

the rise of atmospheric oxygen [15]. However, other hypotheses have been 225

presented in the literature following the genomic characterisation of Vampirovibrionia

and Sericytochromatia. These are summarised in the next section.

3. The evolution of oxygenic photosynthesis in Cyanobacteriota

There is an ongoing debate concerning the timing and mechanism of the origin of 230

oxygenic photosynthesis [31]. Some groups propose an early origin (3.0-3.8 Ga) that

potentially preceded the radiation of extant bacteria [32], [33], whereas others argue

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that the origin directly preceded the Great Oxygenation event at 2.35 Ga [15], [30],

[34], [35]. Cyanobacteriota is the only known phylum with members capable of

oxygenic photosynthesis via coupling of type 1 and type 2 reaction centres. These 235

reaction centres are found individually in anoxygenic phototrophic members of seven

other lineages, RC1 in the Chlorobi, Firmicutes and Acidobacteria, and RC2 in the

Proteobacteria, Chloroflexi, Gemmatimonadetes and most recently in candidate

phylum WPS-2 [36], [37].

240

3.1. Current theories

There are three classes of theories concerning the evolution of oxygenic

photosynthesis in the Cyanobacteriia; the fusion, selective loss and cyanobacterial

export models [1], [31]. The fusion model first proposed in 1990 [38] suggests that

RC1 and RC2 were obtained by a non-photosynthetic cyanobacterial ancestor via 245

horizontal gene transfer (HGT) from two different anoxyphototrophic bacteria,

although there is no compelling evidence for which lineages developed the original

reaction centres [1]. The selective loss model posits that both RCs were present in a

single unknown photosynthetic ancestor and thereafter all photosynthetic lineages

inheriting those genes either vertically or horizontally, lost either RC1 or RC2 with the 250

exception of the Cyanobacteriia [39], [40], [41]. The export hypothesis was first

proposed by Mulkidjanian and colleagues in 2006 [42]. They compared 15 complete

cyanobacterial genome sequences (all members of the class Cyanobacteriia)

revealing over 1,000 protein families that were core to at least 14 of them. Only a few

components of the photosynthetic machinery were represented in the anoxygenic 255

phototrophs, suggesting that photosynthesis originated in the cyanobacterial lineage.

In this hypothesis, anoxygenic photosynthetic lineages acquired their reaction

centres via HGT from anoxygenic ancestors of the extant cyanobacteria referred to

as “procyanobacteria”. Magnabosco et al. (2018) [43] used molecular clock analyses

to explore the different scenarios using what they consider to be the three most 260

ancient phototrophic groups, the Cyanobacteriota, Chloroflexi and Chlorobi. They

concluded that the stem Cyanobacteriota are unlikely to be the recipient of an RC1

from Chlorobi but could still have been either the donor or recipient of RC2 proteins

from the Chloroflexi prior to the rise of O2. Another molecular clock analysis

concluded that phototrophy was acquired in Chloroflexi significantly after the Great 265

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Oxygenation Event and therefore the Chloroflexi could not have donated

photosynthetic genes to the ancestor of the Cyanobacteriia [44].

3.2. Recent debate in light of discovery of basal non-photosynthetic

cyanobacterial lineages 270

The recent discovery of basal non-photosynthetic Cyanobacteriota adds to the long-

standing debate on how oxygenic photosynthesis evolved in this phylum by providing

potential new time constraints. The complete absence of photosynthetic and CO2

fixation genes in the currently available Vampirovibrionia and Sericytochromatia

genomes point to at least three possible scenarios (Fig. 2). The first posits that RCs 275

were acquired by the immediate ancestor of the class Cyanobacteriia after diverging

from the other two classes (Fig. 2a). This implies that the Vampirovibrionia and

Sericytochromatia were never photosynthetic. Shih et al. [35] estimated that the

Cyanobacteriia and Vampirovibrionia diverged approximately 2.5 to 2.6 billion years

ago (Ga), before the rise of oxygen estimated at ~2.3 Ga, and that the crown group 280

Cyanobacteriia postdate the rise of oxygen, diverging ~2.0 Ga. Matheus-Carnevali et

al. [45] noted that two uncultured candidate phyla robustly affiliated with the

Cyanobacteriota, the Margulisbacteria (previously known as RBX-1 or ZB3) and

Saganbacteria (WOR-1), also completely lack photosynthetic and CO2 fixation

genes, which they suggested further supports ancestral absence of these traits in the 285

Cyanobacteriota.

By contrast, Martin et al. [46] suggest that the Cyanobacteriota ancestor was

chlorophototrophic and that the classes Vampirovibrionia and Sericytochromatia lost

their photosynthetic capability (scenario Fig. 2c) akin to the subsequent inferred loss 290

of aerobic respiration in some Vampirovibrionia lineages, notably the order

Gastranaerophilales. Thiel et al. [47] also suggested that it is equally possible that

the Vampirovibrionia and Sericytochromatia lost their photosynthetic capacity and

enzymes for CO2 fixation following divergence from the Cyanobacteriia, although this

scenario requires independent losses of these genes in the two basal classes (Fig. 295

2c). A third and intermediate possibility is that the ancestor of the Cyanobacteriia

and Vampirovibrionia acquired RCs after diverging from the Sericytochromatia

followed by a subsequent gene loss in only the Vampirovibrionia (scenario Fig. 2b).

The complete absence of RC and CO2 fixation genes in the available representatives

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of the basal cyanobacterial classes means that we cannot presently rule out any of 300

these scenarios, or even more complex variations, highlighting the uncertainty of

these ancient events. If no photosynthetic genes or gene remnants are discovered in

the non-photosynthetic classes we may never be able to deconvolute this aspect of

the history of photosynthesis in the Cyanobacteriota. It is, however, unequivocal that

aerobic respiration evolved independently in each of the three classes presumably in 305

response to rising atmospheric oxygen (Fig. 1).

4. Conclusion

The rapid genomic elucidation of uncultured microbial lineages through high

throughput metagenomics is providing fresh perspectives on long debated topics 310

such as the origin of oxygenic photosynthesis and aerobic respiration. While the

current dataset does not definitively resolve the timing and evolutionary history of

these traits in the Cyanobacteriota, it does provide additional constraints which can

help to refine hypotheses. Additional cyanobacterial MAGs will certainly refine our

emerging picture of aerobic respiration in the Cyanobacteriota and other parts of the 315

bacterial domain, however, the origins of oxygenic photosynthesis may never be

resolved via this route, unless photosynthetic members of the basal cyanobacterial

lineages come to light.

Acknowledgements 320

We thank Maria Chuvochina for etymological advice. This work was supported by an

Australian Laureate Fellowship (FL150100038) from the Australian Research

Council.

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Fig. 1. Evolution of oxygenic photosynthesis and aerobic respiration in the

Cyanobacteriota. A cladogram of the phylum Cyanobacteriota taken from a larger

tree of the bacterial domain based on phylogenetic inference of 120 concatenated

single copy marker proteins scaled according to relative evolutionary divergence 565

(shown at base of figure; 0 = root of bacterial tree, 1 = extant taxa) [15]. Bootstrap

resampling analyses (100 times) with maximum likelihood was performed with

FastTree [48]. Black circles represent interior nodes with ≥90% bootstrap support,

grey circles ≥70% bootstrap support and white circles ≥50% bootstrap support.

Current proposed nomenclature based on GTDB [14] is shown to the right of the 570

cladogram. Previously proposed names [12], [13], [26] for the same groups are

shown in parentheses. Genes for Complexes III & IV, Calvin cycle and phototrophy

are distinguished by shape according to the legend at left. Different colours indicate

phylogenetically distinct versions of a given complex. Square brackets denote

operon fusions, and a red “X” indicates putative loss of ETCs. PRK, 575

phosphoribulokinase. In this representation, we infer that the Cyanobacteriota were

ancestrally nonphototrophic and that the class Cyanobacteriia acquired the ability for

photosynthesis after they diverged from the Vampirovibrionia. However, alternative

scenarios have also been proposed (see Fig. 2). The three Cyanobacteriota classes

likely acquired aerobic respiration independently after the rise of oxygen 580

(atmospheric oxygen is represented by a dashed green line). Figure is adapted from

Soo et al., 2017 [15].

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Fig. 2. Three possible scenarios for the evolution of oxygenic photosynthesis

in Cyanobacteriota. A. Acquisition of RCs by the Cyanobacteriia after primary 585

divergence of classes [15, 35]. B. Acquisition of RCs by the ancestor of the

Cyanobacteriia and Vampirovibrionia, with subsequent loss of RCs in the

Vampirovibrionia [46]. C. Acquisition of RCs prior to divergence of all three classes,

with subsequent loss of RCs in the Vampirovibrionia and Sericytochromatia. Green

arrows represent acquisition events and red arrows represent loss [46, 47]. 590

595

600

Vampirovibrionia

Cyanobacteriia

Sericytochromatia

A. Acquisition of RCs

B. Acquisition and loss of RCs

Vampirovibrionia

Cyanobacteriia

Sericytochromatia

C. Loss of RCs

Vampirovibrionia

Cyanobacteriia

Sericytochromatia

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Table 1 Phylogenetic distribution of complex III and IV genes in GTDB Cyanobacteriota genomes*

Order Number of

genomes

petBC* actAB coxAB* ccoNO cydAB

bc-

complexes

ACIII A-family C-

family

bd

oxidase

Cyanobacteriales 329(0.45) 324(0.98) 0(0) 325(0.99) 3(0.01) 68(0.21)

Phormidesmiales 10(0.01) 10(1) 0(0) 10(1) 0(0) 0(0)

Pseudophormidiales 4(0.005) 4(1) 0(0) 4(1) 0(0) 0(0)

Leptolyngbyales 10(0.01) 2(1) 0(0) 10(1) 0(0) 0(0)

Neosynechococcales 2(0.003) 2(1) 0(0) 2(1) 0(0) 0(0)

PCC-7407 1(0.001) 1(1) 0(0) 1(1) 0(0) 0(0)

Thermosynechococcales 7(0.01) 7(1) 0(0) 7(1) 0(0) 0(0)

Synechococcales_B 2(0.003) 2(1) 0(0) 2(1) 0(0) 0(0)

Synechococcales 3(0.004) 3(1) 0(0) 3(1) 2(0.67) 1(0.33)

Limnotrichales 3(0.004) 3(1) 0(0) 3(1) 0(0) 0(0)

Synechococcales_A 246(0.34) 187(0.76) 0(0) 217(0.88) 3(0.01) 0(0)

Gloeomargaritales 1(0.001) 1(1) 0(0) 1(1) 0(0) 0(0)

Pseudanabaenales 12(0.02) 9(0.75) 0(0) 10(0.83) 1(0.08) 0(0)

Eurycoccales 9(0.01) 9(1) 0(0) 9(1) 0(0) 6(0.67)

Gloeobacterales 2(0.003) 2(1) 0(0) 2(1) 0(0) 0(0)

Gastranaerophilales 74(0.10) 0(0) 0(0) 0(0) 0(0) 0(0)

Vampirovibrionales 1(0.001) 1(1) 0(0) 0(0) 1(1) 0(0)

Caenarcaniphilales 4(0.005) 3(0.75) 0(0) 0(0) 1(0.25) 0(0)

Obscuribacterales 2(0.003) 2(1) 0(0) 0(0) 2(1) 0(0)

S15B-MN24 4(0.005) 2(0.5) 2(0.5) 2(0.5) 2(0.5) 0(0)

UBA7694 5(0.01) 0(0) 3(0.6) 5(1) 3(0.6) 0(0)

Total 731 582(0.80) 5(0.01) 613(0.84) 18(0.02) 75(0.10)

* Some genes maybe absent in incomplete Cyanobacteriota MAGs