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S1 of there, Fig. originate evolution Appendix, pathways the (SI 2) core (Fig. metabolic reconstructed we Prochlorococcus, 1073/pnas.1619573114/-/DCSupplemental at online information supporting contains article This option. access open 1 PNAS the through online available Freely interest. of conflict no U.K. declare Dundee, authors of The model University J.A.R., to and paper. Institute; contributed the Weizmann wrote M.J.F. S.W.C. R.M., and Reviewers: M.J.F., model; R.B., and the data; analyzed developed S.W.C. and R.B. R.B. development; tools; reagents/analytic new uhrcnrbtos ..dsge eerh ..promdrsac;RB contributed R.B. research; performed R.B. research; designed R.B. contributions: Author owo orsodnemyb drse.Eal oirramngalcmor [email protected] Email: addressed. be may correspondence whom To [email protected]. tteeoytmlvl epooeteeouinr self- evolutionary to Earth. contributed the of ecosystems oxygenation propose the microbial drive We oceanic evolutionary of level. feed- organization this ecosystem positive reinforce the promotes that at turn, The species in biomass. among cel- carbon, ecosystem backs increases organic thereby this of and how release uptake describe We nutrient excre- carbon. lular and organic rate evolu- metabolic of their the tion increased Specifically, steadily flux. cells energy of tion cellular driven increasing self- and an its coupled by that were suggests Reconstructing self-amplification system and system. this organization of model and evolution addressed a metabolic photosynthetic We the as challenge. oceanic major microbes dominant a heterotrophic using is challenge arises it this how sys- complex and in tems self-organization drives what Understanding Significance xadn nteesuis esree ynbcei o the for cyanobacteria surveyed we studies, these on Expanding a,c,1 n eaiet te ynbcei.Freape ohhave both example, For cyanobacteria. other to relative and 2 oeaietediigfre hpn h vlto of evolution the shaping forces driving the examine To cnetaigmcaimwt rtoatra variants proteobacterial with mechanism -concentrating Synechococcus c eateto ilg,MsahstsIsiueo Technology, of Institute Massachusetts Biology, of Department ctps(,23). 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EARTH, ATMOSPHERIC, EVOLUTION PNAS PLUS AND PLANETARY SCIENCES That the evolution of Prochlorococcus permanently increased mixed the excretion of organic carbon in its late-branching strains is HL II light layer also consistent with limited laboratory observations. At mod- erate light levels in nutrient replete medium, strains from HL HL I clades that dominate surface waters (Fig. 1) excrete up to 20% of fixed carbon, and recently diverging strains excrete the most (37). Cells excrete significant amounts of glycolate (37), one of LL I the dead ends in the metabolic network (SI Appendix, Fig. S1). LL II/III P-starved cells excrete slightly less carbon (37), but this could be LL IV due to the coincident cessation of growth. Nevertheless, a higher nutrients fraction of the carbon excreted by P-starved cells consists of gly- colate and other small carboxylic acids (37). Many phytoplank-

depth ton excrete glycolate under intense light or nutrient limitation marine Synechococcus (24, 38), but retain the capacity to recycle it by using the three- to cyanobacteria subunit iron–sulfur protein glycolate oxidase (GlcDEF, rxn 13 in SI Appendix, Fig. S1), shown to be the enzymatic workhorse Fig. 1. Typical relative abundance distributions of Prochlorococcus eco- for this function in cyanobacteria (23). However, this gene is types as a function of depth and accompanying light intensity and nutrient absent in all but the deepest-branching LLIV clade of Prochloro- concentration profiles in stratified oceanic waters. Ecotype populations are coccus (SI Appendix, Table S1), suggesting a permanent opening geographically and temporally dynamic, but in warm, stable water columns of this pathway early in its evolution (Fig. 3). Finally, the bulk return to this same depth-differentiated state (12). The deepest branching ecotypes are most abundant at the bottom of the euphotic zone, where of organic carbon excreted by Synechococcus consists of polysac- nutrient concentrations are high and light energy low. The most recently charides (39), which are commonly excreted by nutrient-limited diverging ecotypes are most abundant near the surface, where the reverse microbes (26, 40), suggesting that these compounds could simi- is true (10–14). HL, high-light-adapted; LL, low-light-adapted. larly act as a redox safety valve in Prochlorococcus, which dom- inates in one of the most nutrient-poor environments on Earth (7–9) (Fig. 1). The remodeling of Prochlorococcus’ metabolic core includes We further examined the possibility that the evolution of the disruption of photorespiration and the TCA cycle (Fig. 3 Prochlorococcus increased its excretion of organic carbon as and SI Appendix, Fig. S1), raising the possibility that interme- an outlet of excess reducing power (Fig. 3) through additional diates of truncated pathways are excreted from the cell. Phyto- genomic analyses. Functionally related and coexpressed genes are plankton commonly excrete organic carbon as an outlet of excess commonly located near each other, so we searched the genomic reducing power under nutrient limitation or intense light (24). neighborhoods of core metabolic genes for transporters across Analogously, when facing a large energy supply from organic clades (SI Appendix, Fig. S2). We identified chromosome rear- carbon, some heterotrophs will effectively drain reducing power rangements repositioning a series of transporters, including three into the environment by excreting partially oxidized organic car- export and one import transporters, near key metabolic genes, bon rather than fully oxidizing it to CO2 (25–28), whereas some consistent with selection acting to fortify the control of transport photoheterotrophs use CO2-fixation as a sink for excess reduc- pathways (SI Appendix, Fig. S2). Chromosome rearrangements ing power (29). For photosynthetic cells in the oligotrophic sur- are seen in freshwater picocyanobacteria (SI Appendix, Fig. S2), face oceans, where the solar energy supply may commonly out- again suggesting that these pathways came under selection before pace the nutrient supply (Fig. 1), the combination of increased the emergence of the marine lineages and the loss of photores- CO2 fixation and increased excretion of organic carbon could piration in the LLII/III clade of Prochlorococcus (Fig. 3 and SI thus well be under strong selection. This is consistent with Appendix, Fig. S1). This analysis suggests that, in addition to gly- observations that Prochlorococcus has the most efficient carbon- colate, pyruvate and citrate (or isocitrate) are exported, whereas concentrating mechanism (30) and highest known rate of CO2 malate is imported (SI Appendix, Figs. S1 and S2). fixation per photosynthetic pigment (31) of any phytoplankton, Furthermore, because environmentally driven variations in the even though its small size and slow growth (6, 32) suggest a rel- expression of genes can give insight into their function [i.e., atively small carbon flux requirement. Lastly, selection to rid the “reverse ecology” (41)], and the metabolism of Prochlorococ- system of excess reducing power is consistent with the acquisition cus is highly choreographed to the diel light:dark cycle (42, 43), of the Plastoquinol Terminal Oxidase (PTOX) in the LLI and we examined the gene expression of a HL-adapted strain grown HL clades of Prochlorococcus (Fig. 3 and SI Appendix, Table S1), under a diel cycle (42). All putative export pathway genes have which adds an additional outlet for excess reducing power in their mRNA expression maxima at sunrise (SI Appendix, Fig. S2), photosynthetic electron transfer chain (SI Appendix, Fig. S1) consistent with their acting as redox safety valves, activated (33–36). when the supply of reducing power increases at dawn. Similarly,

Biochemical differences between pathways (e.g. ATP/trace

metal requirements or O2 sensitivities of enzymes) can suggest evolutionary driving forces

= metabolite New pathway variants must be added before ancestral variants can be lost to root = reaction maintain continuity of flux ancestral pathway trait distribution

Fig. 2. Illustration of approach to metabolic reconstructions. Phylometabolic trees reflect the evolution of metabolic network phenotypes because they integrate constraints from phylogenetics and metabolism (15, 16). All sequenced genomes within a given clade are searched for the presence/absence of enzymes catalyzing the reactions of different pathways. Mapping pathway variability patterns onto phylogenies of the clade suggests the order of metabolic innovations. In this example, three alternative pathways (pink, yellow, and blue) connect essential and universal pathways (black). Genes for the yellow pathway are nearly universally distributed (Inset), suggesting that it is the ancestral pathway, with the pink and blue pathways deriving from it. Maintaining continuity of flux results in trees of functional phenotypes. Biochemical differences between alternative pathways (e.g., ATP/trace metal requirements or oxygen sensitivities of their enzymes) suggest evolutionary driving forces (15, 16).

E3092 | www.pnas.org/cgi/doi/10.1073/pnas.1619573114 Braakman et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 are u ial rcino h rmr rdcini these in cooccur- production with primary interdependencies the metabolic of perhaps fraction Because ecosystems, sizable 47)? use a (46, photosynthesizers out imbalance carries many redox which prevent photosyn- 45), lower to (44, to flux ability the electron enhance thetic not was why balance force, redox maintaining driving If the evolution? its of course the the over of rest the to that carbon organic suggesting night. some at S2 ), return Fig. peaks may Appendix, pathway ecosystem uptake (SI malate carbon. dusk putative organic at the of of excretion expression increased Lastly, to of leading outlets, evolution redox the Fig. that Appendix, suggests excess (SI for chain transfer outlet electron S1 immediate the most in the power reducing PTOX, of expression the darkening a by direct indicated of as protection/repair shade. damage, to pink UV related inno- protective including are Key clades 53), ). S1 HLII Fig. (52, and Appendix , photodamage HLI (SI the ratio PSII:PSI in the vations absorption reflects and major cross-section, size the absorption relative reflect their their colors reflects thickness their line rep- and their are 1’s, repre- wavelengths, Photosystems and dots. are 2’s blue boxed drains as as pathways electron resented uptake and Cellular dots red lines. as orange sented dashed flux with electron of highlighted ratio the flux of nutrient gradient a to along form simplified in represented 3. Fig. ramne al. et Braakman ν ν htdrove What ,i aia tmda 3,4) olcieeiec thus evidence Collective 42). (35, midday at maximal is ), n e evolutionary time ancestral cyanobacteria eaoi vlto of evolution Metabolic Cyanobium gracile absorption cross-section reconstructed intensity ν specific growth rate e nrae xrto forganics of excretion increased Prochlorococcus’ light cell nutrientquota 2 2 /ν freshwater n are S1 ) Table and S1 Fig. Appendix, (SI Innovations . 1 1 efficiency quantum h eaoi ainsare variants metabolic The Prochlorococcus. Synechococcus marine Prochlorococcus 2 2 PTOX 1 1 5.1B/5.2/5.3 5.1A Prochlorococcus Prochlorococcus Prochlorococcus taiyadded steadily 2 2 2 2 2 PTOX PTOX PTOX 1 1 1 1 1 LL IV LL II/III LL I HL I HL II ty(6 7 ftepooytm Fg and stoichiom- 3 and (Fig. 55) photosystems 54, 9, the (6, of S1 pigments 57) the (56, in etry level changes light in given fested a at elec- flux increased an tron experience also strains diverging recently more [m cross- section absorption II photosystem whole-cell mass-normalized the where weight) dry time (g pho- electrons [µmol higher density a flux experience electron columns, tosynthetic ecotypes This water (HL) stable diverged cells. in recently of First, more trends. composition evolutionary two elemental suggests and and physiology, light macromolecular and growth the 1), and (Fig. 3 structure population (Fig. its metabolism its of of vations evolution the of in standing Evolution Unify- a of Suggests View Organization ing of Levels Across Evidence Integrating discussion). fully further not for aerobic do but for to clues, relevant hypothesized translate provide carbon 49–51) (26, organic interactions. heterotrophs excreting cross-feeding explain individual of to by Benefits needed for carbon therefore benefits are organic pro- Direct cells outcompete of (48). Nonproducer would excretion cross-feeding production dilemma: suppress the of and goods” cost cells “public excret- the ducer and a avoid fixing that to of cells cost leads apparent carbon the ing microbes, oceanic typi- of populations interac- cal unstructured in cross-feeding because by however, carbon Invoking organic problematic, of play. excretion explain in to tions are heterotrophs ring fisatramxn vn nteoe ca Fg ) ihthe How- with 1), (10–14). (Fig. waters ocean surface open the the dominating ever, in always event strat- cells column mixing HL water a the abun- when relative after emerges the ifies that of ecotypes layering its the of by dance obviously the most along suggested is density flux electron photon lular (electrons PSII of efficiency nrni rwhrate growth intrinsic than smaller cantly light— blue ocean—in chococcus of open absorption increase the or typifying maintain but light, yellow inof tion of [moles weight) nutrients limiting of quota cellular normalized where conditions, stant n surface of the evolution the cus suggests near levels across fluxes evidence ing photon increased highest an generate the to 3). at (Fig. cells fortify- allow flux to to related electron machinery primarily cellular were damage divergences ing light of later of clades that HL repair gesting in and added protection been the Var- have to 3). 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Prochlorococcus Prochlorococcus Q stequantum the is Synechococcus σ Prochlorococ- n 5,5) sug- 53), (52, PSII −1 ν e stemass- the is n ,integrat- ), phylogeny ), mlsof [moles ssignifi- is smani- is n σ IText SI | PSII gdry (g Syne- PSII E3093 [1] [2] −1 is is )

EARTH, ATMOSPHERIC, EVOLUTION PNAS PLUS AND PLANETARY SCIENCES (6, 32) and an increased efficiency in its use of limiting nutri- tively rapid and tightly coupled, microbial strains with the lowest ents. The latter is inferred from many features of Prochlorococ- [n]∗ dominate (72), suggesting that selection should favor such cus, including less investment of N and P in the genome by reduc- innovations. To understand how selection to lower [n]∗ shapes ing its size and guanine–cytosine content (60), decreased use of cells, we can substitute an expression for Vmax that assumes amino acids with N-rich side chains (61), a switch from P- to reversible kinetics (SI Appendix, SI Text) into Eq. 3: sulfolipid membranes (62, 63), and less Fe use in metabolism and photosynthetic machinery (33, 34) (SI Appendix, Fig. S1). KM ,n KM ,n νn [n] = νn = + ∆ G/RT , [4] These changes suggest a decreasing Qn for N, Fe, and P, the Vmax [E]k 1 − e r three main limiting nutrients in the oligotrophic oceans (64). An increasing νe coinciding with a decreasing νn over the course of where [E] is the enzyme concentration [moles of enzyme Prochlorococcus evolution can be expressed as a single variable: (g dry weight)−1]. k + is the rate constant [moles of n (moles −1 −1 −1 −1 the electron-to-nutrient flux ratio, νe /νn (Fig. 3). of enzyme) time ], R is the gas constant (J K mol ), and T is temperature (K). Eq. 4 suggests that there are two strate- Electron Flux and Nutrient Acquisition. What are the selective pres- gies for lowering [n]∗ (SI Appendix, Fig. S3). First, cells can mod- ν /ν + sures maximizing e n in Prochlorococcus? Photosynthetic elec- ify the kinetics (decreasing KM ,n and/or increasing k , [E]) and tron flux transfers solar energy into metabolism via cofactors thermodynamics (decreasing ∆r G) of their metabolism, the lat- µ such ATP and NAD(P)H (65). An increased electron flux [ mol ter by increasing νe as we have just argued. Second, cells can −1 −1 electrons (g dry weight) time ] thus suggests an increased lower their [n]∗ by lowering their required flux of limiting nutri- −1 metabolic rate [kJ of absorbed solar energy (g dry weight) ents νn (Eq. 4), which can be achieved by lowering their minimal −1 time ]. This principle is exemplified by the increasing metabolic Qn [i.e., streamlining (73)] or their µ (Eq. 2), both of which are activity and ATP/ADP ratios of plant chloroplasts and cyanobac- apparent in the evolution of Prochlorococcus as discussed above. teria when they shift from darkness into light (66, 67). Further- Optimizing kinetics/thermodynamics and decreasing νn can work more, the highest νe /νn phenotypes are the most recently diverg- synergistically, and both are helped by a decrease in cell mass, as ing clades (Fig. 3), dominating near the surface where the solar is observed along the Prochlorococcus phylogeny (6, 59). That energy supply is greatest and nutrient levels are lowest (Fig. 1), is, selection to lower νn allows a decrease in total cell mass by suggesting an advantage to higher metabolic rates at lower nutri- minimizing the mass dedicated to nonessential components (73). ent concentrations. If the amount of metabolic enzyme and photosynthetic machin- How metabolic rate affects nutrient uptake can be understood ery is kept fixed, this decrease in total cell mass would increase from Michaelis–Menten kinetics, which under strong nutrient both [E] (68) and σPSII (and thus νe , which makes ∆r G more limitation ([n]  KM ,n ) simplifies to (68) (SI Appendix, SI Text): negative). For nitrogen, increasing νe in turn provides an addi- tional avenue for lowering QN (and thus νN ), because path- 0 ways with a more negative ∆r G require less protein biomass νn = [n]an = Vmax [n]/KM ,n , [3] ∗ for a given flux (74). Thus, maximizing νe /νn lowers the [n] of where [n] is the nutrient concentration (moles of n L−1) and cells by making the ∆r G more negative (driving kinetic bottle- 0 −1 −1 neck reactions forward), thereby increasing the nutrient affinity an is the specific nutrient affinity [L (g dry weight) time ]. Specific affinity indicates how strongly cells absorb limiting nutri- (68, 69). As a result, the evolution of Prochlorococcus has driven ents (analogous to the pumping speed of a vacuum pump) and is nutrients to vanishingly low levels (<0.1 nM) in the oligotrophic oceans (64). equivalent to the saturated maximal nutrient uptake rate Vmax −1 −1 [moles of n (g dry weight) time ] over the Michaelis con- ∗ −1 Benefits of Excreting Organic Carbon. If selection to lower [n] stant KM ,n (moles of n L ) (68) (SI Appendix, Fig. S3). Vmax drives the maximization of νe /νn , why should it lead to an reflects the maximum handling rate for absorbed nutrients and increased excretion of organic carbon? We argue this ultimately is proportional to metabolic rate (68, 69). The free energy cost −1 emerges from mass and energy conservation. That is, in the pres- ∆r G (kJ mol ) of nutrient transport scales with natural log of ence of kinetic bottlenecks, cells can drive up their ATP/ADP the ratio of internal to external nutrient concentrations (65) (SI ratio by increasing their ATP supply rate, but to maintain steady Appendix, SI Text) and can become very large in the extremely state, they must also increase ATP consumption rates (49, 69). nutrient-poor oligotrophic oceans (69). For example, the free [The same argument applies to the NAD(P)H/NAD(P) ratio.] energy cost of phosphate uptake in the oligotrophic oceans is far This is consistent with observations that some nutrient-limited greater than the free energy gain of ATP hydrolysis unless the aerobic chemoheterotrophs have increased glucose uptake rates, ATP/(ADP × Pi ) ratio is increased drastically from commonly increased levels of respiration, increased excretion of polysaccha- assumed metabolite concentrations of 1 mM for ATP, ADP, and rides (whose synthesis from glucose requires ATP consumption), Pi (SI Appendix, SI Text). An analogous, but slightly different, sit- and a high flux through various ATP-consuming futile cycles + uation occurs for ammonia (NH4 ) uptake, which has the poten- (25, 26, 49). For photosynthetic cells CO2-fixation is a major sink tial for a major futile cycle in the oligotrophic ocean, because for ATP and NAD(P)H, but cells are limited in the carbon flux its conjugate base NH3 passively diffuses out of the cell (70) (SI density, νC , they can accommodate. This limit is proportional to Appendix, SI Text). It is therefore thought that cells poise their growth rate (Eq. 2), and, because selection to lower [n]∗ favors + internal [NH4 ] at the minimal viable value (71), which in turn relatively slow-growing cells (Eq. 4), it is lower in the oligotrophic similarly requires a significant increase in the ATP/(ADP × Pi ) oceans. Thus, we argue that excreted organic carbon represents ratio to drive forward glutamine synthesis (glutamate + NH3 + a kind of “carbon exhaust” that allows cells to maximize their ATP glutamine + ADP + Pi ), the central highway for nitro- nutrient affinity by increasing their metabolic rate above limits gen into metabolism (65). (See SI Appendix, SI Text for detailed arising from carbon saturation. calculations and discussion.) Finally, while kinetic bottleneck This general mechanism is further illustrated by expanding reactions can be driven forward by increasing the ATP/(ADP × Eq. 4. We assume that the electron flux must support car- Pi ) ratio, this simultaneously increases the free energy cost bon fixation sufficient to build biomass at a rate dictated by of ATP synthesis, therefore requiring a greater proton motive νn , that biomass has an elemental stoichiometry Qn /QC , that force, and thus ultimately a greater photosynthetic electron the efficiency of fixation is #C/#e (carbon atoms electron−1), flux (69). and that a fraction (0<β<1) of carbon is excreted/respired. The principles just outlined suggests that innovations increas- #C/#e depends on the oxidation states of the carbon source, ing νe may lower the minimal subsistence concentration of lim- biomass, and excreted carbon. For example, if the carbon source ∗ iting nutrients [n] (the lowest value of [n] at which net positive is CO2 and biomass and excreted carbon have the oxidation state − + growth is possible). When growth and loss processes are rela- CH2O, then #C/#e is 1/4 (i.e., CO2 + 4e + 4H CH2O +

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Fig. > [β excreted than is faster ing carbon excess unless density, flux also (see expression Text): the SI to lead assumptions these H ctps(8 n ntoewt hc titrcs(0.Lastly, (80). interacts it which with those in feed- within and ecological differentiation (78) produced further ecotypes have drove an could that that flux suggesting backs bacteria), electron other in or increase grazers, sink, interactions phage, electron reduc- biological with an providing excess (e.g., mediate to of also addition outlet polysaccharides In recognized 40). membrane 26, a stress, (25, is Increased power redox latter ing (78). transfer, the polysaccharides of electron membrane production in of synthesis involved and/or small genes of acquisition of the cassettes by (79). defined 4) clusters (Fig. form environment Subpopulations new own because their arises modify landscape chemically adaptive radia- ecotypes the adaptive in shift constructing” the “niche millions since are diverged tions, These that (78). subpopulations ago years stable of of of observa- hundreds ecotypes with of defined consistent to sist broadly contributor is the dominant This that the (77). tions be diversity to argued microbial been global radiations has adaptive and evolutionary produces 76) of landscape expand (75, to type adaptive population shifted This a a causes 4). innovation along key eco- (Fig. a waters which ancestral in deeper restricting dynamic ever and to rates, nutrients types metabolic limiting increasing with down each new drawing water surface, of evolution stable the sequential near in the ecotypes reflects observed (10–14) structure 1) of (Fig. population columns evolution layered Eq. the the of consequences that on ecological Focusing and evolutionary 5? Dynamics. the Coevolutionary are and What Biomass, Ecosystem Rate, Metabolic Prochlorococcus β in decrease a favor thus [n new ecotype 2 cro xrto) xcl sw bevdfrteeouinof evolution the for observed we as exactly excretion), (carbon hs hl nraigmtblcrt ie,lowering (i.e., rate metabolic increasing while Thus, = ] ) htpoetv ehnsslk h ae–ae cycle water–water the like mechanisms Photoprotective O). ctp bnac concentration abundance ecotype [n [E K h mrec of emergence The ancestral ecotype ] M 5 ]k ∗ ugssta yeceigicesn mut ffie carbon, fixed of amounts increasing excreting by that suggests tas cst increase to acts also it , ,n + 1 ν ν − e e Fg 3). (Fig. #/e](Eq. (#C/#e)] /ν e ν n ∆ adaptive radiation ν h vlto of evolution the , n e r /ν G /RT n ν Prochlorococcus rge h mrec fnweoye (pur- ecotypes new of emergence the trigger n n iutnosicessin increases simultaneous and = [E K depth Together, #C/#e. lower to act ) .Slcint lower to Selection 5). nutrients M ]k [n ,n Prochlorococcus tsuggests it Prochlorococcus, + ] ∗ Q Q ctps Eq. ecotypes. yicesn h carbon the increasing by C n ν Prochlorococcus e (#C 1 − Prochlorococcus /#e a nrae the increased has e n increas- and 0 5 ∆ dissolved IAppendix, SI organic carbon r ugssthat suggests G [n )(1 mixed layer /RT ] ∗ − ν would ∆ e con- β r and [5] G ) [n] . ) vle hog euneo innovations of and sequence 5 a through (Fig. evolved S4) Fig. Appendix, in for (SI As trans- look genes. for and metabolic 3 looking and of (Fig. by vicinity lineages controls the ocean in pathway porters on open selection of metabolic of evidence their innovations and of the 5 SAR11 evolution resolve marine (Fig. the of (96–98) reconstruct phylogeny core further the onto to S2) clades Table Appendix, (SI in pathways recon- excretion metabolic coccus as the our emerged exactly that suggest on glycolate) structions dependency and a (pyruvate oceanic evolved compounds Thus, (98). have SAR11 populations of are strains SAR11 adaptations freshwater Similar in observed (97). not pyruvate an have for and same requirement glycolysis coastal lack obligate the strains via ocean Furthermore, into metabolized open glucose whereas feed (96). with glycolysis, pyruvate all glycolate replace can four from strains SAR11 latter starts serine, the that glycine, (96); pathway glycolate, betaine either glycine and 95). or pyruvate (73, SAR11 requires abundant and SAR11 ubiquitous the cooccurring like for heterotrophs, opportunities new produce could it environment, Ecosystems. lowering Microbial Oceanic in Mutualism of ocean Emergence in force driving evolutionary general (Eq. a concentrations been ecosystems. nutrient has 4), steady-state Fig. of lowering a ihtento httemxmzto of envi- consistent maximization aquatic all the any are that of observations lowest notion These the the con- (94). is of with Earth that one on environment is the ronment biomass the from into efficiency—the up in taken verted over growth carbon rate electron of heterotrophic metabolic respiratory fraction general, favors their In oceans in oligotrophic (93). drains proteorodhopsins chains electron with transfer possess sunlight and capturing by (92) sup- organics 91). energy from 90, their supplement (33, ply turn picoeukaryotes in and heterotrophs diatoms oceanic in Many seen machinery also photosynthetic oligotrophic are of 3) the flux (Fig. electron the of the increased Some in that 87–89). modifications microbes 73, membranes— (61, heterotrophic nonphospholipid oceans and of genomes autotrophic oceanic streamlined use all of size, features and of dynamic signature cell features collective proteomes, small convergent this and broadly growth, that of the Slow conclusion in evo- imprint microbes. over the seen The increases be to ecosystems (86).] can led of time biomass also and lutionary animals flux marine and energy distribution the of the dynamic of size ecosys- evolutionary survey the body paleontological of the a biomass, here, [Similarly, thereby evolu- (85), tem. and which capture, game in resource innovations. zero-sum hypothesis,” increases similar a Queen adopt is “Red and classic tion pushes suit the 4) follow in (Fig. to the Unlike concentrations increases lineages nutrient that other lineage lowers all any and in environ- flux innovation coevolving energy Any chemically 84): open the (79, the via in ment cells interconnected microbial all is of evolution ocean the how of view anistic bioavailabil- metal trace buffering in environments. these role in key ity a may, play it also (83), citrate turn, like in acids carboxylic and (82) to that polysaccharides up compounds of several Because cus factor (81). a ocean (by which the 4), higher (Fig. oceans much oligotrophic are the in dissolved (DOC) of carbon concentrations organic steady-state of of long-term by-product evolution the a the increased as affinity, carbon nutrient organic the of increasing amounts excre- increasing and fixation of the tion promoting by that suggests framework this lto ftegyxlt hn n h eldcmne switch well-documented the and shunt glyoxylate the of pletion empe h itiuino eaoi ee cosclades across genes metabolic of distribution the mapped We hnetne oteeoytmlvl Eq. level, ecosystem the to extended When a eeceigaekonio-idn iad,including ligands, iron-binding known are excreting be may Fg and 3 (Fig. [n ,temtblccr fSAR11 of core metabolic the S1), Table Appendix, SI al S2 Table Appendix , SI ] ∗ nrae h xrto fognccro nothe into carbon organic of excretion the increases i.S1). Fig. Appendix, SI aeosre cosboth across observed Prochlorococcus—are PNAS .W ie to aimed We S4). Fig. Appendix, SI | ulse nieMrh2,2017 27, March online Published ,icuigtese-iecom- step-wise the including ), )ta lehr in elsewhere than ∼2) ν e 5 /ν rvdsamech- a provides Prochlorococcus Prochlorococcus Prochlorococcus n n thereby and , Prochlorococ- Prochloro- ial,if Finally, | 5 E3095 and

EARTH, ATMOSPHERIC, EVOLUTION PNAS PLUS AND PLANETARY SCIENCES IA.3 These observations suggest that metabolic mutualisms are self- IA.1/IA.2 IA.1/IA.2 IA.3 amplifying feedback loops (110) that maximize the collective IB νe /νn , and thus total productivity, of ecosystems. Specifically, IIA recycling otherwise wasted electrons through complementary IIB ν e excretion/uptake pathways increases the average νe of partici- IIIB (freshwater) ν n pating cells. Similarly, the loss of functions that are shared and IIIA IIB freshwater IV require limiting nutrients (e.g., iron in catalase) in some mem- SAR11 ν V SAR11 IIIA bers of the community decreases the average n . Mutualisms Rickettsiales thus raise the νe /νn of ecosystems—and lower the subsistence nutrient requirements of their cells (Eq. 5 and Fig. 4)—beyond to alphaproteobacteria V what is possible for individual lineages in isolation. Because excreting organic carbon lowers the minimal subsistence nutri- Fig. 5. Metabolic evolution of SAR11. Variants are shown in simplified ent requirements of individual cells, mutualisms of this kind are form along a gradient of νe/νn. Dashed orange lines highlight innovations (SI Appendix, Fig. S4 and Table S2), which include disruptions or replace- emergent properties of ecosystems and avoid public goods dilem- ments of glycolysis (all branches), the step-wise completion of the glyoxy- mas (48). An upper bound may exist on the maximization of late shunt (from group V to IIB to all groups IA) and the gain and loss of νe /νn due to the minimal requirements of being an autonomous excretion/uptake pathways. See main text for details. Excretion pathways cell. As the smallest photosynthetic cell (6), Prochlorococcus may are represented as red dots and uptake pathways as blue dots. This recon- be closest to this limit, reinforcing the notion that it has a cen- struction is less certain than for Prochlorococcus because SAR11 has greater tral role in shaping the features of ecosystems in the surface diversity (99, 100), and as highlighted in the phylogeny (included clades in oceans. green), many clades have no or few sequenced representatives (SI Appendix, Table S2). Several branches were therefore drawn as a polytomy. Are Plant Cells Microscopic Analogs of Oceanic Microbial Ecosys- tems? It occurred to us that the metabolic organization of oceanic microbial ecosystems and green plant cells are simi- from Emden–Meyerhoff–Parnass (EMP) to Entner–Doudoroff lar (Fig. 6): Intermediates of lower glycolysis and photorespira- (ED) glycolysis (97). Glycolysis is disrupted in the 1.3A clade tion are central conduits of electron transfer from Prochlorococ- (97) (Fig. 5 and SI Appendix, Table S2), which is most abun- cus to SAR11 and from chloroplasts to mitochondria in plant dant in surface waters where the Prochlorococcus HLII clade cells, while TCA cycle intermediates facilitate electron trans- dominates (Fig. 1) (99, 100). Glycolate uptake is also lost in this fer in the opposite direction in both systems (111–113). Sim- SAR11 clade (Fig. 5 and SI Appendix, Table S2), which suggests ilar patterns emerge at other levels of organization. Microbes that pyruvate produced by the Prochlorococcus HLII ecotype other than Prochlorococcus/SAR11 in ocean communities, and may have become its central source of carbon and energy (97). organelles other than chloroplasts/mitochondria in plant cells, Finally, similar to Prochlorococcus (SI Appendix, Fig. S2), chro- appear central to peroxide detoxification (108, 113). The het- mosome rearrangements positioned a series of transporter pro- erotrophic bacteria SAR86 and SAR116 may be important for teins near key metabolic genes in SAR11 (SI Appendix, Fig. S5), this function in the oligotrophic oceans, because they both pos- consistent with selection on the control of transport pathways. sess catalase (114–116) and are abundant in warm stratified We identified putative import transporters for pyruvate and gly- waters (117). Prochlorococcus and chloroplasts both have PTOX colate, and putative export transporters for citrate and malate, as an electron drain in their photosynthetic electron transfer the latter exclusive to the 1A clade (Fig. 5 and SI Appendix, Fig. chain, whereas SAR11 and mitochondria both have alternative S5). The emergence of malate export in SAR11 would provide oxidase (AOX) as an electron drain in their respiratory elec- a potential source for the emergent malate uptake pathway of tron transfer chain (93, 113). Furthermore, like chloroplasts, Prochlorococcus that is putatively activated at night (Fig. 3 and Prochlorococcus uses chlorophyll b in addition to chlorophyll SI Appendix, Figs. S1 and S2). a, which has not been observed in cyanobacteria other than Furthermore, the glyoxylate shunt is a TCA cycle bypass acti- Prochloron and Prochlorotrix (6). Finally, organelles of plant cells vated under redox stress in some microbes (101–103), while have also undergone reductive genome evolution, and, for mito- the evolutionary switch from EMP to the higher-rate ED vari- chondria, it has been argued that this increased the cellular ant of glycolysis has, in other microbes, been attributed to an power density of eukaryotes (118)—similar to what we argued increased energy supply (74). These observations are consistent for oceanic microbial ecosystems. The extensive convergence of plant cells and oceanic microbial ecosystems highlights the con- with selection acting to maximize νe /νn in both systems and thereby producing pathways that transfer pyruvate and glyco- straints that metabolism imposes on the large-scale structure of late from Prochlorococcus to SAR11 and malate from SAR11 to evolution (5) and suggests that the metabolic codependencies of Prochlorococcus. Oligotrophic waters have nanomolar concen- eukaryotic organelles can evolve without the physical intimacy of trations of pyruvate and glycolate, with midday maxima for the endosymbiosis. latter, consistent with biological cross-feeding synchronized with the input of sunlight, although abiotic photochemistry may also Biospheric Self-Amplification and the Rise of Atmospheric Oxygen. contribute (104, 105). We have proposed that maximizing the metabolic rate of cells Additional evidence for metabolic mutualism in these sys- lowers their minimal subsistence nutrient requirements and that tems comes from observations regarding hydrogen peroxide this is achieved by maximizing the cellular electron-to-nutrient (HOOH), a by-product of biological electron transport, photo- flux ratio (νe /νn ), while increasing the excretion of organic car- chemistry, and other abiotic processes (106). Prochlorococcus bon (Eq. 5). This leads to an evolutionary dynamic that increases and some later-diverging clades of SAR11 have lost HOOH- total ecosystem biomass (Fig. 4) and paves the way for self- detoxifying catalase (SI Appendix, Tables S1 and S2), and Prochl- amplifying feedback loops that recycle organic carbon (Fig. 6) orococcus grows better in the presence of bacteria retaining and reinforce the maximization of cellular metabolic rate at the catalase (107, 108). This led to the “Black Queen” hypothesis, ecosystem level. It has been argued that the hierarchical orga- which argues that subpopulations of ecosystems can save essen- nization of pathways within metabolism reflects the outgrowth tial nutrients by giving up inevitably shared functions (such as of self-amplifying feedbacks that increased the free energy con- detoxifying the freely diffusible HOOH), so long as they are pre- sumption of the emerging biosphere (5, 119). Our framework served by others in the ecosystem (109). Our reconstructions sug- extends those arguments into the world of phenotypically differ- gest that the loss of catalase in Prochlorococcus and SAR11 coin- entiated cells and microbial ecosystems, and is consistent with cides with increased excretion of organic carbon, which provides the theorem that the flow of energy through the biosphere pro- carbon and energy for catalase-containing bacteria (109). motes its self-organization into chemical cycles (2).

E3096 | www.pnas.org/cgi/doi/10.1073/pnas.1619573114 Braakman et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 ramne al. et Braakman remained H Oxi- oceans in Great (rich Proterozoic the euxinic deep after and that the atmo- anoxic argued largely (GOE) in been Event rise has dation It Neoproterozoic–Phanerozoic oxygen. the spheric into O insights atmospheric in rise major 131–133). (123, second carbon levels burial a modern global carbon and indicate 127), organic 121), sediments enhanced (126, (120, when (128–130), glaciations perturbations 125), global cycle (124, of Phanero- present) occurrence the to to the Ma Ma) transi- (541 (1,000–541 the Neoproterozoic zoic near the emerged from have tion to pic- marine estimated coincidentally, are not ocyanobacteria Perhaps 123). (122, history atmospheric Earth of drawdown their the increasing explain CO by help simply would 121) it burial (120, production, the carbonates enhanced and sun organics the of by driven self-amplification spheric serine; SER, bisphosphate; succinate. ribulose SUC, RBP, phos- PEP, pyruvate; oxaloacetate; PYR, OAC, malate; phoenoylpyruvate; glycine; MAL, GLY, isocitrate; glycolate; ICE, glyoxylate, GLC, GOX, glycerate; GLA, phosphate; DHAP, citrate; dihydroxyacetone CIT, catalase; KatG, acetyl-CoA; ACE, 3-phosphoglycerate; and 3PG, chloroplasts electron and systems, provide both AOX of Prochlorococcus chains and transport PTOX electron genes. the as similarly in KatG is drains (111–113) crossed-out catalase chloroplasts/mitochondria cata- the cells, not of by plant and loss In indicated peroxisome clades. the the branching indicate in later to its located SAR11 of for some dashed in is lase arrow. cross-out dashed this a by while in indicated gene, glycolate catalase is 5) of of (Fig. loss loss clade 1A.3 The The ocean mitochondria. open the and in uptake chloroplasts between and between as SAR11 pathways indicate shown similar arrows through are flux Blue/orange sequences carbon stoichiometry. organic reaction accurate many without simplicity, arrows For single cells. plant green of 6. Fig. (divinyl)chlorophyll a+b h rpsdeouinr yai Fg )myprovide may 4) (Fig. dynamic evolutionary proposed The bio- If history. Earth for implications has framework Our 2 KatG KatG chlorophyll a+b n h ieo topei O atmospheric of rise the and photosystems: photosystems: PTOX PTOX 21 21 h eaoi raiainof organization metabolic The Prochlorococcus chloroplast 3PG 3PG RBP RBP ohuechlorophyll use both 2OG GLA O O CO CO 2 2 MAL GLC GLC Prochlorococcus MAL 2 2 SAR86, SAR116,others? DHAP OAC PYR CIT HOOH HOOH peroxisome GLA L GLY GLC 2OG cellulose MAL OAC KatG PEP KatG ? b Prochlorococcus swl as well as SER CIT sidctdb rse-u KatG crossed-out a by indicated is H H 2 2 O O ? 2 oceanic microbialecosystem 2 PYR PYR OAC )(3) eetstudies Recent (134). 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EARTH, ATMOSPHERIC, EVOLUTION PNAS PLUS AND PLANETARY SCIENCES with the Neoproterozoic Oxidation Event (122, 123, 132, 142). global emergence is imposing on the Earth system is one of Genomic studies estimate that chloroplast endosymbiosis lead- humanity’s greatest challenges. ing to the rise of all photosynthetic eukaryotes occurred between the late Paleoproterozoic (2,500–1,600 Ma) and the early Neo- Materials and Methods proterozoic (151–154). Paleontological studies in turn find a sig- We analyzed 56 genomes of cyanobacteria representative of the diver- nificantly increased fossil diversity of eukaryotes (including pho- sity of this clade (161) obtained from the UniProt website, 56 genomes of tosynthetic eukaryotes) in the Neoproterozoic (155, 156), whose Prochlorococcus and marine Synechococcus (162), and 16 SAR11 genomes increasing body sizes and fecal pellets could have moreover obtained from the National Center for Biotechnology Information website. strengthened the export and burial of organic carbon from the We searched these genomes for the presence and absence of a set of refer- oceans (157, 158). ence enzymes (SI Appendix, Tables S1 and S2) using the BLASTp and tBLASTn As a final note, one could argue that the emergence of mod- algorithms (163). We reconstructed phylometabolic trees (Fig. 2) by mapping ern human societies is a variant of the general framework we the distributions of metabolic genes onto the phylogenies of Prochlorococ- propose. As our populations expanded and extracted ever more cus (Figs. 1 and 3) and SAR11 (Fig. 5). electrons from fossil fuels, we have increased global CO2, while ACKNOWLEDGMENTS. We thank the reviewers for critical and insightful drawing down natural resources and global O2 (albeit a small reviews, and we thank Jamie Becker, Shane Hogle, and John Casey for com- amount relative to the contemporary inventory for the latter) ments on an earlier version of our manuscript. R.B. is a Simons Foundation (159, 160). In the process, we have become increasingly socially, Fellow of the Life Sciences Research Foundation. This work was supported technologically, and economically interconnected, analogous to by Simons Foundation Grant SCOPE 329108 (to S.W.C. and M.J.F.); Gordon what we have observed in the evolution of oceanic microbial and Betty Moore Foundation Grants 3778 (to M.J.F.) and 495.01 (to S.W.C.); and by the NSF Center for Microbial Oceanography Research and Education ecosystems and plant cells. As in those systems, this has increased (S.W.C.). R.B. dedicates this work to the memory of Harold Morowitz, a pio- our collective ability to harvest more difficult-to-access natural neer in the study of biological energy flow, whose theories and approach to resources. Managing the biogeochemical perturbation that our studying life were central in shaping the core ideas of this work.

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