domain: sdv chim

text:
Gabriel V Markov
email: gabriel.markov@sb-roscof.fr

Vincent Laudet
email: vincent.laudet@oist.jp

Small molecules as products of evoluton

The functonal evoluton of ancient proteins has recently been reconstructed using molecular phylogeny, and the actvity of the deduced molecules can be tested. Unfortunately, the world of small molecules escapes such "resurrecton" studies, which rely on ancestral sequence reconstructon. These studies implicitly assume that only the proteins evolve whereas the small molecules are presumed to be identcal now and in the past. Recent evolutonary analysis of biochemical pathways, however, as well as the impressive surge of metabolomics are changing this situaton and it is becoming possible to reconstruct ancient biochemical pathways. We can now begin to infer the chemical structures of key molecules that were present in the past, synthesize those molecules and test their ability to interact with their cognate (ancient) protein partners. In this artcle, we discuss the possibilites ofered by these new methodological developments and provide key examples. We also highlight four principles that are important to consider when studying the evoluton of small molecules: catalytc promiscuity, metabolic reconfguraton, coevoluton and bidirectonal interactons. These new developments call for an alliance between organic chemists and evolutonary scientsts to investgate the diversity of the chemical building blocks of life, and the evoluton of their biosynthetc pathways.

Introducton

Understanding how biodiversity emerged is a major endeavor of biological research and not only concerns the "endless forms most beautful" celebrated by Darwin but also the molecules that bring them into being. Whether at the morphological or molecular level, there are two main approaches to beter understand the origin and diversifcaton of the products of evoluton. The frst is to compare the living forms and to infer, by comparatve anatomy and phylogeny, what has been the evolutonary path that allowed their emergence [START_REF] Harvey | The Comparatve Method in Evolutonary Biology[END_REF] . The second approach is to dig in the past and to identfy the forms that were living previously. Thanks to paleontology and its exquisite reconstructon of ancient plants, animals and even microorganisms, this has considerably enriched our view of life and ofered us unique insights on ancient worlds.

Untl recently, however, these approaches were limited to extnct organisms and there was no obvious possibility to perform such a historical approach at the molecular level. Ancient DNA analysis has ofered fascinatng possibilites but is limited to quite recent periods (ca. 1-million-years BP). It cannot allow the study of events that occurred millions of years ago [START_REF] Leonardi | Evolutonary Paterns and Processes: Lessons from Ancient DNA[END_REF] . Comparison of protein sequences was an important advance as it permited the inference of the sequences of ancestral proteins. These could then be reconstructed to directly test their functon experimentally and thereby follow their functonal evoluton [START_REF] Hochberg | Reconstructng Ancient Proteins to Understand the Causes of Structure and Functon[END_REF] . These methods rely on ancestral protein sequence reconstructon using molecular phylogenetc inference. The world of small molecules, including organic substrates or products, totally escapes such eforts of evolutonary studies. This is partcularly problematc in the context of biochemical evoluton, such as the evoluton of metabolic pathways or the reconstructon of ancestral hormone/receptor couples, in cases where hormones are small molecules rather than proteins or peptdes. Indeed, in these cases, which include most of metabolic regulaton, it has been considered that only the proteins evolve. The small signaling molecules and their biosynthetc precursors have been implicitly assumed to be identcal now and in the past [START_REF] Thornton | Resurrectng the ancestral steroid receptor: ancient origin of estrogen signaling[END_REF] .

Such an anachronistc analysis can give rise to incorrect inferences of evolutonary trajectories (Figure 1). The reason for such a limitaton was obvious: whereas we had both a conceptual and technical framework allowing us to reconstruct proteins that are encoded by DNA, we did not know how to predict and reconstruct the small molecules, which are produced by these ancestral enzymes arranged in metabolic pathways.

With the emergence of genomics and the prodigious amount of sequence informaton -in partcular high quality complete genomes from a variety of organisms -as well as the impressive recent surge of metabolomics, it has become possible to perform evolutonary analysis of biochemical pathways, to reconstruct ancient pathways and therefore to perform relevant historical tests (Figure 1). The resurrecton of ancient proteins starts by sequence alignment of protein sequences from living organisms (1), from this the constructon of a phylogenetc tree (2) allows to reconstruct the evolutonary history and, in a separate step to predict ancestral sequences at each node of the tree. It is then possible using gene synthesis techniques to produce the predicted protein (3) giving rise to a so-called "resurrected" protein. The functon of this protein can then be evaluated but when it is a protein interactng with a small molecule it carries the risk of a anachronistc functonal test: The ancestral protein is confronted with a modern small molecule.

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To avoid this risk, it is necessary to perform the same exercise on the small molecule side, although this is less straightorward than with protein sequence: the comparison of metabolic pathways existng in living organism (A), the inference of the ancestral pathway either by cladistc analysis or genome wide reconstructon of metabolic pathways (B), the chemical synthesis of the inferred molecule of interest giving rise to a "resurrected" small molecule (C). It is only afer these steps that a meaningful historical functonal test can be performed (D).

How to reconstruct an ancestral pathway? The case of steroids.

To illustrate the resurrecton of the biochemical pathways and molecules that were present in the past let us consider the evolutonary history of the vertebrate steroid receptors and their ligands, which provides an excellent case study to illustrate the coevoluton of ligands and their receptors. Vertebrate steroid hormones are all derived from cholesterol and play very important functons in reproducton (androgens, estrogens, progesterone), in the regulaton of metabolism and in the regulaton of water and ion balance (glucocortcoids and mineralocortcoids). Their acton is mediated by a group of six nuclear receptors: two estrogen receptors ERa and ERb and four ketosteroid receptors the androgen, progesterone, glucocortcoid and mineralocortcoid receptors, (AR, PR, GR and MR, respectvely).

Reconstructon of ancestral proteins has suggested that the ancestral steroid receptor was an estrogen receptor and that other steroid receptors exploited molecules that were metabolic intermediates of estrogen synthesis [START_REF] Thornton | Evoluton of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitaton and serial genome expansions[END_REF] . This socalled "ligand exploitaton model" has been challenged mainly on the ground that there was no clear evidence that 17bestradiol, the natural ligand, was actually an ancient molecule [START_REF] Baker | Recent insights into the origins of adrenal and sex steroid receptors[END_REF] . In these analyses, the authors reconstructed an ancient receptor they called AncSR1. The queston arose, however, as to what was the ancestral ligand of this resurrected receptor? The analysis of bilaterian genomes, searching for orthologues of enzymes important for estrogen biosynthesis, produced negatve results 7 . To tackle this queston, therefore, we performed a cladistc analysis of steroidogenesis, focusing not on the sequences of the enzymes in the pathway but on the sequence of the chemical reactons [START_REF] Markov | Origin of an ancient hormone/receptor couple revealed by resurrecton of an ancestral estrogen[END_REF] . This approach, which was frst elaborated in the context of universal amino acid metabolism [START_REF] Cunchillos | Integratng the universal metabolism into a phylogenetc analysis[END_REF] , allowed us to compare the sequence of the biochemical pathways, that is the suite of chemical reactons, even without informaton about the specifc enzymes catalyzing each reacton. The idea here is to consider biosynthetc pathways as products of evoluton that can be compared by 'comparatve anatomy' and cladistcs, just as two morphological structures are compared.

When several vertebrate species are compared using morphological criteria such as the presence or absence or the shape of well-defned morphological structures (e.g., a jaw bone) this comparison is valid from an evolutonary point of view because the formaton of these structures is controlled by genes that may be unknown but are nevertheless following the Darwinian "descent with modifcaton" principle. Exactly like morphological structures, metabolic pathways are controlled by genes, which may be unknown but are targets of evoluton. Therefore, the structure of these pathways can be compared as bone shape is compared in the constructon of an evolutonary tree based on morphology. In the case of steroidogenesis, a character matrix was built to objectvely assess the similarity between biochemical reactons, allowing to measure successions of identcal or similar reactons and therefore similarites between pathways 8 . This matrix was then analyzed by parsimony to generate a tree in which each branch is not a species or a molecule but a metabolic pathway.

We could therefore infer an ancestral pathway and propose that aromatzed steroids in chordates occurred in cholesterol derivatves that did not undergo side-chain cleavage. The simplest molecule of this new class, paraestrol A, was chemically synthesized, and was experimentally confrmed to bind the ancestral steroid receptor AncSR1, albeit with lower afnity than 17b-estradiol, ofering a unique window on what could have been the ancestral steroid hormone precursor [START_REF] Markov | Origin of an ancient hormone/receptor couple revealed by resurrecton of an ancestral estrogen[END_REF] . Further work will be necessary to clarify how additonal hydroxylatons on the paraestrol backbone would have increased its afnity for steroid receptors to a level compatble with hormonal signaling.

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Four principles of metabolic evoluton.

As the example highlighted above shows, it is now possible to infer the chemical structures of key molecules present in the past, synthesize those molecules and test their ability to interact with their ancient proteins. We consider that four principles must be considered when studying small molecule evoluton (Figure 2). Because steroids have been widely studied from an evolutonary point of view, we use them below to illustrate these principles. Ancestral reconstructons of ligands and receptors have been applied successfully to many other metabolic pathways in diferent organisms such as fies, nematodes, or plants. We therefore briefy summarize partcularly illuminatng examples of these other pathways to illustrate those general principles. A. Catalytc promiscuity is the ability of a single enzyme (the blue one) to catalyze the same molecular transformaton (blue disk) on two diferent molecular backbones (in green). Thus, if its catalytc actvity is modifed during evoluton (red arrow), this can lead to changes in several distnct pathways where the concerned reacton was occurring. B. Metabolic reconfguraton occurs when a new pathway is generated by recombinaton of already existng reactons previously occurring in distnct pathways. In the example here during evoluton (red arrow), the loss of two reactons (doted yellow arrows) is compensated by the recruitment of an enzyme enabling the conservaton of the downstream steps of the pathway, being fueled from a distnct precursor. C. Coevoluton illustratng how a change in a ligand/receptor interacton during evoluton (red arrow) must include change in both metabolic enzyme and receptor ligand binding pocket. D. Bidirectonal interacton at the holobiont level. An organism A (here an intestnal cell) can generate a product actve on organism B (here a commensal bacteria) that in turn can modify it to produce a molecule that will act in return on organism A. When an evolutonary change (red arrow) occurred in the metabolic pathway of organism A this afects both organisms.

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The frst of these principles is catalytc promiscuity (Figure 2A). When a molecular actor (for example, an enzyme or a receptor) can bind to diferent types of molecules the phenotype induced by a change in this molecular actor may afect several characters, sometmes quite disparate 10 ,11 . This is well illustrated by the case of the CYP11A enzyme that is responsible for the removal of the vertebrate steroid side chain. Recent studies have established that CYP11A is able to catalyze hydroxylaton of various sterols, not only cholesterol, but also vitamin D3 metabolites suggestng an acton much wider than previously thought [START_REF] Slominski | Novel actvites of CYP11A1 and their potental physiological signifcance[END_REF] . This suggests that complex unantcipated links could exist between an enzyme and small molecules implicated in distnct metabolic regulatons. In other words, a change in a given enzyme (either of expression patern, expression level or specifcity) could impact more than one product or pathway, opening the way to pleiotropic changes. Other similar examples include several 17β-hydroxysteroid dehydrogenases (17b-HSD) enzymes known to be important for estrogen synthesis, but which are also important for retnoic acid and prostaglandin signaling [START_REF] Penning | AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase): Roles in malignancy and endocrine disorders[END_REF] . The well documented catalytc promiscuity of HSD enzymes is acknowledged has a major driver of diversifcaton in vertebrate steroidogenic pathways [START_REF] Baker | Origin of the response to adrenal and sex steroids: Roles of promiscuity and co-evoluton of enzymes and steroid receptors[END_REF] . Promiscuity at the receptor level further reinforces the importance of pleiotropic efects on metabolic pathways. It has been well described for the vitamin D receptor, which in the intestne can behave as a bile acid receptor [START_REF] Makishima | Vitamin D receptor as an intestnal bile acid sensor[END_REF] , and for the estrogen receptor, which binds a great variety of ligands besides 17b-estradiol [START_REF] Baker | The promiscuous estrogen receptor: Evoluton of physiological estrogens and response to phytochemicals and endocrine disruptors[END_REF] .

The importance of convergence to reveal basic principles of the evoluton of small molecules, such as catalytc promiscuity is also well exemplifed by a pheromone system involved in relatonships between buterfies and plants. It has long been known that plants and insects ofen use the same compounds for communicaton and now the underlying biochemical mechanisms are startng to be uncovered [START_REF] Darragh | A novel terpene synthase controls diferences in ant-aphrodisiac pheromone producton between closely related Heliconius buterfies[END_REF] . Even within plants, there are multple examples of convergent evoluton, with promiscuous cytochrome p450s (CYPs) being recruited many tmes into identcal catalytc reactons, either among closely related subfamilies, or even across most distant paralogs [START_REF] Hansen | Plant cytochrome P450 plastcity and evoluton[END_REF] The second principle is metabolic reconfguraton, sometmes also called patchwork evoluton (Figure 2B), which means that chemical reactons and enzymes can be reshufed to generate a given compound [START_REF] Lazcano | On the origin of metabolic pathways[END_REF] . This allows the interpretaton of how steroid aromatzaton was recruited into the pathway of cleaved side-chain steroids afer having previously operated on steroids with a side chain [START_REF] Markov | Origin of an ancient hormone/receptor couple revealed by resurrecton of an ancestral estrogen[END_REF] . This is also well illustrated by the very early steps of cholesterol biosynthesis. Sterols-cholesterol in animals, phytosterols in plants and ergosterols in fungi-are critcal for life. There is even recent evidence that sterols are synthesized de novo in some bacterial phyla [START_REF] Hoshino | Evoluton of bacterial steroid biosynthesis and its impact on eukaryogenesis[END_REF] . Cholesterol is formed from acetyl-CoA by a metabolic pathway with approximately 20 steps, and vertebrates are able to synthesize cholesterol in an autotrophic manner [START_REF] Capell-Hatam | Sterol evoluton: cholesterol synthesis in animals is less a required trait than an acquired taste[END_REF] . But this is not the case for all animals and several invertebrates are in fact auxotrophs for sterols; that is, they rely on an external source. Even though nematode worms use cholesterol as a source of critcal hormonal compounds such as dafachronic acid, which coordinates their life cycle, they cannot synthesize it. Indeed, nematode genomes lack the frst three enzymes required for cholesterol biosynthesis. In a very elegant analysis Shamsuzzana et al., [START_REF] Shamsuzzama | Metabolic Reconfguraton in C. elegans Suggests a Pathway for Widespread Sterol Auxotrophy in the Animal Kingdom[END_REF] have shown that nematodes can transform plant phytosterols and fungal ergosterols into cholesterol by co-optng downstream cholesterol synthesizing enzymes and modifying them to transform phytosterols and ergosterols to an 6/12 intermediary compound, desmosterol. Desmosterol is fnally converted into cholesterol! Furthermore, they also show that the same strategy-loss of key upstream enzymes of the cholesterol synthesis pathway and co-opton of downstream enzymes to use dietary compounds from plants and/or fungi-is used by other invertebrates, suggestng a widespread cholesterol auxotrophy by interkingdom trophic interactons [START_REF] Shamsuzzama | Metabolic Reconfguraton in C. elegans Suggests a Pathway for Widespread Sterol Auxotrophy in the Animal Kingdom[END_REF] .

Analysis of the pheromone signaling system of nematodes also illustrates the principle of metabolic reconfguraton. Pheromone communicaton is critcally important for nematodes, either in the short-term (e.g., mate atracton) or for long-term life history-related responses (e.g., formaton of dauer larvae). Hundreds of extremely diverse but related compounds, collectvely known as ascarosides, that consist of a sugar moiety coupled to a faty acid sidechain, have been characterized [START_REF] Bose | Natural variaton in dauer pheromone producton and sensing supports intraspecifc competton in nematodes[END_REF] . Systematc metabolomic profling of ascarosides in 30 species of Pristonchus revealed evidence of a high level of biochemical innovaton by reshufing of metabolic actvites [START_REF] Dong | Convergent evoluton of small molecule pheromones in Pristonchus nematodes[END_REF] . Analyzing the occurrence of these molecules in the light of species phylogeny allowed the authors to reveal many clade-related compounds, fully consistent with the noton of metabolic pathway "descent with modifcaton". Interestngly, this analysis also revealed a recurrent convergent evoluton of modular molecules in distant branches of the evolutonary tree indicatng that biochemical evoluton is a repeatable process in these worms [START_REF] Dong | Convergent evoluton of small molecule pheromones in Pristonchus nematodes[END_REF] .

Building on the two previous ones, the third principle, coevoluton (Figure 2C), has been elaborated independently in the feld of ligand-receptor evoluton (especially for peptde ligands and their receptors) and, recently, more precisely formalized for enzyme-substrate couples [START_REF] Noda-Garcia | Metabolite-Enzyme Coevoluton: From Single Enzymes to Metabolic Pathways and Networks[END_REF] . According to this principle, ligands and their receptors are not free to evolve since any mutaton in one partner must be compensated by a mutaton in the other to avoid losing the interacton. Even if this is much more complicated to demonstrate in the case of small molecules that are the products of metabolic pathways, examples do exist for small molecules, again among steroids. Bile acids, which are one of the major end products of cholesterol metabolism in vertebrates, play an important role in the digeston of lipids and proteins as well as displaying antmicrobial actvity in the intestne. They form a set of extremely variable compounds that difer widely across species. Their major receptor, the nuclear receptor FXR, changes its specifcity for primary bile acid salt across species by altering the size and shape of its ligand binding pocket [START_REF] Reschly | Evoluton of the bile salt nuclear receptor FXR in vertebrates[END_REF] . For example, in lamprey and zebrafsh the FXR ligand binding pocket is fat and can easily accommodate the planar bile acids found in these species.

In contrast, human FXR has a curved pocket that binds the bent bile acid salts typically found in human [START_REF] Reschly | Evoluton of the bile salt nuclear receptor FXR in vertebrates[END_REF] . This clearly illustrates a case of coevoluton even if, to our knowledge the evoluton of the enzymes responsible for the producton of the various types of bile acids present in various vertebrates has not been studied and has not been yet related to the evoluton of FXR ligand binding ability. Another fascinatng example of coevoluton is provided by the role of progesterone, a precursor in cortcoid biosynthesis, which is also an allosteric modulator of cortcoid binding by the mineralocortcoid receptor (MR) in some vertebrates through binding to a non-canonical binding site located in the Nterminal domain of the receptor [START_REF] Katsu | Transcriptonal actvaton of elephant shark mineralocortcoid receptor by cortcosteroids, progesterone, and spironolactone[END_REF] .

Detailed analysis of a specifc pathway can also reveal ligand-receptor coevoluton, and this has started to

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provide key insights into the evoluton of signaling pathways in plants and animals. In plants, the various components of the faty-acid derived jasmonate pathway (important for stress response and other developmental and physiological regulatons) were characterized in basal lineages including the liverwort Marchanta polymorpha [START_REF] Monte | Ligand-receptor co-evoluton shaped the jasmonate pathway in land plants[END_REF] . The main components of the pathway are conserved, but it was shown that the bioactve signaling molecule in Marchanta was not jasmonoyl-isoleucine as in Arabidopsis, but two isomeric forms of its precursor, called dinor-oxo-phytodienoic (12oxo-phytodienoic acid). This change of ligands requires a single change of a residue in the receptor but also requires the modifcaton of two pre-existng enzymes. Therefore ligand-receptor coevoluton must be accompanied by a metabolic reconfguraton.

The fourth principle, bidirectonal interacton (Figure 2D), refers to the noton that a given molecule can be actve both on the organism that produces it and also on a neighboring species that can in turn alter it. This is partcularly applicable to small molecules and once again is well exemplifed by steroids. Bacteria can metabolize steroids found in their environment (for example in their eukaryotc hosts or, alternatvely, sewage treatment plants) [START_REF] Holert | Metagenomes Reveal 11/12 Global Distributon of Bacterial Steroid Catabolism in Natural, Engineered, and Host Environments[END_REF] .

Recent evidence suggests a real bidirectonal efect, where steroids degraded by a biochemical pathway in a bacterium can, in return, be used as actve components by eukaryotes. For example, whereas the retro-conversion of estrogens into androgens was deemed impossible in animals, given the irreversibility of the aromatzaton reacton, it was recently found to occur in an anaerobic bacterium, opening a previously unexplored microbe-host metabolic interdependency [START_REF] Wang | Retroconversion of estrogens into androgens by bacteria via a cobalamin-mediated methylaton[END_REF] .

The importance of bidirectonal interacton is also well demonstrated in plant and insects by a recent observaton that revealed how wild tobacco, Nicotana atenuata, protects itself against the actvity of the compounds it uses to repel herbivores [START_REF] Li | Controlled hydroxylatons of diterpenoids allow for plant chemical defense without autotoxicity[END_REF] . Like many other plants, wild tobacco uses specialized metabolites to decrease the harmful actvity of herbivores, trying to target tssues or pathways that are present only in their animal enemies to avoid autotoxicity. Nicotne which targets the nervous systems of animals but has no efect on the plant itself is a classic example of this strategy. In tobacco, two cytochrome P450 enzymes actng on 17-hydroxygeranyllinalool diterpene glycosides are critcal for avoiding autotoxicity. The defensive functon of 17-hydroxygeranyllinalool diterpene glycosides on Manduca sexta caterpillars (the main predator of wild tobacco) is achieved by inhibitng sphingolipid biosynthesis in the insect through hydroxylaton of the product they eat [START_REF] Li | Controlled hydroxylatons of diterpenoids allow for plant chemical defense without autotoxicity[END_REF] . The hydroxylaton of the diterpene compound by cytochrome P450 in the plant is also important to prevent autotoxicity, in additon to arming the chemical gun against the insect. The bidirectonal interacton can therefore also be an arms race.

There is of course no reason to think that this type of bidirectonal interacton is confned to those well-studied examples. If we open this principle to other cases, for example regarding the interactons between organisms in the marine world, the perspectves ofered are dizzying [START_REF] Markov | Hormonally actve phytochemicals from macroalgae: A largely untapped source of ligands to deorphanize nuclear receptors in emerging marine animal models[END_REF] . This principle has been discussed previously in the context of environmental signaling and endocrine disrupton [START_REF] Mclachlan | Environmental signaling: what embryos and evoluton teach us about endocrine disruptng chemicals[END_REF] but must be extended outside this feld and generalized: in short, a mutaton in a given metabolic pathway can afect diferent organisms that interact with the organism in which the mutaton takes place. This is one more reason to study metabolic evoluton in a global and systemic perspectve.

Ongoing improvements in methodological approaches necessary to tackle metabolic pathway evoluton

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Inferring ancestral metabolic pathways is stll a great challenge, but the basic tools necessary to develop such approaches are maturing. Complete genome sequences have made genome-scale metabolic modeling a more and more widely used approach [START_REF] Gu | Current status and applicatons of genome-scale metabolic models[END_REF] . This approach enables the reconstructon of an explicit model of whole organism metabolism, based on sequence annotaton data. This can be coupled with standard comparatve genomics studies to integrate specifc pathways in the context of global metabolism [START_REF] Cannell | Multple Metabolic Innovatons and losses are associated with major transitons in land plant evoluton[END_REF] . The development of genome-scale metabolic network reconstructons has also led to atempts to directly infer ancestral metabolic networks [START_REF] Psomopoulos | Ancestral state reconstructon of metabolic pathways across pangenome ensembles[END_REF] .

Another promising development is the frst database of ancestral resurrected proteins [START_REF] Carlet | Revenant: a database of resurrected proteins[END_REF] . This opens the possibility to cross-validate ancestral metabolic network reconstructons by ensuring that enzyme sequences deposited in such databases are correctly inferred to be present in ancestral genome-scale reconstructons and associated with the appropriate biochemical actvity. A critcal point in that respect is ensuring that already described metabolites in a given species are indeed present in the corresponding genome-scale metabolic network reconstructon. There are now databases that make it possible to automatcally assemble species-specifc metabolomes, although they are stll in their infancy and biased towards well established genetc model species [START_REF] Salek | Automated assembly of species metabolomes through data submission into a public repository[END_REF] . Thanks to ongoing work on emerging model species, however, more datasets will certainly be integrated in the future.

Finally, to understand the evoluton of small molecules it is necessary to explore the space of possible metabolite-protein interactons using high-throughput methods. There is promising progress in the mass-spectrometry and NMR approaches (combined with other separaton techniques) to distnguish proteins with or without bound ligands. This should allow the identfcaton of new targets for plant-derived metabolites [START_REF] Venegas-Molina | Why and how to dig into plant metabolite-protein interactons[END_REF] . With more and more data on such interactons integrated in an evolutonary framework, it will be possible to gain a beter understanding of when those interactons arose during the diversifcaton of the lineages concerned.

Conclusion: A new alliance with chemistry

It is tme to build a wider perspectve encompassing the diversity of the chemical building blocks of life and their specifcites in term of evolutonary mechanisms. The use of analytcal techniques such as high-resoluton mass spectrometry, in the feld of ancient proteomics and ancient lipidomics is already providing a very fruitul unifying factor, unearthing new types of molecular fossils [START_REF] Cappellini | Ancient biomolecules and evolutonary inference[END_REF] , but this move will necessitate the partcipaton of organic chemists eager to synthesize ancestral small molecules predicted through evolutonary analysis that will be used for biochemical experiments on ancient proteins.

Another area in which such an alliance between evolutonists and chemists could produce fruitul conceptual developments is the illustraton of metabolic pathways. Just as phylogenetc trees only provide a schematc view of the relatonships between animal or plant populatons, we believe that the metabolic reactons depicted as they currently are, as a suite of ordered reactons, is probably an oversimplifcaton. Developing network-like representatons may allow beter capture of the essence of the evolutonary process. We believe that it may be fruitul to think of a new way to represent, and therefore conceptualize, metabolic pathways. We strongly believe that incorporatng the evolutonary 9/12 reasoning as discussed here will be helpful for such an aim. All these developments, and probably many others, can only be done through a close collaboraton between molecular evolutonists, biochemists, and chemists. Afer Evo-Devo, let us start Evo-Chemo!

Figure 1 :

 1 Figure 1: Resurrecton of ancient protein and small molecule couples

Figure 2 .

 2 Figure 2. Four general principles regarding evoluton of metabolic pathways involved in small molecule biosynthesis.

Acknowledgements

We thank Tim Hunt, Catherine Jessus and Catherine Leblanc for stmulatng discussions that greatly helped us to sharpen our ideas. We also thank Marleen Klann, Sam Reiter, Ryota Kabe, Dino Moras, Agneesh Barua, Simon Ditami and Jonas Collén for critcal reading of this manuscript. Work from our laboratories is supported by OIST, Academia Sinica, CNRS and Sorbonne Université. GVM partcipated to the Natonal Research Agency investment expenditure program IDEALG (ANR-10-BTBR-04).