Richard G Dorrell
email: richard.dorrell.algae@gmail.com

Charlotte Nef

Setsen Altan-Ochir

Chris Bowler

Alison G Smith

Presence of vitamin B12 metabolism in the last common ancestor of land plants

Keywords: bryophytes, Anthocerotophyta, Marchantiophyta, OneKp, Chlamydomonas, phylogenomics

Vitamin B12, or cobalamin, (hereinafter B12) is an essential organic micronutrient, required by humans as a cofactor for methionine synthase (METH) and for methylmalonyl CoA mutase (MCM), involved in the propionate shunt. B12 is a complex corrinoid molecule made only by a subset of bacteria. Plants and fungi have an alternative methionine synthase (METE) that does not need a B12 cofactor, so these organisms are typically considered to neither synthesise nor utilise B12. In contrast many algal species utilise B12 if it is available, because they encode both METE and METH. Moreover, a large proportion of algal species encode METH only, and so are like animals in being dependent on an external source of the vitamin. Here, we performed a detailed phylogenetic analysis of the distribution of METE, METH and eleven further proteins implicated in B12 metabolism in eukaryotic cells across an exhaustive library of over 1,500 plant and algal genomes and transcriptomes. The results reveal the hitherto undetected existence of B12-associated metabolism deep into the streptophytes. The B12-dependent synthase METH, and the accessory proteins MTRR, CblB, CblC, CblD and CblJ were detected in the basally divergent plant lineage of hornworts, and CblB and CblJ were further identified in liverworts. Using phylogenetic and PFAM analysis we demonstrate this is due to retention of ancestral B12metabolism pathways in the last common ancestor of land plants, followed by at least two independent complete losses in mosses and vascular plants. We further show more limited distributions of genes encoding B12-related proteins across the algal tree of life, including MCM and type II ribonucleotide reductase, alongside an obligate B12-dependency across several major marine algal orders. Finally, by considering the functional biology of early-diverging land plants, together with the collection sites of ten further algal species inferred to have lost B12-dependent metabolism, we propose freshwater-to-land transitions and symbiotic associations to have been major constraining factors in B12 availability in early plant evolution.

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Introduction

Vitamins are organic micronutrients that are taken up by organisms from the environment and are essential for central metabolic processes [START_REF] Schneider | A dataset on trophic modes of aquatic protists[END_REF][START_REF] Croft | Algae need their vitamins[END_REF] . In particular, Bvitamins are the precursors to enzyme cofactors, providing additional chemical reactivity to that of amino acids. Plants, fungi and microorganisms also require these compounds for their metabolism, but are able to synthesise them de novo. The exception is for vitamin B12, a cobalt-containing corrinoid molecule that is not synthesised by any eukaryote [START_REF] Warren | The biosynthesis of adenosylcobalamin (vitamin B12)[END_REF][START_REF] Shelton | Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics[END_REF]. In humans, B12 is essential for two enzymatic activities: B12-dependent methionine synthase (METH) in the C1 cycle [START_REF] Yamada | Human methionine synthase reductase is a molecular chaperone for human methionine synthase[END_REF][START_REF] Banerjee | Cobalamin-dependent methionine synthase[END_REF] and methylmalonyl-coA mutase (MCM), responsible for the metabolism of odd-chain fatty acids and branched-chain amino acids via the propionate shunt [START_REF] Dobson | Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria[END_REF][START_REF] Banerjee | Methylmalonyl-CoA mutase[END_REF]. B12 deficiency and its associated pathologies (pernicious anaemia, methylmalonic acidemia) may be chronic in subsistence economies, with particularly adverse impacts on child development and during pregnancy [START_REF] Stabler | Vitamin B12 deficiency as a worldwide problem[END_REF][START_REF] Taneja | Cobalamin and folate status in infants and young children in a lowto-middle income community in India[END_REF][START_REF] Obeid | Cobalamin Status from pregnancy to early childhood: lessons from global experience[END_REF]. Addressing how to mitigate B12 deficiency in plant-based diets, which may have otherwise substantially lower environmental impacts than omnivorous nutrition, is therefore a key challenge to sustainably feeding a growing planetary population [START_REF] Niklewicz | The importance of vitamin B(12) for individuals choosing plantbased diets[END_REF][START_REF] Pimentel | Sustainability of meat-based and plant-based diets and the environment[END_REF][START_REF] Marrero | Reclaiming traditional, plant-based, climate-resilient food systems in small islands[END_REF].

Historically, B12 in the human diet has been obtained from different sources. These include from ruminant animals and their derivatives (i.e., dairy products) [15], the direct consumption of B12 from soil particles (e.g., via geophagia [START_REF] Reilly | Geophagia: why do humans consume soil?[END_REF], from fortified (nutritional yeast) and from edible seaweeds from across the algal tree of life [15,[START_REF] Marques De Brito | Vitamin B12 sources in non-animal foods: a systematic review[END_REF]. Indeed, many microalgae, microscopic photosynthetic eukaryotes, also encode METH and/or MCM and many are obligately dependent on B12 [START_REF] Croft | Algae acquire vitamin B12 through a symbiotic relationship with bacteria[END_REF][START_REF] Tang | Most harmful algal bloom species are vitamin B1 and B12 auxotrophs[END_REF]. In one study for example, 171/326 sampled algal species were found to be unable to grow in the absence of supplemented B12 [START_REF] Croft | Algae acquire vitamin B12 through a symbiotic relationship with bacteria[END_REF]. B12 auxotrophy has been subsequently documented to be widespread in specific algal phyla (e.g., haptophytes, dinoflagellates) across the Tree of Life [START_REF] Nef | How haptophytes microalgae mitigate vitamin B12 limitation[END_REF][START_REF] Lin | Vitamin B12-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes (metE)[END_REF]. Some microalgal lineages may encode further B12-dependent enzymes (e.g. form II ribonucleotide reductase (RNRII), first documented in the green microalga Euglena) [START_REF] Dobson | Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria[END_REF], which is not found in humans.

B12 biosynthesis involves over twenty discrete enzymatic steps from the common tetrapyrrole intermediate uroporphyrinogen III [START_REF] Raux | Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum[END_REF][START_REF] Raux | Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli[END_REF]. To date, complete B12 biosynthesis has only been described in a subset of bacteria and archaea [START_REF] Raux | Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum[END_REF][START_REF] Roessner | Multiple biosynthetic pathways for vitamin B12: variations on a central theme[END_REF]. Algae may acquire synthesised B12 by scavenging from the environment [START_REF] Droop | Vitamins, phytoplankton and bacteria: symbiosis or scavenging?[END_REF], phagotrophic consumption of B12-containing organisms [START_REF] Sanders | Relationship between phototrophy and phagotrophy in the mixotrophic chrysophyte Poterioochromonas malhamensis[END_REF] or via symbiotic exchanges with B12producing commensals [START_REF] Croft | Algae acquire vitamin B12 through a symbiotic relationship with bacteria[END_REF]. Additionally, there are a number of B12 variants with different axial ligands, which affects the bioavailability of the vitamin. Intrinsic factor, the B12-binding protein found in the human ileum, has a much higher affinity for cobalamin, where the lower axial ligand is 5,6-dimethylbenzimidazole (DMB), than for pseudocobalamin, which has adenine as the lower axial ligand [START_REF] Helliwell | Cyanobacteria and eukaryotic algae use different chemical variants of vitamin B12[END_REF]. Eukaryotic microalgae have a similar preference for cobalamin over pseudocobalamin [START_REF] Helliwell | Cyanobacteria and eukaryotic algae use different chemical variants of vitamin B12[END_REF], although a B12 remodelling pathway has been documented in some species [START_REF] Helliwell | Cyanobacteria and eukaryotic algae use different chemical variants of vitamin B12[END_REF][START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF], which enables them to convert pseudocobalamin to cobalamin if supplied with DMB.

Little is known of the uptake, cellular transport or metabolism of B12 in algae, but the pathways are well documented in humans. Following its endocytic internalisation into the cell, cobalamin is released into the cytosol via the co-operative activity of a cytosolic chaperone CblC and the lysosomal transporters CblF/ CblJ [START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF] (Fig. 1). This involves the reductive removal of any preceding upper-axial ligands associated with the cob(III)alamin and conversion into cob(I)-and cob(II)alamins complexed with CblC [START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF]. Cob(I,II)-alamins complexed with CblC can then be transferred to the conjugate protein CblD, which subsequently directs the synthesis of different cobalamin-dependent enzymes [START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF][START_REF] Coelho | Gene identification for the cblD defect of vitamin B12 metabolism[END_REF]. METH requires methyl-cobalamin, produced from CblD-associated B12 by the activity of methionine synthase reductase (MTRR) [START_REF] Yamada | Human methionine synthase reductase is a molecular chaperone for human methionine synthase[END_REF] in the cytosol, whereas synthesis of MCM involves adenosyl-cobalamin, produced in the mitochondria via the conjugate activity of the probable B12 transporter protein CblA [START_REF] Dobson | Identification of the gene responsible for the cblA complementation group of vitamin B12-responsive methylmalonic acidemia based on analysis of prokaryotic gene arrangements[END_REF] and the adenosyltransferase CblB [START_REF] Dobson | Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria[END_REF]. A small number of additional proteins (e.g., CblX, epi-CblC) have been shown to have epistatic effects on cobalamin uptake in humans, but are of unknown function [START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF]. Finally, in the algal species in which it occurs, RNR-II is cytosolic and utilises adenosyl-cobalamin. B12 produced by bacteria may be scarce in the environment, including in soil [START_REF] Hutner | Biochemical geography: some aspects of recent vitamin research[END_REF] and large portions of the contemporary ocean [START_REF] Bertrand | Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein[END_REF][START_REF] Barber-Lluch | Variability of vitamin B12 concentrations in waters along the Northwest Iberian shelf[END_REF], and uptake and processing of the vitamin is energetically costly [START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF][START_REF] Froese | Vitamin B12, folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation[END_REF]. In contrast to the widespread utilisation of B12 by aquatic algae, several ecologically successful groups of eukaryotes, including all previously documented land plants, have lost their metabolic dependence on B12 [START_REF] Helliwell | Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes[END_REF]. This is possible because of a B12-independent methionine synthase (METE) that arose separately, which can replace METH, albeit with a lower catalytic activity [START_REF] Gonzalez | Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem[END_REF]. The METE gene shows a discontinuous presence across the tree of life, and is also present in many METH-containing species, including a substantial proportion of eukaryotic algae, which renders them facultative B12 users [START_REF] Helliwell | Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes[END_REF]. The most parsimonious explanation is an ancestral presence of both METE and METH genes in eukaryotes, with the repeated loss of METE from obligately B12-dependent species, and the occasional loss of METH from plants, fungi, and other species that do not have B12-associated metabolism.

In the last five years, the dramatic expansion in plant and algal genomic and transcriptomic resources, such as through the OneKp project [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Pierella Karlusich | Modèles biogéographiques et génomes des photoautotrophes aquatiques[END_REF], have provided unprecedented insights into the foundational events underpinning plant evolution. This includes evidence for the stepwise accumulation of evolutionary innovations (e.g., homeobox domain transcription factors, auxin transport pathways and water-stress tolerance factors) associated with the colonisation of land in the closest streptophyte relatives of land plants [START_REF] Wang | Genomes of early-diverging streptophyte algae shed light on plant terrestrialization[END_REF][START_REF] Buschmann | Understanding the algae to land plant transition[END_REF][START_REF] Skokan | PIN-driven auxin transport emerged early in streptophyte evolution[END_REF], and the importance of gene losses as well as gene family expansions for the post-terrestrial diversification of both bryophyte (mosses, liverworts, and hornworts) and vascular plants [START_REF] Harris | Divergent evolutionary trajectories of bryophytes and tracheophytes from a complex common ancestor of land plants[END_REF][START_REF] Li | Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts[END_REF]. These expanded genomic resources prompted us to revise our current understanding of the distribution of B12dependent metabolism across the photosynthetic eukaryotic tree of life. Here we investigate the present of B12 uptake and associated metabolism in over 1,600 published plant and algal genome and transcriptome libraries, and consider in which ecological contexts loss of B12-dependent metabolism has occurred.

Results

Distribution of METH/ METE genes indicate B12 presence in hornworts

We identified homologues of the thirteen B12-associated proteins, illustrated in Fig. 1, from the algae Chlamydomonas reinhardtii, Euglena gracilis and Phaeodactylum tricornutum and from humans (Table 1), and used these to search a composite library of 1,666 plant and algal genomes and transcriptomes. The library contained: 44 algal and plant genomes, including the recently published draft genomes of the hornworts Anthoceros agrestis and A. punctigera [START_REF] Li | Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts[END_REF][START_REF] Grigoriev | PhycoCosm, a comparative algal genomics resource[END_REF]; decontaminated versions of the 1,000 plant transcriptomes (OneKp ; 1,292 libraries) [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Carpenter | Access to RNA-sequencing data from 1,173 plant species: The 1000 Plant Transcriptomes initiative (1KP)[END_REF] and the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP; 300 libraries) [START_REF] Keeling | The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing[END_REF][START_REF] Marron | The evolution of silicon transport in eukaryotes[END_REF]; and 26 further eukaryotic transcriptomes, in particular sampling ecologically abundant marine diatom genera [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF] and freshwater chrysophyte lineages [START_REF] Beisser | Comprehensive transcriptome analysis provides new insights into nutritional strategies and phylogenetic relationships of chrysophytes[END_REF] (Table 2). Homologues were retrieved by reciprocal BLAST best-hit (RbH), single-gene RAxML trees, and PFAM domain analysis (Materials and Methods) [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF][START_REF] Mistry | Pfam: the protein families database in 2021[END_REF][START_REF] Moreno-Hagelsieb | Choosing BLAST options for better detection of orthologs as reciprocal best hits[END_REF], and were categorised taxonomically following recently published multigene phylogenies of plant and algal diversity [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. Full outputs are provided in Dataset S1.

The phylogenetic distributions of putative homologues are shown schematically in Fig. 2 for each protein. The patterns underline the widespread occurrence of the B12dependent form of methionine synthase, METH, across eukaryotic algae, found in all major marine groups, and in both early-branching (Klebsormidiaceae, Mesostigmatophyceae, Chlorokybophyceae) and close relatives of land plants (Zygnematophyceae, Charophyceae, Coleochaetophyceae) within the streptophyte lineage [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Hori | Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation[END_REF]. METH was further detected in multiple distantly related hornwort genera, suggesting widespread conservation of B12-dependent methionine synthases across the class Anthoceratophyta (Dataset S1, sheet 5) [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Villarreal | Hornwort pyrenoids, carbon-concentrating structures, evolved and were lost at least five times during the last 100 million years[END_REF]. These included OneKp homologues from the hornwort genera Megaceros (M. tosanus, OneKp transcript-UCRN-2004435; M. vincentianus-TCBC-2004163), Nothoceros (N. aenigmaticus DXOU-2038410), Paraphymatceros (Paraphymato. hallii: FAJB-2057847) and Phaeoceros (Phaeo. carolinianus, RXRQ-2022853, RXRQ-2022854), as well as probable METH genes from the Anthoceros agrestis Bonn (geneID: Sc2ySwM_228_5027); A. agrestis Oxford (geneID: utg000003l_252) and A. punctigera (geneID: utg000098l_165) genomes. METH was not found elsewhere within the land plant lineage, except for one potential METH homologue identified by RbH in the liverwort Blasia sp. (OneKp transcript: AEXY-2015053; Dataset S1, sheet 5), but that resolved during preliminary phylogenetic analyses with bacterial sequences and showed high sequence similarity with a Mucilaginibacter METH (NCBId: WP_129570389.1; >96% by BLASTp) [START_REF] Dixon | The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12[END_REF]. Previous studies have documented high levels of bacterial contamination in the Blasia transcriptome, [START_REF] Carpenter | Access to RNA-sequencing data from 1,173 plant species: The 1000 Plant Transcriptomes initiative (1KP)[END_REF], and we therefore consider that this is unlikely to correspond to a liverwort METH.

The phylogeny of METH sequences robustly resolved a position for the hornwort sequences within the streptophytes (RAxML bootstrap support : 100%), in a weakly supported sister-group position to homologues from the charophytes / coleochaetales (Chara vulgaris, Chaetosphaeridium globosum, Coleochaete scutata, Col. irregularis) (Fig. 3 ; Dataset S1). This group was positioned more deeply within Viridiplantae sequences, as a sister to equivalent sequences from Chlorophytes (RAxML bootstrap support: 100% ; Fig. 3). Each of the phylogenetically validated Megaceros, Nothoceros, Phaeoceros and Paraphymatoceros hornwort OneKp sequences possessed all five PFAMs typically associated with METH (homocysteine S-methyltransferase, PF02574 ; pterinbinding, PF00809 ; B12-binding, PF02607 and PF02310 ; and the activation domain, PF02965) with similar e-values to functionally characterised equivalents from algae (Fig. S1) [START_REF] Lin | Vitamin B12-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes (metE)[END_REF]. The three identified Anthoceros homologues possessed all PFAMs apart from the first B12-binding (PF02607) domain. However, this PFAM is detected in alternative gene models in each genome (A. agrestis Bonn: 362_443; A. agrestis Oxford: utg000003l_664; A. punctigera, utg000145l_284), suggesting that Anthoceros is likely to also possess a functionally active METH. The hornwort sequences all had conserved active sites (e.g., the substrate-binding pocket of the pterin-binding site) associated with METH activity (Fig. S1) [START_REF] Lin | Vitamin B12-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes (metE)[END_REF][START_REF] Drennan | How a protein binds B12: a 3.0 Å X-ray structure of B12-binding domains of methionine synthase[END_REF].

We also considered the distribution of the methionine synthase reductase (MTRR) necessary for METH activity in our dataset. Well-conserved homologues of MTRR were detected across hornwort genomes and transcriptomes, but were absent from other plant groups (Fig. 2). As per the situation for METH, the hornwort MTRR genes grouped with other streptophyte sequences within the Viridiplantae (Fig. 3), and had well-conserved PFAM domains (flavodoxin 1-PF00258 , FAD-binding-PF00667 , oxidoreductase-PF00175 ; Fig. S1), suggesting vertical origin and functionality. We therefore conclude that the hornwort METH sequences are likely to be catalytically functional, and vertically inherited from the streptophyte ancestor of terrestrial plants.

Widespread occurrence of two distinct METE isoforms in plants

All plant libraries studied possessed METE genes, including hornworts, indicating that both genes were present in the last common plant ancestor (Fig. 2). The hornwort METE genes resolved with other plant homologues (Fig. 3) and possessed well-conserved N-terminal (PF08267) and C-terminal catalytic (PF01717) PFAM domains (Fig. S1), indicating their likewise probable vertical origin and functionality.

The plant METE enzymes resolved phylogenetically in two discrete families: a conventional isoform with 1,768 recovered examples (labelled "Clade 1"); and a less frequently observed isoform ("Clade II") with 125 observed examples (Fig. 3 ; Dataset S1). Both isoforms contained credible occurrences of both Meth_synt_1 (PF08267) and Meth_synt_2 (PF01717) domains, and thus are likely to function as methionine synthases (Fig. S1 ; Dataset S1). Both Clade I and Clade II isoforms were furthermore detected in higher plant, moss, liverwort, hornwort and streptophyte sequences (Fig. 3), suggesting their presence in the last plant common ancestor. All three characterised Arabidopsis methionine synthases (ATMS1-At5g17920; ATMS2 At3g03780; ATMS3-At5g20980) resolved within Clade 1, but the Clade II enzymes all showed greater proximity by BLASTp analysis to these than to the four closely related Arabidopsis adenosyl-methionine synthases (SAM1-At1g02500; SAM2-At4g01850; SAM3-At3g17390; and SAM4-At2g36880), implying that they likewise correspond to probable methionine synthase isoforms (Dataset S1, sheet 6) [START_REF] Joshi | Arabidopsis methionine gamma-lyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase[END_REF].

Both Clade I and Clade II isoforms distantly positioned in the phylogeny to chlorophyte METE sequences, which formed a sister-group to red algae, cryptomonads and chromerids (Fig. 3). The most parsimonious explanation for this distribution would be the ancestral replacement of the streptophyte METE sequence, still retained in chlorophytes, with the Clade I and Clade II isoforms, preceding the loss of METH within specific plant groups.

Distribution of METE genes confirms widespread B12 auxotrophy in algae

In contrast to the widespread distribution of both METE isoforms in land plants, METE was absent from many of the algal libraries searched (Fig. 2). Transcriptome libraries may under-report the presence of METE, as it may be transcriptionally repressed in B12-supplemented cultures (most common algal growth media include exogenous B12, either in the form of soluble vitamins or soil extract) [START_REF] Gonzalez | Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem[END_REF]59], and therefore we limit our consideration to lineages with at least one sequenced genome included [START_REF] Pierella Karlusich | Modèles biogéographiques et génomes des photoautotrophes aquatiques[END_REF]. Consistent with previous studies [START_REF] Nef | How haptophytes microalgae mitigate vitamin B12 limitation[END_REF], we project B12 auxotrophy in all haptophytes (including three genomes included in the study : Emiliania huxleyi, Chrysochromulina tobinii, and Pavlovales sp. CCMP2436) [START_REF] Dorrell | Convergent evolution and horizontal gene transfer in Arctic Ocean microalgae[END_REF]. Only one potential haptophyte METE homologue (from Pavlova lutheri UTEX LB1293) was found by RbH, but was excluded as a probable green algal contaminant (Dataset S1). No occurrences of METE were found in the prasinophyte class Mamiellophyceae (including the Micromonas and Ostreococcus sp. genomes) or the ochrophyte class Pelagophyceae (including two genomes : Aureococcus anophageferrens, and Pelagophyceae sp. CCMP2097) [START_REF] Dorrell | Convergent evolution and horizontal gene transfer in Arctic Ocean microalgae[END_REF] (Fig. 2). The shared absence of METE from haptophytes, pelagophytes and Mamiellophyceae is particularly interesting given their environmental abundance as assessed e.g., via the Tara Oceans expedition [START_REF] Penot | Genomic and meta-genomic insights into the functions, diversity and global distribution of haptophyte algae[END_REF][START_REF] Pierella Karlusich | A robust approach to estimate relative phytoplankton cell abundances from metagenomes[END_REF], underlining the importance of B12 acquisition for marine photosynthesis.

Further instances of obligate B12 requirements, due to the complete absence of METE, were found in less ecologically abundant algal groups and at more phylogenetically localised scales, including most immediate relatives of the model green algal species C. reinhardtii [START_REF] Grant | Direct exchange of vitamin B12 is demonstrated by modelling the growth dynamics of algal-bacterial cocultures[END_REF][START_REF] Bunbury | Responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation[END_REF] and the glaucophytes and Palmophyllphyceae, respectively considered as basally-divergent members of primary chloroplastcontaining algae and the Viridiplantae [START_REF] Price | Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants[END_REF][START_REF] Li | The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants[END_REF]. Finally, dinoflagellates (including three genomes from Symbiodinium sp.) were found to possess only partial METE sequences, which lacked the conventional N-terminal PFAM domain (MetH_synt_1 ; PF08267). The function of this partial METE sequence isoform awaits experimental validation, although B12 dependency has been proposed to be widespread across the dinoflagellates [START_REF] Lin | Vitamin B12-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes (metE)[END_REF][START_REF] Nef | Cobalamin scarcity modifies carbon allocation and impairs DMSP production through methionine metabolism in the haptophyte microalgae Tisochrysis lutea[END_REF] (Dataset S1). We did, however, identify complete METE genes containing both PFAM domains in the chromerid algae Chromera velia and Vitrella brassicaformis, which are the closest obligately photosynthetic relatives of dinoflagellates within the alveolates [START_REF] Oborník | Morphology, ultrastructure and life cycle of Vitrella brassicaformis n. sp., n. gen., a novel chromerid from the Great Barrier Reef[END_REF]. The dinoflagellate METE-like genes were distantly related to all other equivalents and instead resolved with Archaea (Fig. 3), which likewise lacked the N-terminal PFAM domain. This may suggest a horizontal acquisition of the current dinoflagellate METE-like gene that may have been accompanied by the loss of the complete METE isoform.

Finally, the METE genes of the red algal genera Porphyridium and Cyanidioschyzon, as well as in the chrysophyte Poteriospumella, resolved with homologues from bacteria (Dataset S1 ; Fig. 3), independent to all other plant and algal proteins. In each case, these acquisitions were shared between multiple related species or strains, and are therefore likely to be genuine bacterial horizontal gene transfers as opposed to contaminants. Overall, these data indicate complex patterns of METE gene transfer and loss across the photosynthetic eukaryote tree of life.

MCM and RNR II enzymes are restricted to individual algal lineages

Alongside methionine synthase, we considered the distributions of the B12dependent MCM and RNR II enzymes across our dataset (Fig. 2). Neither enzyme was detected in hornworts or more broadly across streptophytes, and were absent from the chlorophytes, except for some sporadic occurrences that are likely consistent with horizontal acquisition as opposed to an ancestral presence in Viridiplantae (discussed below). Homologues of MCM were primarily detected in eukaryotic algae with secondary red chloroplasts (e.g., diatoms. cryptomonads, haptophytes, ochrophytes), where they typically possessed well-conserved PFAM domains, and in many cases predictable mitochondrial targeting sequences consistent with mitochondrial function (Dataset S1 ; Fig. S2). MCM is implicated in the catabolism of branched-chain amino acids in diatoms under nitrate exhaustion [START_REF] Kroth | A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis[END_REF][START_REF] Ge | Methylcrotonyl-CoA Carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum[END_REF], and has further been proposed to participate in the dissipation of excess diatom plastidial reducing potential [START_REF] Ait-Mohamed | PhaeoNet: a holistic RNAseq-based portrait of transcriptional coordination in the model diatom Phaeodactylum tricornutum[END_REF]. We note that a previous RNAi study of Phaeodactylum MCM failed to identify a clear mutant phenotype [START_REF] Ge | Methylcrotonyl-CoA Carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum[END_REF], so it remains to be determined to what extent MCM activity is essential to algae with secondary red chloroplasts, and therefore whether it provides a reason alongside METH for widespread B12 dependence across these groups in the environment.

We additionally detected MCM in a small number of glaucophytes, red algae (Galdieria sulphuraria) and green algae within the genera Tetraselmis and Chlorella (Fig. 2). In contrast to green algal methionine synthase-associated enzymes, whose phylogenies show typically vertical inheritances, the MCM phylogeny retrieve a sistergroup relationship between green algae and haptophytes (Fig. 3), implying a probable ancestral loss of the propionate shunt within the Viridiplantae, and subsequent reacquisition by horizontal gene transfer in specific lineages.

Similarly, the B12-dependent type II ribonucleotide reductase (RNR II), first detected in eukaryotes in the secondary green chloroplast-containing alga Euglena gracilis [START_REF] Helliwell | Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes[END_REF][START_REF] Carell | Ribonucleotide reductase activity in vitamin B12-deficient Euglena gracilis[END_REF] was also found in the distantly related haptophytes and chlorarachniophytes, which have secondary green chloroplasts, reflecting possible ancestral horizontal gene transfers inferred between all three lineages [START_REF] Vanclová | Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid[END_REF]. Intriguingly it is also present in the Chlamydomonadales (although not C. reinhardtii), where it has most likely been acquired by an independent horizontal gene transfer from bacteria (Figs. 2,3).

Distribution of uptake proteins suggests deeper retention of B12-dependent metabolism within the bryophytes

We further considered the distribution of six proteins associated with the uptake and intracellular transport of B12 (CblA, B, C, D, F, and J) and two known epistatic regulators in humans (CblX, epi-CblC) [START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF] across our dataset. Consistent with the distribution of METH and MTRR, we could identify widespread presence of CblC, D and J proteins in hornwort libraries (Fig. 2). The hornwort sequences resolved within clades of other Viridiplantae sequences, and typically contained well-conserved PFAM domains (Fig. 4 ; Fig. S1), suggesting vertical origin and conserved function. The exact location of these enzymes, and precise cellular trafficking pathway in which they may be involved remain to be determined experimentally, as very few carried credible signal peptides that might suggest targeting to the endomembrane system, and participation in a B12 endosomal uptake pathway (Fig. S2).

The only protein for which we could identify consistent endomembrane and/ or vacuolar targeting predictions amongst our recovered homologues was for CblF (Fig. S2), but this was almost exclusively detected amongst dinoflagellates in our dataset (Figs. 2,4). We therefore tentatively propose that the B12 uptake and intracellular trafficking pathways may not be identical across different branches of the eukaryotic tree of life. We could detect potential homologues of both CblX and epi-CblC in hornworts, but found these also in vascular plants and with no clear underlying phylogenetic signal, so we are not confident of the function of these enzymes in B12associated hornwort metabolism (Figs. 2,4).

Surprisingly, homologues of CblB, an enzyme responsible for the synthesis of adenosyl-cobalamin [START_REF] Dobson | Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria[END_REF], were found in hornworts (Figs. 2,S1). This was despite the absence of MCM, which typically uses adenosyl-cobalamin, alongside the CblB partner enzyme CblA from the streptophytes (Fig. 2) [START_REF] Dobson | Identification of the gene responsible for the cblA complementation group of vitamin B12-responsive methylmalonic acidemia based on analysis of prokaryotic gene arrangements[END_REF]. We could further detect homologues of CblB and indeed CblJ in liverworts, and in both cases the homologues from these lineages grouped monophyletically with hornworts (Fig. 4) and possess well-conserved PFAM domains (Fig. S1). It is therefore possible that some form of adenosyl-cobalamin dependent metabolism persisted in the last common plant ancestor, and that this metabolism is retained not only by hornworts but by liverworts within the bryophyte lineage. The validation of this pathway will depend on the functional characterisation of putative adenosyl-cobalamin dependent enzymes, e.g., from published liverwort and hornwort genomes [START_REF] Li | Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts[END_REF][START_REF] Bowman | Insights into land plant evolution garnered from the Marchantia polymorpha genome[END_REF].

Rare occurrences of B12-independent algae are biased towards terrestrial and symbiotic species

Finally, we considered across our global datasets which photosynthetic eukaryotes, other than plants, may have secondarily lost B12-dependent metabolism completely. Considering B12 independence as the presence of METE but absence of METH/ MTRR, RNR II or MCM activities, we identify seven phylogenetically validated occurrences of the loss of B12-associated metabolism (Table 2). For four species, the same distributions could be identified from PFAM domains only. Finally, we identified four species that possess METE and either MCM or RNR II, but lack METH/MTRR activities, suggesting a loss of B12-related methionine synthesis (Table 2).

The widespread but seemingly random loss of METE led us to consider what environmental factors might be involved in driving the distribution of this trait, which might be considered a priori deleterious. An interactive map of all collection sites reported for our dataset is directly available at https://tinyurl.com/3esfpkmv. The B12independent species in our dataset (blue points on interactive map, and listed in Table 2) show characteristic distributions considering their collection sites, mainly originating from freshwater (e.g., Leptosira obovata, Porphyridium cruentum, Poteriospumella JBC07) or terrestrial habitats (Coccomyxa pringsheimii, Cyanidioschyzon merolae, Galdieria sulphuraria). B12-producing bacteria are abundant in some freshwater habitats (e.g., in sediments, as fish gut commensals) but appear to be present in much lower quantities in the water column (e.g., 10 3 -fold less than in sediments), calling into question the bioavailability of B12 to freshwater algae [START_REF] Sugita | The vitamin B12-producing bacteria in the water and sediment of a carp culture pond[END_REF][START_REF] Tsuchiya | Novel ecological niche of Cetobacterium somerae, an anaerobic bacterium in the intestinal tracts of freshwater fish[END_REF]. It is possible in some habitats that this is due to cobalt scarcity [START_REF] Facey | Assessing the importance of cobalt as a micronutrient for freshwater cyanobacteria[END_REF], but is not universally the case. We similarly note that Geminigera cryophila, which not only lacks METH but is also the only cryptomonad within our dataset to encode METE, was collected from Antarctic water masses, which have been previously shown to be characterised by vitamin B12limitation and co-limitation (Fig. 1) [START_REF] Browning | Nutrient co-limitation at the boundary of an oceanic gyre[END_REF][START_REF] Taylor | A new cryptomonad from antarctica: Cryptomonas cryophila sp[END_REF]. Finally, many of the B12-independent algal species are endobiotic to other organisms (Leptosira obovata and two members of the order Trentepohliales, isolated from lichens [START_REF] Kosecka | Trentepohlialean Algae (Trentepohliales, Ulvophyceae) show preference to selected mycobiont lineages in lichen symbioses[END_REF]; Vitrella brassicaformis, a coral symbiont [START_REF] Oborník | Morphology, ultrastructure and life cycle of Vitrella brassicaformis n. sp., n. gen., a novel chromerid from the Great Barrier Reef[END_REF]; and Brandtodinium nutriculum, a foraminiferan symbiont [START_REF] Probert | Brandtodinium gen. nov. and B. nutricula comb. Nov. (Dinophyceae), a dinoflagellate commonly found in symbiosis with polycystine radiolarians[END_REF]), and may be subject to chronic B12 limitation in their native habitats, or even potentially receive methionine and other key metabolites such as folate directly from their hosts.

Discussion

In this study, we query the distribution of thirteen B12-associated proteins across more than 1,600 genomes and transcriptomes from plastid-containing eukaryotes (plants and eukaryotic algae), in particular profiting from the substantial advance in plant genomics made through the OneKp transcriptome project [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF] (Fig. 1, Table 2). Our data suggest the hitherto undocumented retention of vertically inherited B12associated methionine synthesis pathways (METH, MTRR Figs. 2,3) and B12 uptakeassociated proteins (CblB, C D, J ; Figs. 2,4) in hornworts, and the further retention of potential CblB and CblJ homologues in liverworts (Fig. 4). Formally, the active use of B12 by hornworts awaits functional characterisation. This may be best performed by bioassay of B12 content in hornwort tissue [START_REF] Raux | Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli[END_REF], but might also come from the experimental inference of B12 use, via B12-dependent downregulation of hornwort METE expression [START_REF] Gonzalez | Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem[END_REF], or the uptake of fluorescently-labelled B12 analogues [START_REF] Lawrence | Construction of fluorescent analogs to follow the uptake and distribution of cobalamin (vitamin B12) in bacteria, worms, and plants[END_REF] by hornwort cells.

Considering the predicted functionality and vertical origin of the hornwort homologues, our data indicate the probable presence of B12-dependent metabolism in the last common plant ancestor following the transition to land, with subsequent losses in mosses, vascular plants, and (dependent on the functions of the detected CblB and CblJ homologues), liverworts (Fig. 5). It remains to be determined at what point B12dependent metabolism was lost during land plant evolution. Some phylogenetic studies have proposed hornworts as the earliest-diverging land plant group [START_REF] Wickett | Phylotranscriptomic analysis of the origin and early diversification of land plants[END_REF][START_REF] Cox | Land plant molecular phylogenetics: a review with comments on evaluating incongruence among phylogenies[END_REF], which would imply a single loss of METH in a common ancestor of liverworts, mosses and vascular plants. Other studies, including the multigene phylogenomic analyses performed as part of the Anthoceros genome and OneKp transcriptome projects [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Harris | Divergent evolutionary trajectories of bryophytes and tracheophytes from a complex common ancestor of land plants[END_REF][START_REF] Li | Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts[END_REF], recover well-supported monophyly of bryophytes, to the exclusion of vascular plants (Fig. 5). This topology would indicate independent losses of B12-dependent metabolism in vascular plants and mosses, with a potential third loss of use of methylcobalamin, but retention of MCM and its requirement for adenosyl-cobalamin, in liverworts (Fig. 5). In either case, the presence of a metabolic activity in hornworts previously considered unique to algae further blurs the biological distinctions between aquatic streptophytes and plants. This is alongside the retention of biophysical carbon concentrating mechanisms in hornworts [START_REF] Villarreal | Hornwort pyrenoids, carbon-concentrating structures, evolved and were lost at least five times during the last 100 million years[END_REF][START_REF] Meyer | To concentrate or ventilate? Carbon acquisition, isotope discrimination and physiological ecology of early land plant life forms[END_REF], and the preceding evolution of plantlike auxin transporters and homeobox transcription factors in charophytes [START_REF] Buschmann | Understanding the algae to land plant transition[END_REF][START_REF] Skokan | PIN-driven auxin transport emerged early in streptophyte evolution[END_REF].

Finally, it remains to be determined why B12-dependent metabolism was lost in early plant lineages. Many hornwort species are characterised by the presence of cyanobacterial symbionts, which might produce pseudocobalamin; this is not bioavailable to microalgal species [START_REF] Helliwell | Cyanobacteria and eukaryotic algae use different chemical variants of vitamin B12[END_REF][START_REF] Adams | Cyanobacteria in symbiosis with hornworts and liverworts, in Cyanobacteria in Symbiosis[END_REF]. It remains to be determined whether hornworts can assimilate pseudocobalamin and/ or remodel it into cobalamin, and if this is more bioavailable to them than other plant lineages from which METH has been lost. Or indeed if the plant microbiome has played a broader role in the loss of cobalamin-dependent metabolism. Considering the widespread distribution of B12-independent metabolism across the algal tree of life (Table 2), we note repeated losses of METH and its associated enzymes in other freshwater, terrestrial and symbiotic lineages. Thus comparative genomic and physiological studies of these species may elucidate the ecological reasons for the loss of B12-associated metabolism both in algae and land plants. Understanding why plants do not utilise B12-dependent enzymes is particularly important due to the prevalence of vitamin B12 insufficiency/deficiency in populations consuming plant-based diets [START_REF] Stabler | Vitamin B12 deficiency as a worldwide problem[END_REF]. Ultimately, understanding the significance of B12 utilisation and acquisition across algal biology may faciliate the synthetic reintroduction of B12 uptake into transformable crop species, or the mass cultivation of algae for dietary consumption [START_REF] Lawrence | Construction of fluorescent analogs to follow the uptake and distribution of cobalamin (vitamin B12) in bacteria, worms, and plants[END_REF][START_REF] Mozafar | Enrichment of some B-vitamins in plants with application of organic fertilizers[END_REF], sustainably feeding the human planetary population [START_REF] Pimentel | Sustainability of meat-based and plant-based diets and the environment[END_REF]. Euglena RNR II : Q2PDF6.1) [START_REF] Helliwell | Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes[END_REF] were searched across a composite library of 1,663 non-redundant plant and algal genomes and transcriptomes (Table 2 ) [START_REF]One Thousand Plant Transcritomes Initiative, One thousand plant transcriptomes and the phylogenomics of green plants[END_REF][START_REF] Keeling | The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Beisser | Comprehensive transcriptome analysis provides new insights into nutritional strategies and phylogenetic relationships of chrysophytes[END_REF] by BLASTp with threshold e-value 10 -05 [START_REF] Altschul | Basic local alignment search tool[END_REF]. Potential matching sequences were extracted and searched by BLASTp against the complete Arabidopsis thaliana genome [START_REF]Analysis of the genome sequence of the flowering plant Arabidopsis thaliana[END_REF], which uniquely encodes METE, supplemented with the query sequences defined above. Sequences that retrieved a best-scoring match to a query sequence were retained for downstream phylogenetic analysis (Dataset S1). For METE which is retained in b-plants, genes that retrieved one of the three Arabidopsis methionine synthases (ATMS1-At5g17920; ATMS2 At3g03780; ATMS3-At5g20980) were likewise retained for downstream analysis [START_REF] Joshi | Arabidopsis methionine gamma-lyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase[END_REF][START_REF] Yan | METHIONINE SYNTHASE1 is involved in chromatin silencing by maintaining DNA and histone methylation[END_REF].

Materials and Methods

Homologue

Phylogeny : Inferred homologues were aligned against the query sequence, and bestscoring homologues obtained from parallel BLASTp searches of 51 combined genome and transcriptome libraries corresponding to different prokaryotic and nonphotosynthetic eukaryotic taxonomic groups from across the tree of life [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF] by MAFFT v 7.487 using the -gt auto setting [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. The resulting alignments were imported into GeneIOUS v 10.0.9 and initially screened using the in-built NJ tree function with 100 replicates and random starting seeds; and highly divergent branches (defined visually as branches with > 1.0 calculated substitutions/ site) were iteratively removed [START_REF] Kearse | Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF]. The curated alignment was then trimmed with trimal v 1.4 using the -gt 0.5 setting, prior to a second round of tree building and manual curation [START_REF] Capella-Gutiérrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF]. Curated trimmed alignments were then inspected with RAxML v 8.0 using the PROTGAMMAJTT subsitution matrix and 350 bootstrap replicates, following the methodology of previous studies [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]; and the best-scoring tree was then inspected for branches of contaminant origin (defined as sequences from one single library that resolved within or as a sistergroup to a distantly related clade, e.g. algal sequences that resolved with bacterial homologues) [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF]. The full length sequences of homologues that passed the initial RAxML phylogenetic curation were finally passed through a second iteration of mafft alignment, manual curation, and RAxML phylogeny, and confirmed to resolve in topologies coherent with protein function and taxonomy prior to enumeration of homologue presence/ absence (Dataset S1). Functional, targeting and biogeographical annotation : PFAM domains were searched in each homologue retrieved by the initial RbH search by HMMER v 3.3.2 against the Pfam v 35.0 library [START_REF] Mistry | Pfam: the protein families database in 2021[END_REF][START_REF] Potter | HMMER web server: 2018 update[END_REF]. Sequences that retrieved PFAM domains associated with each query protein were recorded (METE : Meth_synt_1-PF08267, Meth_synt_2-PF01717; METH : B12-binding-PF02310, B12-binding_2-PF02607, Met_synt_B12-PF02965, Pterin_bind-PF00809, S-methyl_trans-PF02574 ; CblA : MeaB-PF03308 ; CblB : Cob_adeno_trans-PF01923 ; CblC : MMACHC-PF16690 ; CblD : MMADHC-PF10229 ; CblF : LMBR1-PF04791 ; CblJ : ABC_tran-PF00005 ; CblX : Kelch_1-PF01344, Kelch_2-PF07646, Kelch_3-PF13415, Kelch_4-PF13418, Kelch_5-PF13854, Kelch_6-PF13964 ; epi-CblC : AhpC_TSA-PF00578, Redoxin-PF08534, 1-cysPrx_C-PF10417MCM : MM_CoA_mutase-PF01642 ; MTRR : Flavodoxin_1-PF00258, FAD_binding_1-PF00667, NAD_binding_1-PF00175 ; RNR II-Ribonuc_red_lgC-PF02867, RNR II_Alpha-PF17975) with threshold e-value 10 -05 . Localisations for each protein were inferred, considering both the full length of the protein sequence and the protein sequence trimmed to the first annotated methionine, using WolfPSort v 1.0 with plant substitution matrix [START_REF] Horton | WoLF PSORT: protein localization predictor[END_REF]; SignalP v 5.0 under eukaryotic settings [START_REF] Almagro Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF]; TargetP v 2.0 with plant substitution matrix [START_REF] Almagro Armenteros | Detecting sequence signals in targeting peptides using deep learning[END_REF]; and HECTAR v 1.0 under default settings [START_REF] Gschloessl | HECTAR: a method to predict subcellular targeting in heterokonts[END_REF]. Collection sites were recorded for each algal species using the corresponding culture collection records considering strain synonyms, and where appropriate direct information from the literature or collector [START_REF] Gachon | The CCAP KnowledgeBase: linking protistan and cyanobacterial biological resources with taxonomic and molecular data[END_REF][100][101][102][103][104][105][106][107]. PFAM boxplots in Fig. S1 are displayed using BoxPlotR [108].

Data deposition : All strain information, recovered homologues by RbH, alignments, phylogenetic topologies, PFAM and targeting annotations are provided in Dataset S1. An interactive map of all algal collection sites identified within the dataset, shadeable either via phylogenetic affiliation or inferred Vitamin B12 metabolic status, is available via https://tinyurl.com/3esfpkmv .

Acknowledgments :

The authors thank Dr. Fabio Rocha Jimenez Vieira (IBENS) for assistance with the decontamination of the OneKp transcriptome dataset; and Ms. Cathy Johnston (CSIRO), Dr. Maike Lorenz (Georg-August-Universität Göttingen), Prof. Michael Melkonian (Universität Köln) and Mr. Stephen Peña (UTEX Culture Collection) for the assistance of strain isolation sites tabulated in Dataset S1. both METH and METE, by combined RbH, PFAM domain and single-gene phylogenetic analysis ; and the number of species from these groups for which at least one B12associated enzyme was detected (the following, plus METE or METH) inferred to possess homologues of MTRR, MCM, RNR II, CBLA, CBLB, CBLC, CBLD, CBLF, CBLJ, CBLX or epi-CBLC via the same methodology. The total number of species assessable in each case is given to the right-hand side of each plot. Abbreviations : KMC-Klebsormidiaceae, Mesostigmatophyceae, Chlorokybophyceae ; ZCC-Zygnematophyceae, Charophyceae, Coleochaetophyceae. Complete tabulated occurrences per lineage and species are provided in Dataset S1, sheets 1-2 ; and individual homologue lists per gene in Dataset S1, sheets 5-17. 2. Overview of the libraries searched for presence of B12-containing homologues. Totals are provided for the unique numbers of species searched in different taxonomic groups, divided by library type (genome, 1kp transcriptome, MMETSP transcriptome, and other including single-cell genome and independent transcriptome libraries). Totals are provided by species as opposed to strain, and therefore e.g. three searched hornwort genomes (Anthoceros agrestis Oxford, A. agrestis Bonn, A. punctigera) [START_REF] Li | Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts[END_REF] are tabulated as two distinct species. Species for which both genome and 1kp/ MMETSP transcriptomes have been sequenced (e.g., A. agrestis) are tabulated in both columns. Acronyms : ZCC-Zygnematophytes, Coleochaetales, Charophytes ; KMC-Klebsormidiophyes, Mesostigma, Chlorokybus.

  detection : Potential homologues of thirteen Vitamin B12-associated enzymes (Chlamydomonas METE with GBID : AAC49178.1, METH : XP_042923308.1; human CblA : NP_001362573.1, CblB : NP_443077.1, CblC : NP_056321.2, CblD : NP_056517.1, CblF : NP_060838.3, CblJ : NP_001340531.1, CblX : NP_005325.2, epi-CblC : NP_001189360.1, MTRR : AAF16876.1 ; Phaeodactylum MCM : XP_002179504.1 ;

Funding information :

 information RGD acknowledges an ERC Starting Grant (« ChloroMosaic » ; grant number 101039760), awarded 2023-2027. Work at the IBENS is supported by the Investissements d'Avenir programmes PSL and MEMOLIFE: MEMO LIFE (ANR-10-LABX-54), and PSL*Research University (ANR-11-IDEX-0001-02).

Fig. 1 .Fig. 2 .

 12 Fig.1. Known B12 uptake and utilisation pathways in eukaryotes. This figure shows a schematic eukaryotic cell, with potential B12-associated proteins detected in previous studies across the eukaryotes, labelled following[START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF] and[START_REF] Helliwell | Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes[END_REF]. The geneIDs, origins and PFAM domains associated with each query protein for the phylogenetic analysis are tabulated below.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Phylogenetic trees of B12-dependent metabolism across photosynthetic eukaryotes. This figure shows best-scoring RAxML trees realised across 350 bootstrap replicates with the PROTGAMMAJTT substitution matrix for METE, METH, MTRR, MCM and RNR II enzymes. Suspected dinoflagellate and archaeal METE sequences are shown in a separate panel to the remaining tree topology, due to the high divergence (> 2.0 substitutions per site) observed from all other aligned sequences. Taxa are coloured by phylogenetic origin: dark green-vascular plants and mosses; light green-liverworts;

Fig. 5 .

 5 Fig. 5. Vertical inheritance of hornwort B12 metabolism. This figure shows a concatenated multigene tree topology for all OneKp transcriptomes, taken from [38]. Taxa names are shown by taxonomic affiliation: dark green-vascular plants and mosses; light green-liverworts; cyan-hornworts; dark yellow-other streptophytes; orange-chlorophytes; dark brown-other algae. Branches are shaded by inferred B12 status: red-presence of B12-dependent metabolism only; purple-presence of B12dependent and -independent metabolism; light blue: B12-independent metabolism only; grey-unknown B12 status. For concision, vascular plants (universally lacking B12dependent metabolism) are collapsed to a single branch. Liverworts are marked as « unknown » due to the uncertain function of their encoded CblB and CblJ proteins. Major algal lineages are labelled, alongside invididual species of interest and algal clades with inferred losses of B12-associated metabolism. ; and a nexus format of the schematic OneKp concatenated topology in Dataset S1, sheet 21.

Fig. S1 .

 S1 Fig. S1. Metabolic functions of Vitamin B12-associated metabolism in plants. This figure shows boxplots, displayed using BoxPlotR [108], of the -log10 of the calculated hmmer e-value scores of key PFAM domains observed for each protein [94] of all phylogenetically reconciled homologues of eight potential B12-associated proteins found in hornworts : METE, METH, MTRR, CBLB, CBLC, CBLD, CBLJ and epi-CBLC. Values are shown for four taxonomic groups: eukaryotic algae ; hornworts ; liverworts ; and mosses and vascular plants (grouped together by the absence of B12 metabolism as

Vascular_plants Mosses Hornworts Liverworts Charophytes:_ZCC_clade Charophytes:_KMC_clade Chlorophytes Glaucophytes Red_algae Cryptomonads Haptophytes Ochrophytes

						MetH only	MetE and MetH	MetE only						# species
	Secondary_green_lineages																		11
	Alveolates																		47
	Ochrophytes																		165
	Haptophytes																		26
	Cryptomonads																		19
	Red_algae																		33
	Glaucophytes																		4
	Chlorophytes																		136
	Charophytes:_KMC_clade																		3
	Charophytes:_ZCC_clade																		36
	Liverworts																		24
	Hornworts																		7
	Mosses																		37
	Vascular_plants																		642
		0%			10%			20%				30%				40%			50%
	MTRR	0%			20%			40%	MCM	60%				80%	RNR RNR-II	100%
	Secondary_green_lineages
	Alveolates
	Ochrophytes
	Haptophytes
	Cryptomonads
	Red_algae
	Glaucophytes
	Chlorophytes
	Charophytes:_KMC_clade
	Charophytes:_ZCC_clade
	Liverworts
	Hornworts
	Mosses
	Vascular_plants	0%	20%	40%	60%	80%	100%	0%	20%	40%		60%	80%	100%	0%	20%	40%	60%	80%	100%
	CblA									CBlB							CblC
	Secondary_green_lineages
	Alveolates
	Ochrophytes
	Haptophytes
	Cryptomonads
	Red_algae
	Glaucophytes
	Chlorophytes
	Charophytes:_KMC_clade
	Charophytes:_ZCC_clade
	Liverworts
	Hornworts
	Mosses
	Vascular_plants														0%	20%	40%	60%	80%	100%
		0%	20%	40%	60%	80%	100%	0%	20%	40%		60%	80%	100%
	CblD									CblF							CblJ
	Secondary_green_lineages
	Alveolates
		0%	20%	40%	60%	80%	100%
								0%	20%	40%		60%	80%	100%
		CblX								epiCblC					0% 20% 40% 60% 80% 100%
	Alveolates Secondary_green_lineages																Key
	Ochrophytes															n Species with MetE
	Haptophytes																homologue only
	Glaucophytes Cryptomonads Red_algae															n Species with MetH homologue only
	Charophytes:_ZCC_clade Chlorophytes Charophytes:_KMC_clade															n Species with MetE and MetH homologue
	Mosses Liverworts Hornworts															n Species with MetE/ MetH and protein homologue
	Vascular_plants															Species with MetE/MetH
		0% 20% 40% 60% 80% 100%	0%	20% 40% 60% 80% 100%	but no protein homologue

  , CBLC, CBLD, CBLF, CBLJ, CBLX and epi-CBLC proteins. Taxa are phylogenetic origin: dark green-vascular plants and mosses; light green-liverworts; cyanhornworts; dark yellow-other streptophytes; orange-chlorophytes; dark brown-other algae; black-other lineages. Branch thickness corresponds to bootstrap support.

				Dinoflagellates
				Glaucophytes
	Cryptomonads, Phaeophytes	Chlorodendrophytes,		(Phaeoceros,Noteroclada, Plant Clade III epi-CblC 0.3
		Pycnococcus,		Monoclea, Calpygoeia)
		Chromochloris
		Liverworts
		Hornworts	CblX 0.2	Plant Clade II (Phaeoceros)
	CblJ
	0.3		Plant Clade I (Phaeoceros,
				Scapania)
			Pycnococcus,
	Pyramimonadales	Mesostigma
		Skeletonema

CBLB

Table 1 .

 1 to phylogeny). Greater values imply greater fidelity to the PFAM hmm ; groups for which no homologues were identified are plotted as a -1 value. For the METH sequences, an alignment is additionally provided of a 20 aa region of the first substrate binding pocket of the pterin binding domain of hornwort METH against the equivalent Chlamydomonas sequence following[START_REF] Lin | Vitamin B12-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes (metE)[END_REF], and showing broad conservation of key residues and inferred functions. An overview of the PFAM values is given in Dataset S1, sheet 3; with individual scores for each homologue provided in Dataset S1, sheets 5-17. SignalP and TargetP predictions. Except for MCM which shows broadly mitochondrial targeting predictions and CblF with predominantly endomembrane localisations, no patterns are consistently observed. B : wheel plots of the taxonomic composition of mitochondria-targeted MCM sequences, showing broad conservation of this localisation across algae with secondary red chloroplasts ; and of endomembrane-targeted CblF sequences, which are restricted to dinoflagellates. Overview of the query sequences used for phylogenetic searches, as obtained from human[START_REF] Hannibal | Intracellular processing of vitamin B12 by MMACHC (CblC)[END_REF] and algal gene sequences[START_REF] Helliwell | Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes[END_REF].

	This

phylogenetically reconciled homologues of eight potential B12-associated proteins found in hornworts : METE, METH, MTRR, CBLB, CBLC, CBLD, CBLJ and epi-CBLC. Values are shown for four taxonomic groups: eukaryotic algae ; hornworts ; liverworts ; and mosses and vascular plants (grouped together by the absence of B12 metabolism as opposed

Table

Table 3 .

 3 Identified species inferred to have lost Vitamin B12-associated metabolism, based on the retention of METE and minimally the absence of METH and MTRR sequences as verified by phylogenetic and PFAM analysis. A detailed map of the recorded collection sites of all algae included in the dataset is provided at https://tinyurl.com/3esfpkmv .

	Taxonomic Group	1kp	Genome	MMETSP	Other
	Vascular plants	968	3	0	0
	Mosses	48	1	0	0
	Hornworts	11	2	0	0
	Liverworts	34	1	0	0
	Charophytes: ZCC clade	43	0	0	0
	Charophytes: KMC clade	3	1	0	0
	Chlorophytes	114	14	52	2
	Glaucophytes	5	3	1	0
	Red algae	32	6	10	0
	Cryptomonads	5	1	19	0
	Haptophytes	4	2	29	0
	Ochrophytes	24	12	133	30
	Alveolates	1	5	46	0
	Secondary green lineages	1	2	15	0